Stereospecificity of amino acid side chains in deltorphin defines binding to opioid receptors. A series of individual D-amino acid replacement analogues of deltorphin A, several of which were in combination with a His4 deletion, were used to probe alterations of side-chain orientation on peptide binding parameters with rat brain opioid receptors. Peptides with D-amino acids in residues 1, 3, and 5 exhibited diminished affinities primarily for delta receptors (88-1200-fold) with selectivity decreasing by factors of 13-64-fold relative to deltorphin A (Ki delta = 0.45 nM; Ki mu/Ki delta = 764): the aromatic side chains Tyr1 and Phe3, which lie in the N-terminal "message" domain and the aryl side chain of Leu5 in the C-terminal "address" domain, appear to play essential roles in conferring high delta affinity and selectivity. Although D-His4 only decreased delta affinity by 6-fold and selectivity by a factor of 4, His appears to be involved as an integral component of both domains: [des-His4]deltorphin A and [des-His4] analogues containing consecutive D-amino acid replacements in the remaining residues exhibited weak binding to delta receptors and poor delta selectivity. Substitution of D-Met2 in deltorphin A by D-Ala or D-Nle decreased delta selectivities 3-6-fold through an elevation in mu affinities; however, the converse replacement, D-Met for D-Ala2 in deltorphin B, diminished beta selectivity by an order of magnitude only through the loss in delta affinity. The data show that the high delta affinity and selectivity of deltorphins correlate with and require a strict stereospecificity of the amino acid residue side chains.


Development of 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (L-670,630) as a potent and orally active inhibitor of 5-lipoxygenase. Leukotrienes are potent biological mediators of allergic and inflammatory diseases and are derived from arachidonic acid through the action of the 5-lipoxygenase. In this study, the syntheses and comparative biological activities of three series of 2,3-dihydro-2,6-disubstituted-5-benzofuranols with various substituents on position 3 are described. Compounds from each series were evaluated for their ability to inhibit the production of leukotriene B4 (LTB4) in human peripheral blood polymorphonuclear (PMN) leukocytes and the 5-lipoxygenase reaction in cell-free preparations from rat PMN leukocytes. The structure-activity relationships of each series in vitro and in vivo are presented. The bioavailability, metabolism, and toxicity profile of each series are discussed. The series with no substituent at position 3 was the most potent and among the compounds in that series 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (46, L-670,630) was chosen for further development.


Evidence for independent molecular identity and functional interaction of the haemagglutinin and cysteine proteinase (gingivain) of Porphyromonas gingivalis. The sequence of events involved in haemagglutination and lysis of erythrocytes by washed cells, vesicles and the culture supernate of Porphyromonas gingivalis strain W83 was monitored by 51Cr release and transmission electronmicroscopy. All preparations, except capsular material and lipopolysaccharide, caused haemagglutination and, by a slow process of attachment and specific attack on the surface structures of the red blood cells, produced minute pores and eventual leakage of cellular contents. N-acetylglucosamine, N-acetylgalactosamine and several other sugars such as glucose and sucrose had no effect on haemagglutination. Antiserum raised against a cloned haemagglutinin of P. gingivalis strain 381 inhibited the activity of strain W83 cells, vesicles and supernate. The antiserum-neutralised supernate lost 70-80% of its hydrolytic activity towards alpha-N-benzoyl-L-arginine-4-nitroanilide but the residual activity behaved in a manner similar to the native supernate in that it was completely inhibited by the addition of 2,2'-dipyridyl disulphide and was fully restored upon addition of a low-Mr mercaptan. Binding of the antiserum to the haemagglutinin epitope of P. gingivalis still permitted titration of the active centre cysteinyl thiol group of the proteinase. Purified gingivain caused lysis of erythrocytes and was not neutralised by antiserum to the haemagglutinin. These results suggest that, although the haemagglutinin and gingivain are probably separate molecules, they are closely associated on the outer membrane of P. gingivalis and may be functionally related.


Effect of bioamines on uptake of promastigotes of Leishmania donovani by hamster peritoneal macrophages. Epinephrine and norepinephrine inhibit attachment of Leishmania donovani promastigotes to cultured hamster peritoneal macrophages. The inhibition was significant at catecholamine concentrations of 10(-4) and 10(-5) M and occurred when they were added to the cell mixtures, or after pre-treatment of either macrophages or parasites. Inhibition of attachment after pre-treatment was less marked than when the catecholamines were added to parasite-cell mixtures. Similar results were obtained with dibutyryl cyclic AMP, cholera toxin, theophylline, and cadaverine which raise intracellular cyclic AMP (cAMP). Pretreatment of parasites or macrophages with the bioamines elevated the intracellular cAMP concentration. It is suggested that the inhibitory effect on the host-parasite interaction is mediated through cAMP.


Upstream curved sequences influence the initiation of transcription at the Escherichia coli galactose operon. The two overlapping promoters that control mRNA synthesis at the galactose operon contain three phased stretches of adenine residues, located around positions -84.5, -74 and -63, with respect ot the start of the P1 promoter. As a result, the corresponding DNA sequence is bent, an anomaly that is relieved by the addition of small concentrations of drugs like distamycin A or netropsin. By abortive initiation assays performed on several DNA fragments derived from the wild-type promoter or from various mutants we show that the curved sequence increases the strength of the P1 promoter. In the absence of cyclic AMP (cAMP) and of the corresponding receptor protein (CRP), the upstream curved sequences enhance the rate of isomerization from the closed to the open complex at P1. This effect is abolished when distamycin A is bound in the bent region. In the presence of cAMP-CRP, a more drastic change is observed: activation of the gal P1 promoter takes place at a different formal step, depending whether the upstream curved sequence is present or not (enhancement of the rate of conversion from a closed to an open complex instead of an increase in the affinity of the enzyme during closed complex formation). These data, together with previous results obtained with other mutants of the gal control region, suggest that several closed complexes corresponding to different nucleoprotein arrangements are formed during open complex formation at gal P1, in the presence of CRP.


Biogenesis of outer membrane protein PhoE of Escherichia coli. Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein. Efficient in vivo translocation of the precursor of Escherichia coli outer membrane protein PhoE across the inner membrane is shown to depend on SecB protein. A set of mutants, carrying internal deletions in the phoE gene, was used to locate a possible SecB-binding site and/or a site that makes the protein dependent on SecB for export. Except for two small mutant PhoE proteins, the in vivo and in vitro translocation of all mutant proteins was more efficient in the presence of SecB. The interaction of SecB protein with wild-type and mutant PhoE proteins, synthesized in vitro, was further studied in co-immunoprecipitation experiments with anti-SecB protein serum. The efficiencies of co-immunoprecipitation of precursor and mature PhoE were very similar, indicating the absence of a SecB-binding site in the signal sequence. Moreover, all mutant proteins with deletions in the mature moiety of the PhoE protein were co-immunoprecipitated in these assays, albeit mostly with reduced efficiency. Taken together, these results indicate the existence of multiple SecB-binding sites in the mature portion of the PhoE protein.


Three-dimensional structure of acylphosphatase. Refinement and structure analysis. We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.


Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin. The effects of solvation and charge-charge interactions on the pKa of ionizable groups in bacteriorhodopsin have been studied using a macroscopic dielectric model with atom-level detail. The calculations are based on the atomic model for bacteriorhodopsin recently proposed by Henderson et al. Even if the structural data are not resolved at the atomic level, such calculations can indicate the quality of the model, outline some general aspects of electrostatic interactions in membrane proteins, and predict some features. The effects of structural uncertainties on the calculations have been investigated by conformational sampling. The results are in reasonable agreement with experimental measurements of several unusually large pKa shifts (e.g. the experimental findings that Asp96 and Asp115 are protonated in the ground state over a wide pH range). In general, we find that the large unfavorable desolvation energies of forming charges in the protein interior must be compensated by strong favorable charge-charge interactions, with the result that the titrations of many ionizable groups are strongly coupled to each other. We find several instances of complex titration behavior due to strong electrostatic interactions between titrating sites, and suggest that such behavior may be common in proton transfer systems. We also propose that they can help to resolve structural ambiguities in the currently available density map. In particular, we find better agreement between theory and experiment when a structural ambiguity in the position of the Arg82 side-chain is resolved in favor of a position near the Schiff base.


Spontaneous production of growth factors for human lymphocytes from a human papillomavirus type 18-contained foreskin fibroblast cell line. An immortalized fibroblast cell line, designated as CCFS-1/KMC, derived from human neonatal foreskin fibroblasts, contained human papillomavirus (HPV) type 18 DNA. Since this newly established cell line could spontaneously secrete activating factors for normal human blood lymphocytes, the synthesis and release of potent inflammatory cytokines from this cell line were checked. To determine the presence of cytokines in the supernatant collected from the cell line, tests by a cytokine-specific ELISA and a mitogenesis bioassay were done. The cell line could spontaneously produce several immunoreactive cytokines, such as tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6). It also could stimulate the mitosis of human blood lymphocytes and Raji lymphoblast cell line. These cytokines were present in the same fraction of isoelectric points (pI) from 5.4 to 5.6. This study suggests that non-immune bystander cells may exert immunomodulatory effect on the immune cells via the production of potent inflammatory cytokines during viral infection.


