Type I interferon (IFN) induces antiviral responses through the activation of the ISGF3 transcription factor complex that contains the subunit proteins STAT1, STAT2, and p48/ISGF3 gamma/IRF9. The ability of some human paramyxoviruses to overcome IFN actions by specific proteolysis of STAT proteins has been examined. Infection of cells with type 2, but not type 1 or type 3 human parainfluenza virus (HPIV) leads to a loss of cellular STAT2 protein. Expression of a single HPIV2 protein derived from the V open reading frame blocks IFN-dependent transcriptional responses in the absence of other viral proteins. The loss of IFN response is due to V-protein-induced proteolytic degradation of STAT2. Expression of HPIV2 V causes the normally stable STAT2 protein to be rapidly degraded, and this proteolytic activity can be partially alleviated by proteasome inhibition. No V-protein-specific effects on STAT2 mRNA levels were observed. The results indicate that the V protein of HPIV2 is sufficient to recognize and target a specific cellular transcription factor for destruction by cellular machinery.

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-␣/␤) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI؊) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-␣/␤ signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI؊ had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI؊ were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI؊ mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI؊ but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-␤ promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI؊ but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI؊ mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI؊ growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI؊ by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI؊ induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-␣/␤ synthesis, and virus-induced apoptosis.Alpha and beta interferons (IFN-␣ and IFN-␤) are important cytokines that are produced during virus infection (reviewed in references 5 and 46). IFN-␣/␤ signaling is initiated when secreted IFN binds to its receptor on the cell surface, resulting in the phosphorylation of a family of latent transcription factors called STAT1 and STAT2 (signal transducers and activators of transcription). IFN-activated STAT1 and STAT2 heterodimerize and associate with p48 (also known as IRF-9) to form the transcription factor ISGF3. This transcription factor binds to interferon-sensitive response elements (ISRE) located in the promoter region of IFN-inducible genes (reviewed in reference 24). Many of the identified IFN-induced gene products have potent antiviral activity and can contribute to the inhibition of host and viral protein synthesis, induction of apoptosis, and clearance of viral infections. As such, viruses have evolved mechanisms that counteract the induction of IFN, the activation of the IFN signaling pathways, or both of these processes (reviewed in references ...

We have analyzed the effectiveness of Hsp90 inhibitors in blocking the replication of negative-strand RNA viruses. In cells infected with the prototype negative strand virus vesicular stomatitis virus (VSV), inhibiting Hsp90 activity reduced viral replication in cells infected at both high and low multiplicities of infection. This inhibition was observed using two Hsp90 inhibitors geldanamycin and radicicol. Silencing of Hsp90 expression using siRNA also reduced viral replication. Hsp90 inhibition changed the half-life of newly synthesized L protein (the large subunit of the VSV polymerase) from >1 h to less than 20 min without affecting the stability of other VSV proteins. Both the inhibition of viral replication and the destabilization of the viral L protein were seen when either geldanamycin or radicicol was added to cells infected with paramyxoviruses SV5, HPIV-2, HPIV-3, or SV41, or to cells infected with the La Crosse bunyavirus. Based on these results, we propose that Hsp90 is a host factor that is important for the replication of many negative strand viruses.

Mature picornaviral proteins are derived by progressive, posttranslational cleavage of a precursor polyprotein. These cleavages play a role in the control of virus functions. Although the processed termini are separated by as much as 75 A in the native virus capsid, the fold and arrangement of polypeptide chains in a protomer before proteolysis are likely to be similar to that found in the mature virus. The three-dimensional structures of rhinovirus and Mengo virus suggest that the cleavage sites within the protomeric precursor are in structurally flexible regions. The final proteolytic processing event, maturation of the virion peptide VPO (also called peptide 1AB) appears to occur by an unusual autocatalytic serine protease-type mechanism possibly involving viral RNA basic groups that would serve as protonabstractors during the cleavage reaction.Controlled limited proteolysis plays a major role in the regulation of many biological processes. Examples are activation of zymogens to enzymes and prohormones to hormones, assembly of cytoskeletal components, control of cellular differentiation, initiation of cascade mechanisms such as blood clotting, removal of signal peptides in the transport of proteins across membranes, and the recycling of cellular proteins. In a viral life cycle, proteolysis can be involved in the differential control of host versus viral biosynthesis, in posttranslational modification of a precursor polyprotein, and in directing assembly events. Most of these processes utilize one of four different types of enzymic activity: serine proteases, cysteine proteases, acid proteases, or metalloproteases. Both convergent and divergent evolution have been involved in producing the observed mechanisms. Here we present evidence suggesting a viral proteolytic mechanism in which both protein and nucleic acid components participate.Picornaviruses (1) are of major economic and medical importance. The family contains a diverse variety of highly virulent human and animal pathogens, which traditionally are subdivided into four genera on the basis of physical properties of the virions. The subgroups include rhinoviruses (human and bovine), cardioviruses [e.g., Mengo, encephalomyocarditis (EMC) and Theiler viruses], enteroviruses (e.g., polio, hepatitis A, and coxsackie viruses), and aphthoviruses (foot-and-mouth disease viruses). In spite of the disparate afflictions caused by these agents, recent determinations of three-dimensional structures (2-4) and nucleotide sequences have shown that all picornaviruses share remarkable similarity in their particle structure and genome organization. Indeed, the similarity also extends to plant RNA viruses (2) and insect RNA viruses (J. E. Johnson, personal communication).Picornavirus virions contain a single, positive-stranded RNA genome (of between 7000 and 8500 bases) enclosed in a protein capsid shell with an external diameter of around 300 A. The capsids are composed of 60 copies of four nonidentical virion polypeptide chains (VP1, VP2, VP3, and VP4). The RNA c...

The complement system is an important component of the innate immune response to virus infection. The role of human complement pathways in the in vitro neutralization of three closely related paramyxoviruses, Simian Virus 5 (SV5), Mumps virus (MuV) and Human Parainfluenza virus type 2 (HPIV2) was investigated. Sera from ten donors showed high levels of neutralization against HPIV2 that was largely complement-independent, whereas nine of ten donor sera were found to neutralize SV5 and MuV only in the presence of active complement pathways. SV5 and MuV neutralization proceeded through the alternative pathway of the complement cascade. Electron microscopy studies and biochemical analyses showed that treatment of purified SV5 with human serum resulted in C3 deposition on virions and the formation of massive aggregates, but there was relatively little evidence of virion lysis. Treatment of MuV with human serum also resulted in C3 deposition on virions, however in contrast to SV5, MuV particles were lysed by serum complement and there was relatively little aggregation. Assays using serum depleted of complement factors showed that SV5 and MuV neutralization in vitro was absolutely dependent on complement factor C3, but was not dependent on downstream complement factors C5 or C8. Our results indicate that even though antibodies exist that recognize both SV5 and MuV, they are mostly non-neutralizing and viral inactivation in vitro occurs through the alternative pathway of complement. The implications of our work for development of paramyxovirus vectors and vaccines are discussed.