An HIV isolate obtained from an HIV seropositive patient was shown to have a low titer of infectious particles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analyses of proteins associated with this virus, designated HIV (FRE-3), showed that it contained large amounts of the gag viral protein precursor, Pr55. Electron microscopy (EM) of cells from the infected T-cell line, HuT 78, revealed a mixed population of lymphocytes; some cells were releasing only mature extracellular virus particles, while other produced aberrant "immature" virus particles. Individual cells were obtained by cloning HuT 78 on a feeder layer of primary sheep choroid plexus cells. Some of the clones are producing what appears to be "wild-type" HIV (reverse transcriptase-positive, mature gag proteins visualized on SDS-PAGE), which by EM appear normal in all stages of maturation. Other single-cell clones release noninfectious, structurally aberrant, immature virus particles. These latter clones do not have any detectable mature gag proteins and accumulate large amounts of the Pr55 gag precursor; some also lack reverse transcriptase activity. Purified and lysed whole virus preparations lack an intact protease; the addition of partially purified protease isolated from a "wild-type" virus results in the degradation of Pr55 to proteins that comigrate with mature HIV gag proteins. These results suggest that the genetic defect may reside in the protease gene itself. This in vitro assay for HIV protease, using its natural substrate, Pr55, will be used to identify HIV protease-specific inhibitors that may have therapeutic applications in treating HIV-infected patients. The large amount of Pr55 gag precursor protein present in these viruses has been useful in detecting the passive acquisition of HIV antibodies in patients with primary immunodeficiency syndromes receiving large doses of intravenous IgG. Rabbit antisera raised against these viruses have also been useful for detecting the presence of HIV antigen by immunohistochemical staining of routinely fixed autopsy specimens for AIDS patients.
NIH ExPorter
NIH ExPorter