Prevalence of transposons encoding kanamycin, ampicillin and trimethoprim resistance in isolates from urinary tract infections detected using DNA probes. Drug resistant Gram-negative bacteria causing urinary tract infections were collected. Kanamycin, ampicillin or trimethoprim-resistant strains were analyzed separately for the presence of Tn5, Tn3, or Tn7 by colony hybridization. Of these isolates, kanamycin-resistant transposons were present in 38.2% of 60 kanamycin-resistant isolates. A 3.3 kb fragment containing SacI-BamHI transposase of Tn3 and 42.6% showed a positive reaction in 129 ampicillin-resistant clinical isolates. Among the 75 trimethoprim-resistant isolates studied, 52% were shown to contain Tn7 when probed with a 1 kb BamHI fragment of Tn7. Results from Southern hybridizations demonstrated that these antibiotic resistant genes had been born on plasmids in some clinical isolates.


Expression of the Marek's disease virus (MDV) homolog of glycoprotein B of herpes simplex virus by a recombinant baculovirus and its identification as the B antigen (gp100, gp60, gp49) of MDV. A gene encoding a homolog of glycoprotein B of herpes simplex virus (gB homolog) has been identified on the Marek's disease virus (MDV) genome (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989); however, the molecular and immunological characteristics of the gene product(s) are still not clear. In the present study, the gB homolog of MDV was expressed in insect cells by a recombinant baculovirus, and it was characterized to determine its molecular and antigenic properties. The expressed recombinant protein had three molecular sizes (88 to 110, 58, and 49 kDa) and was recognized by antisera from chickens inoculated with each of the three serotypes of MDV. By immunofluorescence analysis, it was shown that the protein was expressed in the cytoplasm and on the surface of the recombinant baculovirus-infected cells. The gB homolog of MDV was processed similarly to pseudorabies virus and varicella-zoster virus with respect to cleavage and the intramolecular disulfide bond between the cleaved products. Interestingly, the expressed protein reacted with monoclonal antibody M51, specific to the B antigen (gp100, gp60, gp49) of MDV, although the locations of the gene encoding the B antigen and of the gene encoding the gB homolog were reported to be different. Moreover, competitive experiments revealed that anti-gB homolog serum and monoclonal antibody M51 recognized the same molecules. From these results, the gB homolog and the B antigen of MDV seem to be the same glycoprotein.


Bovine immunodeficiency-like virus encodes factors which trans activate the long terminal repeat. Lentiviruses are known to encode factors which trans activate expression from the viral long terminal repeat (LTR); the primary trans activator is the tat gene product. One of the putative accessory genes (tat) of the bovine immunodeficiency-like virus (BIV) bears sequence similarity to other lentivirus tat genes. This finding suggests that BIV may encode a trans-activating protein capable of stimulating LTR-directed gene expression. To test this hypothesis in vitro, BIV LTR-chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed and transfected into three cell lines established from canine, bovine, or lapine tissues that are susceptible to BIV infection. The level of BIV LTR-directed CAT gene expression was significantly elevated in BIV-infected cells compared with uninfected cells. The relatively high basal-level expression of BIV LTR-CAT in uninfected canine and bovine cell lines suggests that cellular factors play a role in regulating BIV LTR-directed gene expression. Additionally, by using a clonal canine cell line in which the BIV LTR-CAT plasmid is stably expressed, BIV LTR-directed CAT expression is elevated 15- to 80-fold by cocultivation with BIV-infected cells, supporting the notion that BIV encodes a trans activator. The relative specificity of this viral activation was assessed by coculturing the clonal BIV LTR-CAT cell line with bovine leukemia virus- or bovine syncytial virus-infected cells; these bovine retroviruses increased expression from the BIV LTR only two- to threefold. Thus, BIV LTR regulatory elements in infected cells, like those of human immunodeficiency virus type 1 and other lentiviruses, are trans activated, presumably through the action of a Tat-like protein and cellular factors.


Identification and characterization of the herpes simplex virus type 1 virion protein encoded by the UL35 open reading frame. The UL35 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) has been predicted from DNA sequence analysis to encode a small polypeptide with a molecular weight of 12,095. We have investigated the protein product of the UL35 ORF by using a trpE-UL35 gene fusion to produce a corresponding fusion protein in Escherichia coli. The TrpE-UL35 chimeric protein was subsequently isolated and used as a source of immunogen for the production of rabbit polyclonal antiserum directed against the UL35 gene product. The TrpE-UL35 antiserum was found to recognize a 12-kDa protein which was specifically present in HSV-1-infected cells. By utilizing the TrpE-UL35 antiserum, the kinetics of synthesis of the UL35 gene product was examined, and these studies indicate that UL35 is expressed as a gamma 2 (true late) gene. The 12-kDa protein recognized by the TrpE-UL35 antiserum was associated with purified HSV-1 virions and type A and B capsids, suggesting that the UL35 ORF may encode the 12-kDa capsid protein variably designated p12, NC7, or VP26. To confirm this assignment, immunoprecipitation and immunoblotting studies were performed to demonstrate that the TrpE-UL35 antiserum reacts with the same polypeptide as an antiserum directed against the purified p12 capsid protein (anti-NC7) (G.H. Cohen, M. Ponce de Leon, H. Diggelmann, W.C. Lawrence, S.K. Vernon, and R.J. Eisenberg, J. Virol. 34:521-531, 1980). Furthermore, the anti-NC7 serum was also found to react with the TrpE-UL35 chimeric protein isolated from E. coli, providing additional evidence that the UL35 gene encodes p12. On the basis of these studies, we conclude that UL35 represents a true late gene which encodes the 12-kDa capsid protein of HSV-1.


Hepatitis B virus (HBV)-specific cytotoxic T-cell response in humans: production of target cells by stable expression of HBV-encoded proteins in immortalized human B-cell lines. To analyze the hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response during acute and chronic viral hepatitis, target cells that express HBV-encoded antigens in the context of the appropriate HLA restriction element must be available for each subject studied. Since HBV is not infectious for human cells in vitro, such target cells must be produced by DNA-mediated gene transfer into cultured human primary cells or cell lines. For this purpose, we have developed a panel of Epstein-Barr virus-based episomal expression vectors containing each of the HBV open reading frames under the transcriptional control of the simian virus 40 early promoter. Transfection of Epstein-Barr virus-immortalized B-cell lines with this panel of recombinants consistently leads to stable expression of the HBV envelope, nucleocapsid, and polymerase proteins. The HBV X gene product is transiently expressed following transfection, but stable expression of this protein cannot be maintained on a long-term basis. To assess the suitability of this system for the identification of HBV-specific CTL in humans, a panel of EBO-HBV transfectants of defined HLA haplotype was used to monitor the HBV-specific CTL response in a patient with acute viral hepatitis type B. Transfectants that stably express the HBV nucleocapsid (core) antigen were found to serve as excellent targets for the detection of HLA class I-restricted CTL that recognize endogenously synthesized HBV core antigen in this patient; they were also successfully used to stimulate the specific expansion of these CTL in vitro.


Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. Epstein-Barr virus (EBV) genome-positive nasopharyngeal carcinomas (NPCs) regularly express the virus-coded nuclear antigen EBNA1, but not other EBNAs, and a subset of tumors also appear to be latent membrane protein LMP1 positive; the status of NPCs with respect to a second virus-coded latent membrane protein LMP2 is unknown. In the present work the EBV-NPC cell interaction has been analyzed at the RNA level with reverse transcription and polymerase chain reaction-based amplification to detect specific latent viral mRNAs. All four transplantable NPC cell lines studied and 17 of 18 fresh snap-frozen NPC biopsy specimens expressed an EBNA1 mRNA with a BamHI Q/U/K splice structure exactly like that recently identified in group I Burkitt's lymphoma (BL) cell lines and shown to be driven from a novel viral promoter, Fp. The BamHI Y3/U/K-spliced EBNA1 mRNA characteristic of virus-transformed B-lymphoblastoid cell lines (LCLs) was never found in NPCs. These same NPC biopsy specimens were then analyzed for evidence of the various LMP transcripts which are constitutively expressed in LCLs but down-regulated in BL cells. While only 3 of 18 tumors gave a clear LMP1 mRNA-specific signal after first-round amplification with either of two sets of polymerase chain reaction primers, the majority proved to be LMP1 mRNA positive after second-round amplification with nested primers. A rather similar pattern of results was obtained with respect to LMP2B mRNA expression, such transcripts being detectable only in a subset of tumors, and then at apparently low levels. In contrast, clear evidence of LMP2A mRNA expression was obtained in 17 of 17 fresh biopsies. The predominant form of EBV infection in NPCs, with coexpression of EBNA1 and LMP mRNAs, is therefore quite distinct from that seen in BL cells (in which EBNA1 is the only expressed mRNA) and in LCL cells (in which all six EBNA and three LMP transcripts are present). This third form of EBV latency may not be restricted to NPC but may have more general relevance in the context of EBV infection in vivo.


Mutations within the 5' half of the avian retrovirus MC29 v-myc gene alter or abolish transformation of chicken embryo fibroblasts and macrophages. Avian myelocytomatosis virus MC29 induces a wide variety of neoplastic diseases in infected birds and transforms cells of the macrophage lineage as well as fibroblasts and epithelial cells. A biological and biochemical analysis, carried out on a series of in-frame insertion and deletion mutations within the gag-myc gene of MC29, revealed several mutations within the 5' portion of the v-myc gene that encode proteins either completely defective for transformation or compromised in their ability to transform chicken embryo fibroblasts but not macrophages. Mutations within the 3' end of the v-myc gene which disrupt sequences encoding the basic/helix-loop-helix region were defective for transformation of both fibroblasts and macrophages. Eight variants were cloned into the replication-competent avian expression vector RCAS. Analysis of cells infected with transformation-defective, replication-competent viruses confirmed the expression of functionally defective Myc proteins. Further, expression of the transformation defective variant dl91-137 in chicken fibroblasts inhibited subsequent transformation by wild-type MC29. The results reported herein support the hypothesis that Myc proteins function as regulators of transcription in a variety of cell types and clearly point out the necessity of putative regulatory domains within the amino-terminal half of the Myc protein.


