The Paramyxoviruses Simian Virus 5 and Mumps Virus Recruit Host Cell CD46 To Evade Complement-Mediated Neutralization

ABSTRACT The complement system is a critical component of the innate immune response that all animal viruses must face during natural infections. Our previous results have shown that treatment of the paramyxovirus simian virus 5 (SV5) with human serum results in deposition of complement C3-derived polypeptides on virion particles. Here, we show that the virion-associated C3 component includes the inactive form iC3b, suggesting that SV5 may have mechanisms to evade the host complement system. Electron microscopy, gradient centrifugation, and Western blot analysis indicated that purified SV5 virions derived from human A549 cells contained CD46, a plasma membrane-expressed regulator of complement that acts as a cofactor for cleavage and inactivation of C3b into iC3b. In vitro cleavage assays with purified complement components showed that SV5 virions had C3b cofactor activity, resulting in specific factor I-mediated cleavage of C3b into inactive iC3b. SV5 particles generated in CHO cells, which do not express CD46, did not have cofactor activity. Conversely, virions derived from a CHO cell line that was engineered to overexpress human CD46 contained elevated levels of virion-associated CD46 and displayed enhanced C3b cofactor activity. In comparison with C3b, purified SV5 virions had very low cofactor activity against C4b, consistent with the known preference of CD46 for C3b versus C4b. Similar results were obtained for the closely related mumps virus (MuV), except that MuV particles derived from CHO-CD46 cells had higher C4b cofactor activity than SV5 virions. In neutralization assays with human serum, SV5 and MuV containing CD46 showed slower kinetics and more resistance to neutralization than SV5 and MuV that lacked CD46. Our results support a model in which the rubulaviruses SV5 and MuV incorporate cell surface complement inhibitors into progeny virions as a mechanism to limit complement-mediated neutralization.

A Hyperfusogenic F Protein Enhances the Oncolytic Potency of a Paramyxovirus Simian Virus 5 P/V Mutant without Compromising Sensitivity to Type I Interferon

ABSTRACT Viral fusogenic membrane proteins have been proposed as tools to increase the potency of oncolytic viruses, but there is a need for mechanisms to control the spread of fusogenic viruses in normal versus tumor cells. We have previously shown that a mutant of the paramyxovirus simian virus 5 (SV5) that harbors mutations in the P/V gene from the canine parainfluenza virus (P/V-CPI−) is a potent inducer of type I interferon (IFN) and apoptosis and is restricted for spread through normal but not tumor cells in vitro. Here, we have used the cytopathic P/V-CPI− as a backbone vector to test the hypothesis that a virus expressing a hyperfusogenic glycoprotein will be a more effective oncolytic vector but will retain sensitivity to IFN. A P/V mutant virus expressing an F protein with a glycine-to-alanine substitution in the fusion peptide (P/V-CPI−-G3A) was more fusogenic than the parental P/V-CPI− mutant. In two model prostate tumor cell lines which are defective in IFN production (LNCaP and DU145), the hyperfusogenic P/V-CPI−-G3A mutant had normal growth properties at low multiplicities of infection and was more effective than the parental P/V-CPI− mutant at cell killing in vitro. However, in PC3 cells which produce and respond to IFN, the hyperfusogenic P/V-CPI−-G3A mutant was attenuated for growth and spread. Killing of PC3 cells was equivalent between the parental P/V-CPI− mutant and the hyperfusogenic P/V-CPI−-G3A mutant. In a nude mouse model using LNCaP cells, the hyperfusogenic P/V-CPI−-G3A mutant was more effective than P/V-CPI− at reducing tumor burden. In the case of DU145 tumors, the two vectors based on P/V-CPI− were equally effective at limiting tumor growth. Together, our results provide proof of principle that a cytopathic SV5 P/V mutant can serve as an oncolytic virus and that the oncolytic effectiveness of P/V mutants can be enhanced by a fusogenic membrane protein without compromising sensitivity to IFN. The potential advantages of SV5-based oncolytic vectors are discussed.

Improved growth of enteric adenovirus type 40 in a modified cell line that can no longer respond to interferon stimulation

Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture, to aid in growth of the virus, a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5, which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.

Exchange of P/V genes between two non-cytopathic simian virus 5 variants results in a recombinant virus that kills cells through death pathways that are sensitive to caspase inhibitors

The paramyxovirus Simian virus 5 (SV5) is largely non-cytopathic in human epithelial and fibroblast cells. WF-PIV has been described previously as a naturally occurring SV5 variant that encodes P and V proteins differing from the wild-type (WT) SV5 proteins in eight and five amino acid positions, respectively. In this study, it is shown that WF-PIV is like WT SV5 by being largely non-cytopathic in A549 lung epithelial cells. However, substitution of the WF-PIV P/V gene into the background of WT SV5 resulted in a hybrid virus (P/V-WF) that induced apoptotic cell death not seen with either of the parental viruses. The kinetics of HeLa cell killing and induction of apoptosis by the P/V-WF chimera differed from those of the previously described P/V-CPI− chimera by being slower and less extensive. HeLa cell killing by the P/V-WF chimera was effectively reduced by inhibitors of caspase-9, but not of caspase-8. These results demonstrate that an exchange of P/V genes from two non-cytopathic SV5 variants can produce apoptosis-inducing chimeras, and that the role of the SV5 P/V gene products in limiting apoptosis can be dependent on expression in the context of a native viral genome.

A Simian Virus 5 (SV5) P/V Mutant Is Less Cytopathic than Wild-Type SV5 in Human Dendritic Cells and Is a More Effective Activator of Dendritic Cell Maturation and Function

ABSTRACT Human epithelial cells infected with the parainfluenza virus simian virus 5 (SV5) show minimal activation of host cell interferon (IFN), cytokine, and cell death pathways. In contrast, a recombinant SV5 P/V gene mutant (rSV5-P/V-CPI−) overexpresses viral gene products and is a potent inducer of IFN, proinflammatory cytokines, and apoptosis in these cells. In this study, we have compared the outcomes of wild-type (WT) SV5 and rSV5-P/V-CPI− infections of primary human dendritic cells (DC), important antigen-presenting cells for initiating adaptive immune responses. We have tested the hypothesis that a P/V mutant which activates host antiviral responses will be a more potent inducer of DC maturation and function than WT rSV5, which suppresses host cell responses. Infection of peripheral blood mononuclear cell-derived immature DC with WT rSV5 resulted in high levels of viral protein and progeny virus but very little increase in cell surface costimulatory molecules or secretion of IFN and proinflammatory cytokines. In contrast, immature DC infected with the rSV5-P/V-CPI− mutant produced only low levels of viral protein and progeny virus, but these infected cells were induced to secrete IFN-α and other cytokines and showed elevated levels of maturation markers. Unexpectedly, DC infected with WT rSV5 showed extensive cytopathic effects and increased levels of active caspase-3, while infection of DC with the P/V mutant was largely noncytopathic. In mixed-culture assays, WT rSV5-infected DC were impaired in the ability to stimulate proliferation of autologous CD4+ T cells, whereas DC infected with the P/V mutant were very effective at activating T-cell proliferation. The addition of a pancaspase inhibitor to DC infected with WT rSV5 reduced cytopathic effects and resulted in higher surface expression levels of maturation markers. Our finding that the SV5 P/V mutant has both a reduced cytopathic effect in human DC compared to WT SV5 and an enhanced ability to induce DC function has implications for the rational design of novel recombinant paramyxovirus vectors based on engineered mutations in the viral P/V gene.