Variants of the Paramyxovirus Simian virus 5 with accelerated or delayed viral gene expression activate proinflammatory cytokine synthesis

Abstract

Our previous results have shown that the parainfluenza virus SV5 is a poor inducer of proinflammatory cytokines interleukin-8 (IL-8) and macrophage chemoattractant protein 1 (MCP-1). By contrast, an engineered P/V mutant rSV5-P/V-CPI- and a naturally-occurring variant WF-PIV (Wake Forest-Parainfluenza Virus) are both potent activators of IL-8 and MCP-1. In the present study, we addressed the question of why rSV5-WT is such a poor inducer of host cytokine responses relative to the two SV5 variants, and we used the CC chemokine RANTES as a measure of host responses. Time course experiments showed high-level secretion of IL-6 and RANTES following infections of human A549 lung epithelial cells with the P/V-CPI- mutant and WF-PIV. By contrast, SV5-WT induced very low cytokine responses, with the notable exception of moderate induction of RANTES. The mechanism of RANTES induction by the two SV5 variants shared common properties, since RANTES secretion from infected cells had similar kinetics, depended on virus replication, correlated with increased RANTES mRNA levels and promoter activation, and was reduced by inhibitors of the p38 MAPK, ERK, and PI3K pathways. Despite the similar mechanisms of RANTES induction, the two SV5 variants differed dramatically in their growth and gene expression kinetics. By comparison to the P/V mutant rSV5-P/V-CPI- which has accelerated viral gene expression, WF-PIV infection showed a prolonged delay in viral replication, and infected cells did not show high-level viral RNA and protein expression until approximately 12-24 hpi. Sequence analysis revealed that the N, P, V, and M genes from WF-PIV differed by 3, 8, 5, and 10 amino acids compared to rSV5-WT, respectively. Chimeric viruses harboring the WF-PIV P/V or M genes in the context of the other rSV5 genes had growth properties similar to rSV5-WT but had a RANTES-inducing phenotype similar to that of the bone fide WF-PIV virus. Our data indicate a role for both the P/V and the M gene products as determinants of RANTES induction in response to SV5 infection.

Role for the Paramyxovirus Genomic Promoter in Limiting Host Cell Antiviral Responses and Cell Killing

“…Phase microscopy and fluorescent microscopy were carried out as described previously (50). Analysis of IRF-3 nuclear translocation was performed as detailed elsewhere (13).…”

“…In contrast, we have shown previously that the naturally occurring SV5 variant Wake Forest isolate of parainfluenza virus (WF-PIV) has a delay in viral RNA synthesis but is a potent inducer of IL-8 and IFN-␤ responses (50). WF-PIV is closely related to the CPI strain of SV5 and was isolated in our lab from persistently infected Vero cells (50). Sequence analysis has identified many nucleotide differences between WF-PIV and WT SV5 (50).…”

“…V protein also appears to act as a decoy substrate for kinases that activate IFN regulatory factor 3 (IRF-3) (29) and IRF-7 (36). Similarly, the SV5 V protein is reported to block IL-6 induction (27), although virus-infected cells still respond to tumor necrosis factor alpha-mediated induction of IL-6 (50).…”

“…WF-PIV is closely related to the CPI strain of SV5 and was isolated in our lab from persistently infected Vero cells (50). Sequence analysis has identified many nucleotide differences between WF-PIV and WT SV5 (50). Exchanging the P/V gene from WF-PIV or a second variant, CPI-, into an otherwise WT SV5 background results in a chimeric virus that overexpresses viral gene products and activates cytokine secretion and apoptosis (10,46,50).…”

“…Sequence analysis has identified many nucleotide differences between WF-PIV and WT SV5 (50). Exchanging the P/V gene from WF-PIV or a second variant, CPI-, into an otherwise WT SV5 background results in a chimeric virus that overexpresses viral gene products and activates cytokine secretion and apoptosis (10,46,50). These results illustrate that chimeras can have phenotypes that are very different from those of the two parental viruses (10) and suggest that the trans-acting paramyxovirus gene products function in concert to coordinate viral gene expression and limit host cell responses.…”