Association of ICP0 but not ICP27 with purified virions of herpes simplex virus type 1. Recent studies have shown that ICP4, one of the major immediate-early proteins of herpes simplex virus type 1 is present within the tegument region of the virion (F. Yao and R. J. Courtney, J. Virol. 63:3338-3344, 1989). With monoclonal antibodies to two additional immediate-early proteins, ICP0 and ICP27, and Western blot (immunoblot) analysis, ICP0, but not ICP27, was also found to be associated with purified virus particles. In an effort to localize the ICP0 within the virion, purified virions were treated with trypsin in the presence and absence of detergent. The data suggest that ICP0 is located within the tegument region of the virion and is not localized in the envelope or within the nucleocapsid. The number of molecules of ICP0 per virion was estimated to be approximately 150.


Nucleotide 880 splice donor site required for efficient transformation and RNA accumulation by human papillomavirus type 16 E7 gene. Mutations within coding sequences of the various human papillomavirus type 16 (HPV-16) genes have been used to demonstrate that the HPV-16 E7 gene is necessary and sufficient for transformation of rodent cells. We now provide evidence that, in addition to E7 coding sequences, a small cis-acting region immediately flanking the 3' end of E7 coding sequences is also required for transformation. This was shown by translation termination linker insertion, progressive deletion analysis, and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within the 3'-flanking region by deletion of as few as 4 nt or substitution of 3 nt totally abolished transformation. Regeneration of the wild-type sequence in a previously transformation-incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in a stronger transformation phenotype, while mutating the +2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the amount of E7 mRNA in transformation-deficient splice site mutants was much lower. Nuclear runoff experiments revealed that there was no change in the rate of synthesis of E7 message in the nt 880 splice site mutant. Furthermore, mutations of HPV-16 sequences indicated that the two other early region splice donor sites have no more than minor roles in transformation and efficient RNA accumulation. These results indicate that the specific integrity of the nt 880 splice donor site is essential for both accumulation of E7 RNA and efficient E7-mediated transformation.


Structural and functional characterization of the poliovirus replication complex. Two populations of membrane-bound replication complexes were isolated from poliovirus-infected HEp-2 cells by sucrose gradient centrifugation. The two fractions show similar ultrastructural features: the replication complex is enclosed in a rosettelike shell of virus-induced vesicles and contains a very tightly packed second membrane system (compact membranes). The vesicular fraction, which bands in 30% sucrose, contains replicative intermediate (RI) and 36S RNA. The fraction banding in 45% sucrose contains only minute amounts of RI and contains mainly 36S RNA, two-thirds of which is encapsidated. In vitro, the two fractions show similar RNA synthesizing capacities and produce 36S plus-strand RNA. Dissolving the membranes within and around synthetically active replication complexes with sodium deoxycholate abolishes the completion of 36S RNA but still allows elongation in the RI. Our findings suggest an architecture of the replication complex that has the nascent plus strands on the RI enclosed in the compact membranes and the replication forks wrapped additionally in protein. Plus-strand RNA can be localized by in situ hybridization with a biotinylated riboprobe between the replication complex and the rosette of the virus-induced vesicles. It was found that the progeny RNA strands are set free soon after completion from the replication complex at the sites where the compact membranes within the replication complex are in close contact with the surrounding virus-induced vesicles.


Recombinant vaccine for canine parvovirus in dogs. VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-assemble, forming viruslike particles similar in size and appearance to CPV virions. These viruslike particles have been used to immunize dogs in different doses and combinations of adjuvants, and the anti-CPV responses have been measured by enzyme-linked immunosorbent assay, monolayer protection assays, and an assay for the inhibition of hemagglutination. A dose of ca. 10 micrograms of VP2 was able to elicit a good protective response, higher than that obtained with a commercially available, inactivated vaccine. The results indicate that these viruslike particles can be used to protect dogs from CPV infection.


Stable rescue of a glycoprotein gII deletion mutant of pseudorabies virus by glycoprotein gI of bovine herpesvirus 1. Glycoproteins homologous to glycoprotein B (gB) of herpes simplex virus constitute the most highly conserved group of herpesvirus glycoproteins. This strong conservation of amino acid sequences might be indicative of a common functional role. Indeed, gB homologs have been implicated in the processes of viral entry and virus-mediated cell-cell fusion. Recently, we showed that pseudorabies virus (PrV) lacking the essential gB-homologous glycoprotein gII could be propagated on a cell line expressing the gB homolog of bovine herpesvirus 1, gI(BHV-1), leading to a phenotypic complementation of the gII defect (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65:621-631, 1991). However, this pseudotypic virus could still replicate only on complementing cell lines, thereby limiting experimental approaches to analyze the effects of the gB exchange in detail. We describe here the construction and isolation of a PrV recombinant, 9112C2, that lacks gII(PrV) but instead stably carries and expresses the gene encoding gI(BHV-1). The recombinant is able to replicate on noncomplementing cells with growth kinetics and final titers similar to those of its gII-positive wild-type PrV parent. Neutralization tests and immunoprecipitation analyses demonstrated incorporation of gI(BHV-1) into 9112C2 virions with concomitant absence of gII(PrV). Analysis of in vitro host ranges of wild-type PrV, BHV-1, and recombinant 9112C2 showed that in cells of pig, rabbit, canine, monkey, or human origin, the plating efficiency of 9112C2 was similar to that of its PrV parent. Exchange of gII(PrV) for gI(BHV-1) in recombinant 9112C2 or by phenotypic complementation of gII- PrV propagated on gI(BHV-1)-expressing cell lines resulted in penetration kinetics intermediate between those of wild-type PrV and BHV-1. In conclusion, we report the first isolation of a viral recombinant in which a lethal glycoprotein mutation has been rescued by a homologous glycoprotein of a different herpesvirus. Our data show that in gII- PrV, gI(BHV-1) in vitro fully complements the lethal defect associated with lack of gII(PrV). These results conclusively demonstrate that gI(BHV-1) in a PrV background can execute all essential functions normally provided by gII(PrV). They also indicate that the origin of gB-homologous glycoproteins influences the penetration kinetics of herpesviruses.


Immediate-early RNA 2.9 and early RNA 2.6 of bovine herpesvirus 1 are 3' coterminal and encode a putative zinc finger transactivator protein. Bovine herpesvirus 1 (BHV-1) contains three major immediate-early (IE) genes involved in regulation of the productive cycle of replication. Two spliced IE RNAs, IER4.2 (4.2 kb) and IER2.9 (2.9 kb), are under the control of a single promoter; IER1.7 (1.7 kb) is transcribed from a different promoter in the opposite direction. Examining the kinetics of transcription, we found that the IER4.2/2.9 promoter was turned off at the end of the IE period. An alternative promoter became active, directing synthesis of an unspliced early RNA, ER2.6 (2.6 kb), which was colinear with the second exon of IER2.9 except for its 5' end in the intron about 10 bases upstream of the splice site. Sequence analysis revealed a single open reading frame common to IER2.9 and ER2.6 with a coding potential of 676 amino acids. The putative protein, named p135, contained a cysteine-rich zinc finger domain near the N terminus with homology to ICP0 of herpes simplex virus type 1, to protein 61 of varicella-zoster virus, to early protein 0 of pseudorabies virus, and to other viral and cellular proteins. The remaining parts of p135 exhibited only limited homology, mainly with pseudorabies virus protein 0, but the entire sequence was highly conserved between two strains of BHV-1 (K22 and Jura). The latency-related antisense transcript covered a large portion of ER2.6 excluding the zinc finger coding region. In transient expression assays, p135 activated a variety of promoters, including that for ER2.6, but repressed the IER1.7 promoter. Thus, p135 combines functional characteristics of ICP0, a strong transactivator, and of protein 61, a repressor. BHV-1 seems to have evolved a subtle mechanism to ensure the continued synthesis of p135 while turning off IER4.2, which encodes p180, the herpes simplex virus type 1 ICP4 homolog.


Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen. Mouse C3H 10T1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 10T1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 10T1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.


Formation of heterotrimers between the membrane-integrated and the soluble glycoproteins of vesicular stomatitis virus leads to their intracellular cotransport. BHK cells infected with vesicular stomatitis virus serotype Indiana generate intracellularly two different types of glycoproteins: the authentic membrane-integrated G protein of virions and a smaller soluble Gs protein lacking the transmembrane and cytoplasmic domains which is secreted into the growth medium. A Gs1 protein species which is formed during or shortly after translation in the endoplasmic reticulum lumen is modified in the same way as the G1 protein by endoglycosidase H-sensitive oligosaccharides of the high-mannose type. Both G1 and Gs1 are almost simultaneously transported, trimmed, and processed into G2 and Gs2 species which possess carbohydrate side chains of the complex type, making both glycoproteins resistant to endoglycosidase H cleavage. Secretion of Gs2 protein into the growth medium and arrival of G2 protein on the cell surface occur concomitantly. Membrane-integrated G protein and the soluble Gs protein molecules oligomerize intracellularly into heterotrimers which can be immunoprecipitated after chemical cross-linking. Gs protein seems to contain sufficient structural information for the formation of heterotrimers which are efficiently transported to the cell surface. Heterotrimer formation between G and Gs proteins explains the rapid secretion of Gs molecules.


