Mechanisms leading to translation arrest in Autographa californica multinucleocapsid nucleopolyhedrovirus-infected Lymantria dispar 652Y cells

Infection of the Lymantria dispar 652Y (Ld652Y) cell line with the baculovirus Autographa californica Multinucleocapsid nucleopolyhedrovirus (AcMNPV) results in global translation arrest, initiation of apoptosis in the absence of a functional, virus encoded, apoptotic suppressor and a non-productive infection. While AcMNPV enters Ld652Y cells and transcription of virus genes occurs, virus progeny are not produced. Expression of a single gene, host range factor-1 (hrf-1), isolated from Lymantria dispar Multinucleocapsid Nucleopolyhedrovirus (LdMNPV), precludes translation arrest in AcMNPV-infected Ld652Y cells further allowing permissive replication of the virus. HRF-1 also enables replication of Hyphantria cunea NPV, Bombyx mori NPV and Spodoptera exigua NPV in Ld652Y cells indicating that hrf-1 is an essential host range factor for baculovirus infection in Ld652Y cells. Database and motif searches conducted with the hrf-1 sequence provide no indication to its function. The goal of this study was to determine the mechanism of translation arrest in AcMNPV-infected Ld652Y cells to further identify possible functions for HRF-1. We show that eIF2alpha phosphorylation at serine 51 occurs in AcMNPV-infected Ld652Y cells at 12 h.p.i. correlating to the onset of translation arrest. Ld652Y cells infected with vAchrf-1 show a reduced level of eIF2alpha phosphorylation for the full-length peIF2alphaser51 protein at 6, 12, 24 and 48 h.p.i. as compared to mock-infected cells. PK2, the AcMNPV eIF2alpha kinase inhibitor, was expressed in AcMNPV-infected Ld652Y cells but did not prevent eIF2alpha phosphorylation. An apparent proteolytic cleavage of peIF2alphaser51 was observed beginning at 12 h.p.i.; this cleavage event was investigated further. In Sf21 cells, a permissive cell line for AcMNPV replication, peIF2alpha ser51 was cleaved in both AcMNPV and vAchrf-1-infected cells indicating that HRF-1 was not directly responsible for the cleavage. Cell permeable protease inhibitors did not block cleavage of peIF2alpha ser51. However, the proteasome inhibitor epoxomicin did reduce phosphorylation of eIF2alpha at serine 51, block cleavage of peIF2alphaser51 and inhibit virus replication in AcMNPV and vAchrf-1-infected Sf21 cells and vAchrf-1-infected Ld652Y cells. The proteasome inhibitor MG132 mimicked the effects of epoxomicin in vAchrf-1-infected Ld652Y cells. However, treatment with MG 132 in AcMNPV and vAchrf-1-infected Sf21 cells resulted in reduced virus titers and no block of peIF2alphaser51 cleavage. This study suggests a role for the proteasome in enabling baculovirus replication. (Abstract shortened by UMI.)...