Human parainfluenza virus (HPIV) serotypes 1, 2, and 3 are significant causes of severe respiratory tract disease in infants and young children. The HPIVs are enveloped, non-segmented, single-stranded, negative-sense RNA viruses belonging to subfamily Paramyxovirinae within the Paramyxoviridae family. These serotypes can be further classified as belonging to either the Respirovirus (HPIV1 and HPIV3) or Rubulavirus (HPIV2) genus and are immunologically distinct in that primary infection does not result in cross-neutralization or cross-protection. The HPIV genome encodes three nucleocapsid-associated proteins including the nucleocapsid protein (N), the phosphoprotein (P) and the large polymerase (L) and three envelope-associated proteins including the internal matrix protein (M) and the fusion (F) and hemagglutinin-neuraminidase (HN) transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and are the major viral protective antigens. In addition, the P gene encodes the accessory protein(s) C (HPIV1), V (HPIV2), and C, D, and possibly V (HPIV3). The HPIVs cause respiratory tract disease ranging from mild illness, including rhinitis, pharyngitis, and otitis media, to severe disease, including croup, bronchiolitis, and pneumonia. HPIV1, HPIV2 and HPIV3 have been identified as the etiologic agents responsible for 6.0%, 3.3% and 11.5%, respectively, of hospitalizations of infants and young children for respiratory tract disease. Together these viruses account for approximately 18% of all pediatric hospitalizations due to respiratory disease. Licensed vaccines are currently not available for any of the HPIVs. The major goal of this project is to develop live attenuated vaccines against all three serotypes. Candidate vaccine viruses are recovered from cDNA using reverse genetic systems described in previous reports. This provides the means to develop well-defined live vaccines. Based on previous work, we already have multiple HPIV3 vaccines in clinical trials. Thus, this report focuses on HPIV1 and HPIV2. Human parainfluenza virus type 1 (HPIV1): We studied the effects of the C proteins of HPIV1 (which are a nested set of proteins arising from translational initiation at different start codons in the C ORF) on host innate immune responses. Infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing activation of interferon regulatory factor 3 (IRF3) and NF-kB, which are transcription factors involved in inducing interferon (IFN)-beta and pro-inflammatory cytokines. Both of these inhibitory effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C ORF P(C-), resulting in a potent IFN-beta response. Using murine knockout cells, we found that IFN-beta induction following infection with either mutant relied mainly on the host pattern recognition receptor called melanoma-associated differentiation gene 5 (MDA5) protein rather than retinoic acid-inducible gene I (RIG-I) protein. Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). This appeared to be the result of an increased and imbalanced synthesis of viral genomes, antigenomes, and mRNA by the mutant viruses. We found that the activation of the dsRNA-regulated protein kinase R (PKR) and the induction of IFN-beta followed the kinetics of dsRNA accumulation. We found no evidence that the C proteins directly inhibited the intracellular signaling involved in IFN-beta induction. Instead, the role of the C proteins in preventing IFN-beta induction appears to be at the level of down-regulating viral RNA synthesis to prevent the formation of dsRNA. This illustrates a somewhat unusual mechanism for virus-mediated suppression of innate immunity a mechanism that operates at the level of the virus rather than the host cell. This also is of interest because the F170S mutation is represented in an HPIV1 vaccine candidate presently in clinical trials. Human parainfluenza virus type 2 (HPIV-2): Whereas HPIV1 encodes C proteins from a separate ORF in the P mRNA, HPIV2 encodes a V protein that is unrelated to C and is expressed by RNA editing and frame shifting. Thus, the upstream half of V is identical to that of P while the downstream half is distinct and has a characteristic cysteine-rich motif. We mutated the RNA editing signal to create an HPIV2 mutant (rHPIV2-Vko) in which the P/V gene expressed only the P protein. Loss of expression of the V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. The rHPIV2-Vko virus, unlike wt HPIV2, strongly induced type I IFN and permitted signaling through the IFN receptor, leading to the establishment of a robust antiviral state. rHPIV2-Vko infection induced extensive syncytia and caused dramatic cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-Vko was highly restricted in the upper and lower respiratory tract (URT and LTR) of AGMs and was not detected in differentiated primary human airway epithelial (HAE) cultures, suggesting that the V protein is essential for efficient replication of HPIV2 in vivo and in HAE cultures in vitro. The high degree of restriction of rHPIV2-Vko in AGMs and in HAE cultures suggests that this mutant is over-attenuated and would not be suitable as a live attenuated virus vaccine, but will be useful to study the function of V during HPIV2 infection. The HPIV2 V protein was investigated in further detail. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV2 virus that was engineered to express the V and P coding sequences from separate mRNAs, so that mutations in one coding sequence would not affect the other. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with the pattern recognition receptor MDA5. Like WT HPIV2, these mutants inhibited IFN-beta induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit IRF3 dimerization (a measure of activation) or IFN-beta induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.