Regulation of the Epstein-Barr virus DNA polymerase gene. The gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.


Identification of critical cis elements involved in mediating Epstein-Barr virus nuclear antigen 2-dependent activity of an enhancer located upstream of the viral BamHI C promoter. The six genes encoding the Epstein-Barr virus nuclear antigens (EBNAs) are transcribed from one of two promoters, BamHI C promoter (Cp) or BamHI W promoter (Wp), located near the left end of the viral genome. During the establishment of viral latency in B lymphocytes, Wp is used exclusively before a switch to Cp usage. We and others have previously identified an enhancer in the region upstream of Cp which requires EBNA 2 for activity (M. Woisetschlaeger, X. W. Jin, C. N. Yandava, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991; N. S. Sung, S. Kenney, D. Gutsch, and J. S. Pagano, J. Virol. 65:2164-2169, 1991). Infection of B lymphocytes with a mutant virus lacking the EBNA 2 gene results in prolonged usage of Wp and failure to switch to Cp usage, indicating that EBNA 2 transactivation of the enhancer upstream of Cp may be critical for promoter switching. In this study, we have defined the minimal EBNA 2-dependent enhancer by using a series of deletion mutants. The results of site-directed mutagenesis revealed that there are three regions of the enhancer that are important for activity, two of which appear to bind B-lymphocyte-specific factors.


Driving adenovirus type 12-transformed BALB/c mouse cells to express high levels of class I major histocompatibility complex proteins enhances, rather than abrogates, their tumorigenicity. The tumorigenicity of adenovirus type 12 (Ad12)-transformed cells has been attributed to the low levels of class I major histocompatibility complex (MHC) protein expression by these cells. These levels of class I proteins are thought to be below the threshold critical for cytotoxic T-lymphocyte recognition, a process that may be involved in tumor cell immunosurveillance. We have used gene transfer experiments to investigate the role played by class I protein expression in the tumorigenicity of Ad12-transformed BALB/c mouse cells in naive, syngeneic adult mice. Our Ad12-transformed mouse cells were tumorigenic in adult mice and were similar to other Ad12-transformed mammalian cells in that they expressed low levels of class I MHC mRNA and cell surface proteins. Despite these low levels of expression, the cells were highly immunogenic in syngeneic mice and were rejected as allografts by allogeneic mice. Transfection of genomic H-2Dd or H-2Ld fragments into these cells produced a variety of cell clones that expressed increased levels of cell surface class I proteins. These cells expressing high levels of class I protein were up to 16-fold more tumorigenic than the parental cells in syngeneic adult mice. Thus, by quantitative assays, the tumorigenicity of Ad12-transformed BALB/c mouse cells is not functionally related to the low levels of class I MHC proteins they express. The increased tumorigenicity expressed by H-2Dd- and H-2Ld-transfected cells was not detected in BALB/c nu/nu mice, suggesting that a thymus-dependent mechanism that is not mediated by evasion of cytotoxic T-lymphocyte recognition could contribute to the difference in tumorigenicity of Ad12-transformed BALB/c mouse cells that express low and high levels of class I MHC proteins.


Insertional mutagenesis of flvi-2 in tumors induced by infection with LC-FeLV, a myc-containing strain of feline leukemia virus. LC-FeLV is a myc-containing strain of feline leukemia virus (FeLV) which exhibits only partial transforming activity in vitro and in vivo. LC-FeLV infection in kittens may induce, but does not necessarily induce, thymic lymphosarcoma in viremic animals after a short latency. These observations suggest that infection with LC-FeLV is not sufficient to induce complete transformation and that another genetic event(s) is required. One possibility for such an event is that the integrating provirus acts as an insertional mutagen and thereby disrupts the structure or function of another proto-oncogene. Using a strategy of transposon tagging, this possibility was examined in eight feline T-cell lymphosarcomas, including four induced by experimental infection with LC-FeLV, three induced by natural infection with FeLV, and one FeLV-negative tumor. The analysis demonstrated one locus, termed flvi-2, to be structurally altered in six of the tumors examined, including three induced by LC-FeLV and three in which no activated myc oncogene is apparent. Inverse polymerase chain reaction was used to demonstrate the presence and transcriptional orientation of proviruses integrated at flvi-2 in five of these tumors. The flvi-2 locus does not hybridize to cloned probes representing 21 previously identified proto-oncogenes or common domains of retroviral integration. Thus, the data suggest that interruption of the flvi-2 locus cooperates with the myc oncogene in the induction of T-cell lymphomas by LC-FeLV; indeed, the observations indicate that the insertional mutagenesis of flvi-2 plays a role in T-cell lymphomagenesis even in the absence of feline v-myc.


Use of second-site homologous recombination to demonstrate that Epstein-Barr virus nuclear protein 3B is not important for lymphocyte infection or growth transformation in vitro. Recombinant Epstein-Barr viruses with a stop codon inserted into the nuclear protein 3B (EBNA 3B) open reading frame were generated by second-site homologous recombination. These mutant viruses infected and growth transformed primary B lymphocytes, resulting in the establishment of lymphoblastoid cell lines (LCLs). Polymerase chain reaction analysis and Southern hybridizations with infected cell DNA demonstrated the presence of the mutant EBNA 3B and the absence of wild-type EBNA 3B. Immunoblot analysis of the LCLs with affinity-purified EBNA 3B antibodies confirmed the absence of EBNA 3B cross-reactive protein. Virus was reactivated from two of these infected LCLs and serially passaged through primary B lymphocytes. The newly infected cells contained only the mutant recombinant virus. No difference was noted between mutant and wild-type recombinants, derived in parallel, in latent (other than EBNA 3B) or lytic cycle-infected cell virus protein expression or in the growth of the latently infected transformed cell lines. These data indicate that the EBNA 3B protein is not critical for primary B-lymphocyte infection, growth transformation, or lytic virus infection in vitro.


Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells. The herpes simplex virus type 1 protein, ICP0, can activate expression of all kinetic classes of viral promoters in transient expression assays. To examine the role of ICP0 in the regulation of viral gene expression during productive infection, we characterized the wild-type virus, an ICP0 null mutant (7134), and several ICP0 nonsense mutant viruses with regard to virus replication and protein synthesis in Vero cells. Relative to wild-type virus, 7134 was severely deficient in viral growth and protein synthesis at low multiplicities of infection but exhibited a nearly wild-type phenotype at high multiplicities. The phenotypes of the ICP0 nonsense mutants were intermediate between those of the wild-type virus and 7134 in that the more ICP0-coding sequence expressed by a given nonsense mutant, the more wild type-like was its phenotype. The location of the ICP0 domain responsible for transactivation during productive infection was confirmed to be within the N-terminal portion of the protein, as previously shown in transient expression assays. Immunoprecipitation and immunofluorescence tests were used to detect low-level expression of selected immediate-early (IE), early (E), and late (L) proteins by mutant and wild-type viruses following low-multiplicity infection. The 7134 deletion mutant and several nonsense mutants expressed markedly reduced levels of E and L proteins but wild-type levels of the IE protein, ICP4. Because the latency-associated transcripts (LATs) are specified by the strand opposite that which encodes ICP0, the ICP0 deletion and nonsense mutants are by definition ICP0-LAT double mutants. The ability of a LAT- ICP0+ mutant to replicate as efficiently as wild-type virus at low multiplicities and the ability of ICP0-expressing 0-28 cells to complement the defects of the mutants in E and L protein synthesis indicates that the phenotypes of the mutants are caused by mutations in ICP0 and not the LATs. Thus, we conclude that ICP0 up-regulates E and L but not necessarily IE gene expression during productive infection. The activation of IE gene expression by ICP0 during productive infection is likely overshadowed by the activity of the virion-associated protein, VP16. This hypothesis was tested by transfection of Vero cells with infectious mutant and wild-type viral DNAs. In such tests, no VP16 is present at early times posttransfection. Significantly fewer cells transfected with infectious 7134 DNA expressed ICP4 than cells transfected with KOS DNA. This reduction was fully reversed by cotransfection with an ICP0-expressing plasmid.(ABSTRACT TRUNCATED AT 400 WORDS)


Herpes simplex viruses with mutations in the gene encoding ICP0 are defective in gene expression. Herpes simplex virus type 1 (HSV-1) mutants with codon insertions and deletions in IE-0, the gene encoding ICP0, were constructed. The HSV-1 deletion mutant dl1403 (N. D. Stow and E. C. Stow, J. Gen. Virol. 67:2571-2585, 1986) and an IE-0:lacZ transplacement vector isolated in this study were used to facilitate the construction of mutant viruses. Mutant viruses, all of which produced stable ICP0, were examined for their ability to plaque and grow on both Vero and HeLa cells because previous results showed that HSV-1 immediate-early (IE) gene promoters and their products are differentially expressed in these cells (J. Chen, X. Zhu, and S. Silverstein, Virology 180:207-220, 1991; I. H. Gelman and S. Silverstein, J. Virol. 61:2286-2296, 1987). Viruses with IE-0 genes that only poorly activated reporter genes in transient expression assays plaqued less efficiently on Vero cells and consistently accumulated decreased levels of late proteins. These mutants were also examined in single-step growth curve experiments and for the dependence of virus yield on multiplicity of infection (MOI). At low MOIs, their yields were less in Vero cells than in HeLa cells; by contrast, at high MOIs, there was no apparent difference in yield in either cell type, although each virus produced considerably fewer progeny than wild-type virus. Analysis of steady-state levels of RNA from genes representing each of the three major kinetic classes demonstrated that lower levels of RNAs accumulate in these mutants. We conclude from these studies that while ICP0 is not essential for virus growth in tissue culture, defects in this gene result in impairment of virus replication and delay the expression of early and late gene transcripts.


Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity. The cleavage of the p220 subunit of eukaryotic initiation factor 4F (eIF-4F) that is induced by the poliovirus protease 2A has been shown previously to require another translation initiation factor, eIF-3. The role of eIF-3 in this cleavage reaction, however, is not known. An antiserum was raised against human eIF-3 and used to analyze the eIF-3 subunit composition in poliovirus-infected and uninfected HeLa cells and after incubation of eIF-3 in vitro with viral 2A protease. No evidence for 2Apro-dependent cleavage of any eIF-3 subunit was detected. Infected cells contain an activity that catalyzes the cleavage of p220 to a specific set of cleavage products. This activity is thought to be an activated form of a latent cellular protease. The p220-specific cleavage activity was partially purified. It was resolved from eIF-3 by both gel filtration and anion-exchange chromatography. Neither intact eIF-3 nor any detectable subunits of eIF-3 were found to copurify with the p220-specific cleavage activity. The latter activity behaves as a protein of 55,000 to 60,000 molecular weight and is inhibited by alkylating agents and metals, which indicates the presence of essential thiol groups. When this activity was incubated with partially purified p220, cleavage occurred only in the presence of eIF-3. Thus, eIF-3 appears to play a role in the p220 cleavage cascade which is subsequent to the 2Apro-induced activation of the p220-specific protease.


Effect of genomic location on expression of beta-galactosidase mRNA controlled by the herpes simplex virus type 1 UL38 promoter. To examine the effect of genomic location on the details of expression of selected herpes simplex virus promoters, we have constructed recombination vectors for placing such promoters controlling the beta-galactosidase reporter gene into two regions of the viral genome lacking any nearby promoter or regulatory elements. The first vector generates the promoter-beta-galactosidase reporter gene inverted within the locus of the gC (UL44) translational reading frame; the second replaces the LAT promoter and the first 600 bases of the primary transcript in both copies of the RL region. These locations were chosen to obviate any possible influence of upstream but noncontiguous heterologous or homologous DNA sequence elements upon promoter activity. When the reporter gene controlled by the strict late (gamma) UL38 promoter was placed in the gC location, it was significantly less active than in its normal location; in contrast, promoter activity was comparable to wild-type values when the promoter was recombined into the RL region. The low level of activity in the gC location could be partially alleviated by the incorporation of additional DNA sequences upstream of the UL38 promoter. Despite the effect of genomic location upon the level of expression, the kinetics of expression in either location mirrors the wild-type UL38 strict late kinetics of expression. Finally, we used deletional analysis to demonstrate that no more than 29 bases of DNA sequence 5' of the mRNA cap site are required for promoter activity in either location; this result is consistent with earlier results of transient-expression assays and indicates that the UL38 promoter shares general features with other strict late (gamma) herpes simplex virus promoters.


Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17. Parvoviruses are known to interfere with cellular transformation and carcinogenesis. Since infecting adeno-associated virus (AAV) frequently integrates its DNA into the cellular genome, we analyzed whether this integration influences the transformed phenotype of the human tumor cell line HeLa. Analysis of three independent HeLa cell clones with integrated AAV DNA (HA-3x, HA-16, and HA-28) revealed the following phenotypic changes of these cells: (i) reduced growth rate, (ii) increased serum requirement, (iii) reduced capacity for colony formation in soft agar, (iv) reduced cloning efficiency on plastic, (v) elevated sensitivity to genotoxic agents (N-methyl-N'-nitro-N-nitrosoguanidine, 7,12-dimethylbenz[a]anthracene, human tumor necrosis factor alpha, UV irradiation [256 nm], and heat [42 degrees C]), and (vi) reduced sensitivity to the cytolytic effect of parvovirus H-1. Reduced growth rate and enhanced sensitivity to gamma irradiation were also observed in vivo when tumors from AAV DNA-containing HeLa cells were transplanted into nude mice. This alteration of the biological properties of HeLa cells was independent of the number of AAV genomes integrated, the physical structure of integrated AAV DNA, and the transcription of AAV genes. Integration of AAV DNA was found to occur preferentially on the long arm of chromosome 17 in the three HeLa cell clones analyzed. These findings demonstrate that genomic integration of AAV DNA can alter the biological properties of human tumor cells.


An early event in murine cytomegalovirus replication inhibits presentation of cellular antigens to cytotoxic T lymphocytes. Cytomegalovirus (CMV) infection of simian virus 40 (SV40)-immune mice inhibits priming of SV40-specific helper and cytotoxic T lymphocytes (CTL) in vivo (A. E. Campbell, J. S. Slater, and W. S. Futch, Virology 173:268-275, 1989; J. S. Slater, W. S. Futch, V. J. Cavanaugh and A. E. Campbell, Virology 185:132-139, 1991). We now demonstrate that murine CMV (MCMV) infection of SV40-transformed macrophages and fibroblasts prevents presentation of SV40 T antigen to SV40-specific CTL. MCMV-infected macrophages failed to stimulate SV40-immune CTL precursors in vitro. In addition, MCMV-infected, SV40-transformed macrophage and fibroblast target cells lost their susceptibility to lysis by major histocompatibility complex class I-restricted, SV40-specific CTL clones. MCMV infection did not alter the synthesis of SV40 T antigen in the target cells. MCMV early gene expression was required for inhibition of SV40 T-antigen presentation; immediate-early gene expression was insufficient for this effect. Early viral gene expression also resulted in significant reduction of H-2K and H-2D molecules on the surface of MCMV-infected fibroblasts. However, this reduction occurred independently from suppression of antigen presentation to CTL. The same target cells which were resistant to lysis by SV40 CTL were susceptible to lysis by MCMV-specific CTL. MCMV early gene products therefore interfere with the processing and/or presentation of SV40 T-antigen determinants to CTL independent of alterations in the major histocompatibility complex.


Spleen necrosis virus, an avian immunosuppressive retrovirus, shares a receptor with the type D simian retroviruses. The reticuloendotheliosis viruses (REV) are a family of highly related retroviruses isolated from gallinaceous birds. On the basis of sequence comparison and overall genome organization, these viruses are more similar to the mammalian type C retroviruses than to the avian sarcoma/leukemia viruses. The envelope of a member of the REV family, spleen necrosis virus (SNV), is about 50% identical in amino acid sequence to the envelope of the type D simian retroviruses. Although SNV does not productively infect primate or murine cells, the receptor for SNV is present on a variety of human and murine cells. Moreover, interference assays show that the receptor for SNV is the same as the receptor for the type D simian retroviruses. We propose that adaptation of a mammalian type C virus to an avian host provided the REV progenitor.


Pseudorabies virus envelope glycoprotein gI influences both neurotropism and virulence during infection of the rat visual system. We previously demonstrated that intraocular injections of virulent and attenuated strains of pseudorabies virus (PRV) produce transneuronal infection of functionally distinct central visual circuits in the rat. The virulent Becker strain of PRV induces two temporally separated waves of infection that ultimately target all known retinorecipient neurons; the attenuated Bartha strain only infects a functionally distinct subset of these neurons. In this study, we demonstrate that deletion of a single viral gene encoding glycoprotein gI is sufficient to reproduce both the novel pattern of infectivity and the reduced neurovirulence of the Bartha strain of PRV. Glycoprotein gIII, a major viral membrane protein required for efficient adsorption of virus in cell culture, has no obvious role in determining the pattern of neuronal infectivity, but appears to function with gI to influence neurovirulence. These data suggest that neuroinvasiveness and virulence are the products of an interaction of viral envelope glycoproteins with as yet unidentified cellular receptors.


Identification and characterization of pseudorabies virus glycoprotein H. On the basis of DNA sequence analysis, it has recently been shown that the pseudorabies virus (PrV) genome encodes a protein homologous to glycoprotein H (gH) of other herpesviruses (B. Klupp and T.C. Mettenleiter, Virology 182:732-741, 1991). To obtain antibodies specific for gH(PrV), rabbits were immunized with synthetic peptides representing two potential epitopes on gH(PrV) as predicted by computer analysis. The antipeptide sera recognized the gH precursor polypeptide pgH translated in vitro from an in vitro-transcribed mRNA. Western blot (immunoblot) analyses of purified pseudorabies virions using these antisera revealed specific reactivity with a protein with an apparent molecular mass of 95 kDa. Specificity of the reaction could be demonstrated by competition experiments with respective peptides. Analysis of PrV deletion mutants defective in genes encoding known glycoproteins proved that gH(PrV) constitutes a novel PrV glycoprotein not previously found. Treatment of purified virion preparations with endoglycosidase H reduced the apparent molecular mass of gH(PrV) to 90 kDa, indicating the presence of N-linked high-mannose (or hybrid) carbohydrates in mature virions. Removal of all N-linked carbohydrates by N-glycosidase F resulted in a product of 76 kDa. In summary, our results demonstrate the existence of gH in PrV as a structural component of the virion.


