Replication of Herpes Simplex Virus 1 Depends on the γ ₁ 34.5 Functions That Facilitate Virus Response to Interferon and Egress in the Different Stages of Productive Infection

Abstract

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.

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

“…In the present study, we found that the viral protein ICP34.5 interacted with cellular p32 to form a complex, and the redistribu- tion of this complex was linked to the viral budding from nuclei. Notably, knockdown of p32 by shRNA caused a restriction of HSV-1 nuclear egress by 75% (Table 2), whereas 79% of R3616 was trapped in the nucleus in normal HeLa cells (Table 1), similar to that in MEF 3T6 cells in which the majority of viruses are retained in the nucleus of cells infected with ICP34.5 deletion mutants (23). These results suggest that p32 is a mediator of HSV-1 nuclear egress by interaction with ICP34.5.…”

“…Previous studies suggest that ICP34.5 is required for viral nuclear egress in MEF 3T6 cells (23,24). To further study this phenotype, we examined the nuclear egress of HSV-1 in HeLa cells by electron microscopy (EM) (Fig.…”

“…ing Protein-HSV-1 ICP34.5 is a virulence factor that has multiple roles in virus-infected cells, such as reducing interferon ␤ production (29), promoting protein synthesis (36), blocking autophagy (31), and facilitating viral egress (23). To study ICP34.5, we set out to identify cellular proteins that may interact with ICP34.5.…”

“…(33). Furthermore, ICP34.5 contributes to viral egress, a function independent of its anti-interferon activity (23). Nevertheless, the mechanism of ICP34.5 involved in viral egress has yet to be unveiled.…”

“…Previous studies suggest that nuclear egress involves HSV-1 ICP34.5 (23,24), which is an essential protein in viral pathogenesis (25)(26)(27). HSV-1 ICP34.5 consists of 263 amino acids with an amino-terminal domain, a linker Ala-Thr-Pro repeat, and a carboxyl-terminal domain.…”