Summary auto-generated
This study investigated why HSV-1716, an ICP34.5 null mutant of herpes simplex virus, exhibits severely restricted replication in CV-1 monkey kidney cells, despite replicating well in most malignant cells. The researchers found that HSV-1716 cannot replicate in CV-1 cells due to a translational block caused by the host cell's PKR (protein kinase R) response. However, this block can be overcome by coinfection with simian virus 40 (SV40) or by the presence of SV40 T antigen, as demonstrated by the normal replication of HSV-1716 in COS-1 cells (CV-1 cells transformed with SV40). Viral gene transcription was normal in infected CV-1 cells, indicating the block occurs at the protein synthesis level rather than at transcription. Using metabolic labeling and Western blotting, the researchers confirmed that viral protein translation is severely inhibited in CV-1 cells infected with HSV-1716 but proceeds normally in wild-type HSV or in COS-1 cells. Treatment with okadaic acid, a phosphatase inhibitor, restored the translational block, supporting the hypothesis that HSV ICP34.5 and SV40 T antigen overcome the PKR-mediated translational shutdown at different points in the pathway.
Key findings
- HSV-1716 replication is blocked in CV-1 cells due to PKR-mediated translational inhibition, not transcriptional defects
- SV40 coinfection rescues HSV-1716 replication in CV-1 cells by overcoming the translational block downstream of eIF2α phosphorylation
- HSV-1716 replicates normally in COS-1 cells that constitutively express SV40 T antigen, confirming the virus can bypass restrictions when T antigen is present
- Okadaic acid restores the translational block in permissive cells, supporting a phosphatase-dependent mechanism for both HSV ICP34.5 and SV40 T antigen
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Abstract
Herpes simplex virus (HSV) mutants lacking the gene encoding infected cell protein (ICP) 34.5 exhibit an attenuated phenotype in models of pathogenesis and have been used for experimental cancer therapy. Recently it was shown that the HSV ICP 34.5 protein functions to prevent the host cell-induced double-stranded RNA-activated protein kinase (PKR)-dependent translational block that normally occurs during virus infection. We now report that an HSV ICP 34.5 mutant called HSV- 1716 is unable to replicate in the simian kidney cell-derived line CV- 1, due to a translational block. Moreover, we find that this block can be overcome by simian virus 40 (SV40). This has been shown directly by infecting CV-1 cells with SV40 and HSV-1716 simultaneously, and indirectly via HSV-1716 infection of COS-1 cells (CV-1 cells transformed by an origin-defective mutant of SV40 that codes for wild- type T antigen). The translational block is restored when infections are done in the presence of the phosphatase inhibitor okadaic acid. These results support, but do not directly prove, contentions that HSV ICP 34.5 interacts with the PKR pathway to restore translation in non- permissive cells, and that SV40 large T antigen has a similar functional role, but acts downstream of the site of ICP 34.5 interaction (eIF2alpha) in the pathway. Study of this CV-1/COS-1 system should allow further clarification of the virus-host interactions that underlie the restricted replication of HSV-1 ICP 34.5 gene null mutants.