Infection of HeLa cells with poliovirus results in modification of a complex that binds to the rRNA promoter. In HeLa cells, RNA polymerase I (Pol I)-mediated transcription is severely inhibited soon after infection with poliovirus. We have developed a gel retardation assay to analyze DNA-protein complexes formed at the Pol I promoter. We show here that two complexes (A and C) formed by nuclear extracts from uninfected cells disappear after infection of cells with poliovirus. In contrast, a new, rapidly migrating complex (D) is formed in virus-infected cell extract. This change in the mobility of gel-retarded complexes correlates well with the kinetics of inhibition of rRNA transcription in virus-infected cells. Incubation of nuclear extracts from mock-infected cells with bacterially expressed, purified poliovirus protease 3C results in the disappearance of complexes A and C with concomitant generation of complex D. A partially purified transcription factor fraction derived from uninfected cells that contains complex A is able to restore Pol I transcription when added to virus-infected cell extracts, suggesting that this complex plays an important role in Pol I transcription. These results suggest that poliovirus proteinase 3C may have an important role in the shutoff of Pol I transcription in cells infected with poliovirus.


Expression of Aleutian mink disease parvovirus capsid proteins by a recombinant vaccinia virus: self-assembly of capsid proteins into particles. A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, VV:ADSP. Immunohistochemical staining of VV:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of VV:ADSP-infected cell lystates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified VV:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with VV:ADSP contained ADV-specific antibodies and two of these eight serum samples had neutralizing activity, indicating that the particles produced in VV:ADSP-infected cells were immunogenic. Finally, when lysates from VV:ADSP-infected cells were compared with standard ADV antigens in counterimmunoelectrophoresis assays, a similar pattern of specific reactivity was observed for sera from normal and infected mink.


Specific interactions of HeLa cell proteins with proposed translation domains of the poliovirus 5' noncoding region. To determine which sequences or structures in the poliovirus 5' noncoding region (5'NCR) are involved in binding proteins used for internal ribosome binding and protein synthesis initiation, translation competition assays were performed in rabbit reticulocyte lysates in the presence and absence of HeLa cell extract. The results revealed two functional domains in the poliovirus 5'NCR. One, requiring nucleotides (nts) 457 to 626, binds proteins that are required for translation of all mRNAs and that are present in both reticulocyte lysates and HeLa cell extracts. Another, contained within nts 286 to 456, interacts with proteins that are specific for poliovirus translation and are present in HeLa cells but not in significant amounts in rabbit reticulocyte lysates. In order to detect HeLa cell proteins that interact stably with the 5'NCR of poliovirus, UV cross-linking was used. At least four major protein-RNA complexes were identified, three of which were shown by RNA competition analysis to bind specifically to defined domains within the 5'NCR. Protein A (54 kDa) cross-linked to RNA sequences and/or structures located between nts 457 and 626; proteins B (48 kDa) and C (38 kDa) bound to nts 286 to 456.


Defective synthesis of early region 4 mRNAs during abortive adenovirus infections in monkey cells. Human adenovirus 2 grows poorly in monkey cells, partly because of defects in late gene expression. Since deletions in early region 4 (E4) cause similar defects in late gene expression, we examined E4 mRNA expression in abortive infections. Processing of E4 mRNAs was defective during abortive infections, most likely at the level of splicing. At early times in productive infections in HeLa cells, the major E4 species produced is a 2-kb mRNA; at late times, a shift occurs so that smaller spliced E4 mRNAs are also produced. In CV-1 cells, a nonpermissive monkey cell line, this shift did not take place and only the 2-kb species was produced at late times, suggesting a defect in E4 mRNA splicing during abortive infections. The adenovirus DNA-binding protein (DBP) was required for normal processing of E4 mRNAs, since a host range mutant (hr602) containing an altered DBP gene showed a normal late E4 mRNA pattern in CV-1 cells; in addition, DBP was required during infections in HeLa cells for late E4 mRNA expression. DBP was not required for production of the late E4 pattern in transient expression assays in HeLa or 293 cells, suggesting that a second factor in addition to the DBP, present during infection but not transfection, modulates E4 mRNA processing.


Human papillomavirus type 33 in a tonsillar carcinoma generates its putative E7 mRNA via two E6* transcript species which are terminated at different early region poly(A) sites. Human papillomavirus type 33 (HPV-33)-specific early region transcripts in a tonsillar carcinoma were analyzed by using the RNA polymerase chain reaction method. A total of five cDNA species including species with potential to encode E6*I, E6*II, and E6*III, could be identified. As determined by 3' cDNA end mapping, one E6*I cDNA species was found to utilize a novel early region poly(A) site and was polyadenylated at or near the putative initiation codon of the E1 open reading frame (ORF). Compared with the HPV-16 and HPV-18 E6* mRNAs, the HPV-33 E6*I and E6*II species utilize different splice acceptor sites, the latter being localized within the E7 ORF. Furthermore, HPV-33 E6* mRNAs were found to contain a short overlapping ORF resulting in alternative coding potentials if translation were to start at an internal AUG codon within the E6 region. These results indicate that like HPV-16 and HPV-18, HPV-33 generates E6* mRNAs which may serve as efficient mRNAs for E7. However, HPV-33 has the ability to generate its putative E7 mRNAs by the utilization of two early region poly(A) sites, which offers the possibility of expressing E7 in different ways.


A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating. The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.


Crossover site selection during recombination of polyomavirus replicons. Two hybrid replicons containing polyomavirus (Py) genomes with large duplications of the viral late coding sequences were transfected into various permissive mouse cell lines. In all cell lines, either replicon yielded the sole amplifiable product expected from intramolecular homologous recombination, unit-length Py DNA (P155). In normal and in Py-transformed cells, such recombination was highly effective and involved sequences previously found to act as recombination hot spots (S repeats). In cells transformed by simian virus 40, however, these hot spots were inoperative in the generation of P155, which occurred with a reduced efficiency. These data confirm and extend earlier data indicating that the nature of products arising from recombination in Py replicons is tightly controlled by both cis- and trans-acting factors.


A selectable marker allows investigation of a nontransforming Epstein-Barr virus mutant. The derivation of specifically mutated Epstein-Barr virus (EBV) recombinants is dependent on strategies to identify, enumerate, and clone infected B lymphocytes. In recent experiments, EBV recombinants containing a positive selection marker were identified and cloned in B-lymphoma (BL) cells infected and then plated under selective conditions (F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). We now use BL cells, for the first time, as hosts for assaying and cloning otherwise isogenic EBV recombinants carrying a hygromycin phosphotransferase (HYG) gene linked to either a nontransforming deletion mutant or a transforming wild-type EBV nuclear antigen 2 (EBNA-2) gene. Both types of recombinants converted BL cells to hygromycin resistance with similar efficiency, formed episomes, and usually expressed only EBNA-1. Only the wild-type EBNA-2 HYG gene EBV recombinant transformed primary B lymphocytes. This strategy of assaying virus on BL and primary B lymphocytes makes possible the direct assessment of the transforming efficiency of an EBV recombinant. The resultant infected BL cells are also useful for the characterization of the nontransforming recombinant EBV genomes. The HYG gene insertion in the BHLF1 open reading frame eliminated BHLF1 protein expression. The insertion and resulting BHLF1 mutation did not interfere with primary B-lymphocyte infection, growth transformation, induction of lytic infection, or virus production. Thus, these experiments also indicate that neither the BHLF1 open reading frame nor the HYG gene insertion critically affects B-lymphocyte infection in vitro.


Replication of plasmid-derived human papillomavirus type 11 DNA in cultured keratinocytes. Plasmid-derived human papillomavirus type 11 (HPV11) DNA has been shown to replicate episomally and semiconservatively following transfection of primary human foreskin keratinocytes. HPV11 DNA was excised from the bacterial plasmid, religated to form circular molecules, and cotransfected along with pSV2Neo. Transfectants were selected and shown to contain replicated episomal HPV DNA. Once selected, HPV11 DNA persists in cells through at least two additional passages.


Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. Sequencing of multiple recombinant clones generated from polymerase chain reaction-amplified products demonstrated that the degree of heterogeneity of two well-conserved regions of the hepatitis C virus (HCV) genome within individual plasma samples from a single patient was consistent with a quasispecies structure of HCV genomic RNA. About half of circulating RNA molecules were identical, while the remaining consisted of a spectrum of mutants differing from each other in one to four nucleotides. Mutant sequence diversity ranged from silent mutations to appearance of in-frame stop codons and included both conservative and nonconservative amino acid substitutions. From the relative proportion of essentially defective sequences, we estimated that most circulating particles should contain defective genomes. These observations might have important implications in the physiopathology of HCV infection and underline the need for a population-based approach when one is analyzing HCV genomes.


Transactivation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by the human foamy virus bel1 protein requires a specific DNA sequence. Human foamy virus (HFV) encodes the transcriptional transactivator bel1. The bel1 protein transactivates HFV long terminal repeat (LTR)-directed gene expression by recognizing a region in U3. It also transactivates human immunodeficiency virus type 1 (HIV-1) LTR-directed gene expression in transient transfection assays. To identify the specific region in HIV-1 LTR responsible for bel1 action, we examined the effect of bel1 on chloramphenicol acetyltransferase (CAT) gene expression in transfected cells with a series of mutant HIV-1 LTR/CAT plasmids. The region between -158 and -118 from the transcription initiation site, immediately upstream of the core enhancer element, was identified as responsible for the transactivation by bel1. In addition, bel1 transactivated a heterologous promoter when this region was positioned upstream of it in the sense and antisense orientations. Optimal transactivation of the HIV-1 LTR by bel1 did not require an intact TAR sequence, suggesting that the binding of tat to the TAR sequence is not a prerequisite for bel1 function in HIV-1 LTR-directed gene expression. In the region of the HIV-1 LTR that is necessary for the bel1-mediated transactivation, we have found a sequence which is conserved between HIV-1 and HFV. Our results suggest that the bel1 action on HIV-1 seems to be mediated by a specific DNA sequence which is shared by both the HIV-1 LTR and HFV LTR.


