Replication-independent activation of human plasmacytoid dendritic cells by the paramyxovirus SV5 Requires TLR7 and autophagy pathways.

The paramyxovirus Simian Virus 5 (SV5) is a poor inducer of interferon (IFN) secretion in all cell types tested so far, including primary epithelial cells and primary human myeloid dendritic cells. SV5 is hypothesized to limit induction of antiviral responses through control of viral gene expression and production of the V protein antagonist. Plasmacytoid dendritic cells (pDCs) are known to uniquely express toll-like receptor (TLR)-7 and are a main producer of IFN-alpha among peripheral blood mononuclear cells in response to many viruses. Here, we tested whether SV5 would remain a poor inducer of IFN in primary human pDCs. The efficiency of SV5 infection of pDCs could be increased by an increasing multiplicity of infection. pDCs infected by both live and UV-inactivated SV5 induced large amounts of IFN-alpha secretion and resulted in upregulation of maturation markers CD80 and CD86. However, IL-6 secretion was not induced by SV5 infection. When TLR7 signaling was inhibited, SV5 induced less IFN secretion and CD80 expression, and there was a corresponding increase in number of infected cells. Similar effects were seen with inhibitors of cellular autophagy pathways, suggesting that the SV5 activation of pDC requires access to the cytoplasm and autophagic sampling of cytoplasmic contents. These results have implications for control of SV5 infections in vivo and for development of SV5 as a vaccine vector.

TLR3-dependent upregulation of RIG-I leads to enhanced cytokine production from cells infected with the parainfluenza virus SV5.

Here we address the role of RIG-I and TLR3 in differential cytokine responses against Simian Virus 5 (SV5) and two distinct cytokine inducing SV5 mutants. IFN-beta and IL-6 secretion was induced by infection with P/V-CPI-, an SV5 mutant with P/V substitutions, and were reduced by either siRNA-mediated knockdown of RIG-I expression or by expression of a dsRNA-binding protein. TLR3 overexpression did not alter cytokine secretion induced by P/V-CPI- or by Le-(U5C, A14G), an SV5 promoter mutant. TLR3 signaling by addition of exogenously added dsRNA was not blocked by WT SV5 or either SV5 mutant. Unexpectedly, TLR3 activation in infected cells led to enhanced IL-8 secretion, which correlated with increased RIG-I expression. Dominant negative RIG-I and TRIF supported a model whereby TLR3 activation upregulates RIG-I expression and in turn hypersensitizes cells to RIG-I-mediated cytokine secretion. Implications for crosstalk between different innate immunity pathways in mounting antiviral responses to paramyxoviruses are discussed.

Role for the paramyxovirus genomic promoter in limiting host cell antiviral responses and cell killing.

The parainfluenza virus simian virus 5 (SV5) is a poor inducer of innate immune responses. In contrast, the naturally occurring SV5 variant Wake Forest parainfluenza virus (WF-PIV) activates the synthesis of proinflammatory cytokines and beta interferon (IFN-beta). Comparison of SV5 and WF-PIV genome sequences revealed nine nucleotide differences within the viral genomic promoter, including two substitutions (U5C and A14G) in the most highly conserved 3'-end promoter element. To test the consequences of these promoter variations, a recombinant SV5 mutant [Le-(U5C, A14G)] was engineered to harbor the two WF-PIV genomic promoter substitutions in an otherwise wild-type (WT) SV5 background. Human lung epithelial cells infected with the Le-(U5C, A14G) mutant had higher rates of viral protein synthesis and levels of mRNA than cells infected with WT SV5, but levels of genomic RNA were not changed. Unlike WT SV5, the Le-(U5C, A14G) mutant was a potent inducer of interleukin-6 and IFN-beta synthesis, despite expressing a functional V protein antagonist. Cytokine responses to Le-(U5C, A14G) infection were reduced either by small interfering RNA-mediated knockdown of retinoic acid-inducible gene I (RIG-I) or after infection of cells that were engineered to express the reovirus sigma3 double-stranded RNA-binding protein. Le-(U5C, A14G) induced cytopathic effects not seen with WT SV5, and the extent of cell killing correlated with elevated levels of viral F protein and cell-cell fusion. Our results support a model whereby the SV5 promoter has evolved to function at an attenuated level in order to limit (i) synthesis of aberrant RNAs which induce RIG-I-mediated responses and (ii) overproduction of mRNA for potentially toxic gene products, such as the F protein. Control of genomic promoter activity may be particularly important for viruses such as SV5, that express a V protein targeting mda-5 but do not encode antagonists such as the paramyxovirus C proteins, that specifically target RIG-I.

A hyperfusogenic F protein enhances the oncolytic potency of a paramyxovirus simian virus 5 P/V mutant without compromising sensitivity to type I interferon.

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.

Paramyxovirus-induced shutoff of host and viral protein synthesis: role of the P and V proteins in limiting PKR activation.

The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI(-) mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI(-) mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI(-) virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2alpha. WT SV5 was a poor activator of the eIF-2alpha kinase protein kinase R (PKR). By contrast, the P/V-CPI(-) mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI(-) mutant was restored to approximately 50% that of control HeLa cells. By contrast, the rates of P/V-CPI(-) viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI(-) mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI(-) mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.