The VP35 protein of Ebola virus is a viral antagonist of interferon. It acts to block virus or double-stranded RNA-mediated activation of interferon regulatory factor 3, a transcription factor that facilitates the expression of interferon and interferon-stimulated genes. In this report, we show that the VP35 protein is also able to inhibit the antiviral response induced by alpha interferon. This depends on the VP35 function that interferes with the pathway regulated by double-stranded RNA-dependent protein kinase PKR. When expressed in a heterologous system, the VP35 protein enhanced viral polypeptide synthesis and growth in Vero cells pretreated with alpha/beta interferon, displaying an interferon-resistant phenotype. In correlation, phosphorylation of PKR and eIF-2␣ was suppressed in cells expressing the VP35 protein. This activity of the VP35 protein was required for efficient viral replication in PKR ؉/؉ but not PKR ؊/؊ mouse embryo fibroblasts. Furthermore, VP35 appears to be a RNA binding protein. Notably, a deletion of amino acids 1 to 200, but not R312A substitution in the RNA binding motif, abolished the ability of the VP35 protein to confer viral resistance to interferon. However, the R312A substitution rendered the VP35 protein unable to inhibit the induction of the beta interferon promoter mediated by virus infection. Together, these results show that the VP35 protein targets multiple pathways of the interferon system.

The ability of the ␥ 1 34.5 protein to suppress the PKR response plays a crucial role in herpes simplex virus pathogenesis. In this process, the ␥ 1 34.5 protein associates with protein phosphatase 1 to form a large complex that dephosphorylates eIF-2␣ and thereby prevents translation shutoff mediated by PKR. Accordingly, ␥ 1 34.5 null mutants are virulent in PKR-knockout mice but not in wild-type mice. However, ␥ 1 34.5 deletion mutants, with an extragenic compensatory mutation, inhibit PKR activity but remain avirulent, suggesting that the ␥ 1 34.5 protein has additional functions. Here, we show that a substitution of the ␥ 1 34.5 gene with the NS1 gene from influenza A virus renders viral resistance to interferon involving PKR. The virus replicates as efficiently as wild-type virus in SK-N-SH and CV-1 cells. However, in mouse 3T6 cells, the virus expressing the NS1 protein grows at an intermediate level between the wild-type virus and the ␥ 1 34.5 deletion mutant. This decrease in growth, compared to that of the wild-type virus, is due not to an inhibition of viral protein synthesis but rather to a block in virus release or egress. Virus particles are predominantly present in the nucleus and cytoplasm. Notably, deletions in the amino terminus of the ␥ 1 34.5 protein lead to a significant decrease in virus growth in mouse 3T6 cells, which is independent of eIF-2␣ dephosphorylation. In correlation, a series of deletions in the amino-terminal domain impair nuclear as well as cytoplasmic egress. These results indicate that efficient viral replication depends on the ␥ 1 34.5 functions required to prevent the PKR response and to facilitate virus egress in the different stages during virus infection.

The gamma(1)34.5 protein of herpes simplex virus 1 (HSV-1) is a virus-encoded protein phosphatase 1 (PP1) regulatory protein that contributes to viral resistance to interferon. It functions to block the shutoff of protein synthesis mediated by the double-stranded RNA-dependent protein kinase. This requires the carboxyl terminus of the gamma(1)34.5 protein to recruit PP1, forming a high-molecular-weight complex that dephosphorylates the alpha subunit of translation initiation factor eIF-2 (eIF-2alpha). In the present study, we introduced a series of point mutations into a region in the effector domain of the gamma(1)34.5 protein, which is adjacent to the PP1-binding domain. Analysis of these mutants in virus-infected cells shows that Ser209Ala, Ser209Asp, Ser218Ala, or Trp219Tyr substitution does not affect viral response to interferon-alpha. In contrast, Arg215Leu or Ser218Asp substitution rendered the virus hypersensitive to interferon-alpha, which correlates with the inability of these gamma(1)34.5 mutants to mediate dephosphorylation of eIF-2alpha. However, Arg215Leu or Ser218Asp substitution does not disrupt the formation of a high-molecular-weight complex required for eIF-2alpha dephosphorylation or binding of the gamma(1)34.5 protein to PP1. These results suggest that concerted action of the PP1-binding domain and the effector domain of the gamma(1)34.5 protein is required to confer HSV-1 interferon resistance.