Functional analysis of the true late human cytomegalovirus pp28 upstream promoter: cis-acting elements and viral trans-acting proteins necessary for promoter activation. As a model for analyzing the regulation of human cytomegalovirus late genes, we investigated the 28-kDa phosphoprotein (pp28) gene region. Transcripts of 1.6 and 1.3 kb were expressed in wild-type human cytomegalovirus-infected cells but not in cells infected with a DNA-negative temperature-sensitive mutant (ts66), indicating that DNA replication is absolutely required for pp28 gene expression. Transient promoter activation studies revealed that the pp28 gene region upstream promoter (pp28US) functioned early when expressed independently of the viral genome. However, the promoter was not efficiently activated by immediate-early (IE) proteins but was activated equally well by both wild-type virus and ts66. Deletion analysis of the pp28US promoter indicated that sequences upstream of the CAP site between -107 and -32 were required for activation of the pp28 promoter. Within that region exist a 10-bp sequence at -90 (AGTGAT CGTG) and its inverted repeat at -32 which positively influence pp28 promoter function. Therefore, in the case of the pp28US promoter, viral proteins interact through discrete sequences to facilitate late gene expression.


Transmembrane protein oligomers of caprine arthritis-encephalitis lentivirus are immunodominant in goats with progressive arthritis. To dissect mechanisms of caprine arthritis-encephalitis lentivirus-induced arthritis, an undefined immunodominant viral glycoprotein, gp90 (G. C. Johnson, A. F. Barbet, P. Klevjer-Anderson, and T. C. McGuire, Infect. Immun. 41:657-665, 1983), was characterized. Monoclonal antibody to gp90 and specific antiserum to env gene products demonstrated that gp90 was a transmembrane protein (TM) dimer. Goats with progressive arthritis had high antibody titers to oligomeric and monomeric (38-kDa) TM.


Consistent transcription of the Epstein-Barr virus LMP2 gene in nasopharyngeal carcinoma. Two species of the Epstein-Barr virus-encoded latent membrane protein 2, LMP2A and LMP2B, are generated by alternative splicing, each species having a distinct first exon. LMP2 transcription in undifferentiated nasopharyngeal carcinoma (NPC), which is consistently associated with the Epstein-Barr virus, was investigated. Fifteen NPC specimens were analyzed by Northern (RNA) blot and RNA-based polymerase chain reaction; the LMP2A transcript was present in all specimens except one. In some specimens the LMP2B transcript was also detected. Sequence analysis of LMP2 cDNAs obtained from two NPC specimens revealed four mutations in exon 1 of the LMP2A transcript, which were present in both tumors and which resulted in nonconservative amino acid changes. These data suggest that the LMP2 expressed in NPC is distinct from that which is expressed in lymphoid cells.


Future alternatives to heparin: low-molecular-weight heparin and hirudin. The antithrombotic effects of standard heparin were compared with those of low-molecular-weight heparin (LMWH) and hirudin by use of an in vitro perfusion system. Fresh blood collected from human volunteers was treated with varying doses of these three agents and perfused in a recirculating system over everted porcine vein segments. A low shear rate (100/sec) was selected to simulate conditions in large arteries and veins. Platelet and fibrinogen deposition were evaluated with indium 111 and iodine 125 radiolabels, respectively. Anticoagulant activity was assessed by measuring the activated clotting time (ACT). Anti-Xa activity was assayed to determine the degree to which these agents used antithrombin III pathways. Low-molecular-weight heparin was the weakest anticoagulant, requiring 32 micrograms/ml blood to double the ACT. By contrast, the ACT doubled with only 0.75 and 1.10 micrograms/ml blood of heparin and hirudin, respectively. Heparin and hirudin inhibited platelet and fibrin deposition at equivalent doses. Low-molecular-weight heparin was a less potent inhibitor of fibrin than heparin or hirudin. Hirudin, a direct thrombin inhibitor, exhibited minimal anti-Xa activity, contrasted with 0.14 anti-Xa units/micrograms for LMWH and 0.13 anti-Xa units/mg for heparin. These data suggest that heparin and hirudin are more potent anticoagulants and antiplatelet agents than LMWH.


[Prognostic significance of echogenic lesion within small hepatocellular carcinoma]. To evaluate prognostic significance of echogenic lesion within small hepatocellular carcinoma (SHCC, less than or equal to 2 cm in diameter), clinical and pathological findings of 32 cases with SHCC containing echogenic lesion (echogenic SHCC) were compared with those of 55 cases with non-echogenic SHCC. Compared with the non-echogenic SHCC group, the frequency of clinical stage I was significantly higher, and there were significantly more cases with solitary tumor relative to cases with multiple tumors in the echogenic SHCC group. Histologically, the incidence of the HCC composed of well-differentiated tumor cells corresponding to Edmondson's grade I was significantly higher in the echogenic SHCC group than in the non-echogenic SHCC group. Although HCCs tended to become progressively less differentiated with increasing tumor sizes in the both groups, the process of cellular change appeared to proceed more slowly in the echogenic SHCC group. Survival rate after tumor detection was 73% at three years, 56% at five years and 48% at seven years and nine years in the echogenic SHCC group, while it was 46% at three years, 42% at five years and 0% at seven years in the non-echogenic SHCC group. The present results showed that the presence of echogenic lesion within SHCC could be useful prognostic indicator.


The long-term course of treated alcoholism: II. Predictors and correlates of 10-year functioning and mortality. This study examines factors related to mortality and 10-year posttreatment functioning for a sample of alcoholic patients who return to their families after an index residential treatment episode. Of the 113 patients followed 2 years after treatment, 20 had died by the time of the 10-year follow-up. Mortality risk was greater among patients who, prior to treatment, consumed more alcohol and were unemployed. Mortality was more strongly associated with medical conditions, liver problems, medication use and lack of confidants assessed 2 years posttreatment. The course for the surviving patients between the 2-year and 10-year follow-ups was one of improvement in terms of alcohol consumption, relative stability in terms of physical symptoms and depression, and an aging-related decline in social activities and employment. Life context and coping factors assessed 2 years after treatment were predictive of long-term outcome. Persons in less stressful life situations, in more cohesive and organized families, and who more frequently used active cognitive coping responses at the 2-year follow-up tended to function better at the 10-year follow-up. Overall, the findings support the value of embedding long-term follow-up studies in a theory of the disorder that is the target of the intervention.


Stimulatory effect of tumor necrosis factor-alpha on the growth of CMK, a human megakaryoblastic leukemia cell line. Recombinant human tumor necrosis factor-alpha (TNF-alpha) was found to stimulate the growth of CMK, a human megakaryoblastic leukemia cell line. This stimulatory effect of TNF-alpha was blocked by anti-TNF-alpha antibody, but antibodies to recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and interleukin 6 (all growth factors for CMK cells) did not reduce the stimulatory effect of TNF-alpha. Scatchard analysis showed that CMK cells expressed TNF-alpha receptors on the cell surface. The growth of CMK cells was also stimulated by lymphotoxin, which shares the same receptor as TNF-alpha. These results suggest that TNF-alpha stimulated the growth of CMK cells directly via its specific receptor.


[Severe leukopenia and thrombocytopenia and transient acquired hypogammaglobulinemia in a patient with infectious mononucleosis]. A 30-year-old male with infectious mononucleosis by the Epstein-Barr virus who presented severe neutropenia and thrombocytopenia and type IgG acquired transitory hypogammaglobulinemia as complications during the acute period of the disease is presented. Although the etiopathogenesis of these phenomena is usually associated with an autoimmune basis, the antiplatelet and antileukocyte antibodies were negative. A bibliographic revision of the hematologic alterations of this disease was carried out and it was observed that the combination of the complications described has not been previously referred, thus, the present case may be the first observation with these characteristics.


Chronic lithium treatment prevents the dexamethasone-induced increase of brain polyamine metabolizing enzymes. The paper describes the effects of various regimens of lithium chloride treatment on dexamethasone-induced increases in brain polyamine metabolizing enzymes. In contrast to peripheral tissues where acute lithium treatment suppresses the increase in ornithine decarboxylase activity, in the brain only chronic treatment was effective in preventing this increase and also the increases in the activities of S-adenosylmethionine decarboxylase and spermidine/spermine N1-acetyltransferase. This findings indicate a novel brain target for lithium's action and in turn provide new avenues for exploring polyamine function in the brain.


Changes in responsiveness of the aorta to vasorelaxant agents in genetically diabetic rats: a study in WBN/Kob rats. The effects of various vasorelaxant agents on aortas from control and genetically diabetic rats were examined. The concentration-response curves for the isoproterenol (ISO)-induced relaxation of both aortic strips with and without endothelium are shifted to the right in diabetic rats. The relaxation responses of diabetic aorta to forskolin and vasoactive intestinal peptide did not differ from those of controls. The relaxation responses of diabetic aorta to cromakalim and nicorandil did not differ from those of controls. These results indirectly indicate that ISO-induced relaxation responses of the aortic strips from genetically diabetic rats decreased, and that this decreased relaxation response of the strips to ISO may be due to decreased density or affinity of beta adrenoceptors on the endothelium and vascular smooth muscle.


