| Interferons are a family of cytokines that are secreted by cells and exert antiviral, cell growth inhibitory, and immunoregulatory activities. Responsive cells initiate a signaling cascade that culminates in the induction of interferon-inducible genes, including the double-stranded RNA-dependent protein kinase (PKR). PKR directly limits viral protein synthesis in the host cell by inhibiting mRNA translation initiation via serine 51-phosphorylation of the a subunit of the translation initiation factor 2 (eIF-2alpha) in virus-infected cells. Viruses have evolved numerous mechanisms for evading the cellular interferon-induced PKR response. In order to examine the role of viral interferon antagonists in the context of virus infection, we developed a new approach for the construction of recombinant viruses, based on cloning the herpes simplex virus (HSV) genome as a bacterial artificial chromosome (BAC) in E. coli. Specifically, a herpesvirus deletion mutant lacking the non-essential gamma134.5 gene, which is hypersensitive to the antiviral actions of interferon, was used as a tool for investigating the role of viral antagonists. In this study, a series of point mutations was introduced into a cluster of conserved residues in the carboxy terminal domain of the gamma134.5 protein of HSV-1. Substitutions at serine 209 to alanine, serine 209 to aspartic acid, serine 218 to alanine, and tryptophan 219 to tyrosine do not affect viral response to interferon. In contrast, we found that substitutions at arginine 215 to leucine and serine 218 to aspartic acid are deleterious to the function of the gamma134.5 protein to mediate interferon resistance in virus-infected cells. These results suggest that concerted action of the carboxy domain is required to confer HSV-1 interferon resistance.; In addition, we also examined the role of the VP35 protein of Ebola, a recently identified interferon antagonist, using the surrogate HSV-BAC system. We show that VP35 renders virus replication and viral protein accumulation resistant to the interferon-induced antiviral effects in Vero cells treated exogenously with interferon-alpha. In addition, VP35 is able to confer resistance to interferon-alpha/beta in PKR+/+ and PKR-/- mouse embryo fibroblasts. VP35 also inhibits phosphorylation of eIF-2alpha in virus-infected NIH-3T3 cells. These findings suggest that Ebola VP35 contributes to viral resistance to interferon-alpha/beta by inhibition of the PKR pathway. |
