Increased Readthrough Transcription across the Simian Virus 5 M-F Gene Junction Leads to Growth Defects and a Global Inhibition of Viral mRNA Synthesis

Abstract

Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50-to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ϳ1.5-to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5 end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.

Naturally Occurring Substitutions in the P/V Gene Convert the Noncytopathic Paramyxovirus Simian Virus 5 into a Virus That Induces Alpha/Beta Interferon Synthesis and Cell Death

“…To generate pRSV5-P/V-CPIϪ, a StuI-ClaI DNA fragment was excised from pEF.W3/CPIϪ/V, a plasmid encoding the CPIϪ V gene (7) and inserted into the corresponding region of pBH311. rSV5 was recovered using pRSV5-P/V-CPIϪ as described previously (19) with minor modifications (33,37). Briefly, 3.5-cm dishes of A549 cells were infected at a multiplicity of infection (MOI) of ϳ3 with vaccinia virus MVA and then transfected with plasmids carrying the NP (1.2 g), P (0.15 g), and L (1.5 g) genes along with pRSV5-P/V-CPIϪ (5 g).…”

“…The W3A strain of SV5 and the Greer strain of HPIV2 were grown in MDBK and CV-1 cells, respectively. Single-step growth assays and plaque assays were carried out as described previously (37).…”

A Novel RNA Virus System for Selective Killing of Breast Cancer Cells

“…Vaccinia virus MVA expressing T7 RNA polymerase was the kind gift of B. Moss and was grown and titered on BHK cells (Wyatt et al, 1995). Single step growth kinetics and SV5 plaque assays were carried out as described previously (Parks et al, 2001) Signals from the NP-P and HN-L junctions, respectively ( Fig. 1A; Rassa and Parks, 1999), and the overall length was a multiple of six (Murphy and Parks, 1997).…”

“…Equivalent amounts of protein were serially diluted in PBS and applied to nitrocellulose using a vacuum manifold. Nitrocellulose membranes were blocked (5% milk in PBS), and probed using a rat monoclonal antibody specific for an HA epitope (anti-HA clone 3F10, Roche Molecular Biochemicals) or rabbit antisera to the SV5 NP and P proteins (Parks et al, 2001). Proteins were visualized using HRP-conjugated goat anti-rabbit and ECL (Pierce Chemicals).…”

“…SV5 virus expressing the HA-tagged TK protein was recovered from pRSV5-TK as described previously (He et al, 1997) with minor modifications (Parks et al, 2001). Virus stocks were generated from single plaques by growth in MDBK cells.…”