A novel opioid structure which accepts protonated as well as non-protonated nitrogen: a family of pure, delta receptor selective antagonists. Conventional opioids including opioid peptides require an "opioid" nitrogen which exists in protonated state while interacting with the receptor. In the present paper we demonstrate that the Tyr-Pro-Gly-Phe-Leu-Thr hexapeptide sequence accepts N-terminal substituents such as N-t-Boc, N-phenylacetyl and N-diphenylacetyl where the N cannot become protonated, as well as "traditional" substitutions such as N,N-diallyl, where protonation is likely under physiological conditions. The opioid peptides bearing these substituents are pure antagonists of medium affinity (Ke values in the mouse vas deferens bioassay against [Met5]-enkephalin are in the 3 x 10(-7)-4 x 10(-6) M range) with a high delta receptor preference (50-350-fold delta over mu selectivity ratios).


A murine model of pseudorabies virus latency. The mouse is a useful laboratory animal for studying various aspects of pseudorabies virus (PRV) virulence. Mice are highly susceptible hosts for PRV infection and are unable to survive acute viral infection. Because of this, mouse models have not been useful for studying PRV latent infections. Here, we report an efficient strategy for establishing latent PRV infections in laboratory mice. Passive transfer of high titered neutralizing antibodies to mice prior to inoculation with highly lethal doses of PRV (Bartha) resulted in survival rates of at least 60% with establishment of latent infections in survivors. Latent PRV infection in mice was demonstrated by: (1) recovery of infectious PRV-Bartha from explants of trigeminal ganglion (TG), and (2) detection of PRV nucleic acids in latently infected TGs by in situ hybridization and polymerase chain reaction (PCR), between 2-8 months post-infection. This PRV latency model indicates that attenuated PRV strains, those currently used extensively in vaccination programs worldwide, can establish a reactivatable latent infection in an experimental host. The mouse model may be particularly useful for examining the molecular bases of PRV latency and reactivation.


Control of bacterial DNA supercoiling.

Control of bacterial DNA supercoiling. Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, supercoiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady-state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.


Structure and expression of a human oxytocin receptor. Just before the onset of labour, uterine myometrium becomes extremely sensitive to oxytocin, for which it is a primary target tissue, because of a dramatic increase in the number of oxytocin receptors. We report here the structure and expression of the human oxytocin receptor complementary DNA isolated by expression cloning. The encoded receptor is a 388-amino-acid polypeptide with 7 transmembrane domains typical of G protein-coupled receptors. The oxytocin receptor, expressed in Xenopus oocytes, specifically responds to oxytocin and induces an inward membrane current. Messenger RNAs for the receptor are of two sizes, 3.6 kilobases in breast, and 4.4 kilobases in ovary, uterine endometrium and myometrium. The mRNA level in the myometrium is very high at term. We conclude that the increase in receptor number in the myometrium at labour is, at least in part, due to the increase in mRNA.


SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity. Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.


The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19. Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway. In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K). In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5). Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast. Whether this activity requires additional components similar to those found in mammalian SRP is not known. We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER. Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19). We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.


Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells. Persistent changes in synaptic efficacy are thought to underlie the formation of learning and memory in the brain. High-frequency activation of an afferent excitatory fibre system can induce long-term potentiation, and conjunctive activation of two distinct excitatory synaptic inputs to the cerebellar Purkinje cells can lead to long-term depression of the synaptic activity of one of the inputs. Here we report a new form of neural plasticity in which activation of an excitatory synaptic input can induce a potentiation of inhibitory synaptic signals to the same cell. In cerebellar Purkinje cells stimulation of the excitatory climbing fibre synapses is followed by a long-lasting (up to 75 min) potentiation of gamma-aminobutyric acid A (GABAA) receptor-mediated inhibitory postsynaptic currents (i.p.s.cs), a phenomenon that we term rebound potentiation. Using whole-cell patch-clamp recordings in combination with fluorometric video imaging of intracellular calcium ion concentration, we find that a climbing fibre-induced transient increase in postsynaptic calcium concentration triggers the induction of rebound potentiation. Because the response of Purkinje cells to bath-applied exogenous GABA is also potentiated after climbing fibre-stimulation with a time course similar to that of the rebound potentiation of i.p.s.cs, we conclude that the potentiation is caused by a calcium-dependent upregulation of postsynaptic GABAA receptor function. We propose that rebound potentiation is a mechanism by which in vivo block of climbing fibre activity induces an increase in excitability in Purkinje cells. Moreover, rebound potentiation of i.p.s.cs is a cellular mechanism which, in addition to the long-term depression of parallel fibre synaptic activity, may have an important role for motor learning in the cerebellum.


CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Occupancy of the T-cell antigen receptor is insufficient to induce T-cell activation optimally; a second co-stimulatory signal is required. Exposure of T-cell clones to complexes of antigen with major histocompatibility complex molecules in the absence of the co-stimulatory signal induces a state of clonal anergy. This requirement for two stimuli for T-cell activation could have an important role in vivo in establishing peripheral tolerance to antigens not encountered in the thymus. The receptor on T cells required for the co-stimulatory stimulus involved in the prevention of anergy has not been identified. The human T-cell antigen CD28 provides a signal that can synergize with T-cell antigen receptor stimulation in activating T cells to proliferate and secrete lymphokines. Here we report that a monoclonal antibody against the murine homologue of CD28 (ref. 7; J.A.G. et al., manuscript in preparation) can provide a co-stimulatory signal to naive CD4+ T cells and to T-cell clones. Moreover, we demonstrate that this co-stimulatory signal can block the induction of anergy in T-cell clones.


[Effects of dietary fiber and insulin treatment on serum levels of lipids and lipoprotein (a) in patients with type II diabetes mellitus]. As lipoprotein(a) is an independent risk factor for the development of coronary heart disease we determined the effect on serum lipid and lipoprotein(a) levels of dietary fibre and of treatment with insulin in patients with diabetes mellitus type II. Twelve type II diabetic patients (mean age 62 (SD 10) yrs, body mass index 25.8 (SD 3.5) kg/m2), all treated with oral antidiabetic agents, were studied in a randomised cross-over trial, in which they used breads meals prepared with guar gum (a mean of 11.2 g guar per day) for 3 months in comparison with normal high-fibre bread. Fifteen other patients (age 70 (8), BMI 27.4 (5.6) kg/m2) poorly controlled on oral hypoglycaemic agents, were treated with insulin. The guar treatment of the 12 patients resulted in lower total cholesterol (5.24 vs 5.7 mmol/l, p less than 0.1) and LDL cholesterol (3.77 vs 4.33 mmol/l, p less than 0.001) in comparison with normal high-fibre bread. Lipoprotein(a) levels were not different (76 vs 82 mg/l). Insulin therapy in the 15 other patients decreased HbA1c levels after 6 months from 11.0 to 7.7% (p less than 0.001), total cholesterol from 6.8 to 6.1 mmol/l (p less than 0.05), and LDL cholesterol from 4.4 to 4.1 mmol/l (p less than 0.05). Lp(a) decreased only slightly in 11/15 patients, from 491 to 441 mg/l (p = 0.07). Neither the use of the dietary fibre guar nor improved metabolic control with insulin therapy lowered elevated lipoprotein(a) levels in type II diabetic patients.


Molecular mechanisms for generation of neural diversity and specificity: roles of polypeptide factors in development of postmitotic neurons. Development of postmitotic neurons is influenced by two groups of polypeptide factors. Neurotrophic factors promote neuronal survival both in vivo and in vitro. Neuronal differentiation factors influence transmitter phenotypes without affecting neuronal survival. The list of neurotrophic factors is increasing partly because certain growth factors and cytokines have been shown to possess neurotrophic activities and also because new neurotrophic factors including new members of the nerve growth factor (NGF) family have been identified at the molecular level. In vitro assays using recombinant neurotrophic factors and distributions of their mRNAs and proteins have indicated that members of a neurotrophic gene family may play sequential and complementary roles during development and in the adult nervous system. Most of the receptors for neurotrophic factors contain tyrosine kinase domains, suggesting the importance of tyrosine phosphorylation and subsequent signal transduction for their effects. Molecules such as LIF (leukemia inhibitory factor) and CNTF (ciliary neurotrophic factor) have been identified as neuronal differentiation factors in vitro. At the moment, however, it remains to be determined whether or not the receptors for a group of neuronal differentiation factors constitute a gene family or contain domains of kinase or phosphatase activity. Synergetic combinations of neurotrophic and neuronal differentiation factors as well as their receptors may contribute to the generation of neural specificity and diversity.


Posterior vermal Purkinje cells in macaques responding during saccades, smooth pursuit, chair rotation and/or optokinetic stimulation. Anatomical locations of the Purkinje cells (P cells), showing modulations in activity during either saccadic or smooth-pursuit eye movements, during primate chair rotation, or in response to optokinetic stimulation, were studied in the posterior vermes of monkeys trained to move their eyes with a visual target. The majority (68.3%) of the responsive P cells were saccade-related units. They were located exclusively in vermal lobules VIc and VII: the oculomotor vermis. Most P cells sensitive to chair rotation were located in vermal lobules VIa,b and VIII (91.2%), designated as the paraoculomotor vermis. The P cells which modulated activity during smooth-pursuit eye movements, associated with eye position, or during optokinetic stimulation were found in both the oculomotor and paraoculomotor vermis. There were 25 P cells which modulated their activity during smooth pursuit in the oculomotor vermis. Among them, only three responded also to optokinetic stimulation but none was sensitive to chair-rotation stimulation. These findings suggest that the control of saccadic eye movements is the primary function of the oculomotor vermis.