Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

Nick D Van Skike,Douglas W White,Peter T Benziger,Nana K Minkah,Jarrod B French,Chad H Hogan,Deborah M Kim-Holzapfel,Mehmet Kara,Darby G Oldenburg,Scott A Tibbetts,Gary Wu,Laurie T Krug,Paul D Ling

doi:10.1371/journal.ppat.1006843

Nick D Van Skike, Douglas W White + Show 11 more

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https://doi.org/10.1371/journal.ppat.1006843

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Abstract

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Highlights

Herpesviruses traverse multiple cell types to gain access to host cells that serve as long-term reservoirs of latent infection
We report that a rhadinovirus of rodents requires a previously uncharacterized viral FGARAT (vFGARAT) family member, ORF75A, to support viral growth and persistence in mice
We performed a phylogenetic analysis of the amino acid sequences of the vFGARATs, and identified four major clades across multiple species within the lymphocryptovirus and rhadinovirus subfamilies: lymphocryptovirus ORF75, rhadinovirus ORF3, rhadinovirus ORF75, and murine rhadinovirus ORF75 homologs (Fig 1A)

Summary

Introduction

Herpesviruses traverse multiple cell types to gain access to host cells that serve as long-term reservoirs of latent infection. The successful colonization and maintenance inside the host lies in the evasion of cellular intrinsic and host immune defenses. A unique adaptation of the gammaherpesvirus subfamily (γHVs) is the capture and repurposing of the cellular purine metabolism enzyme, formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT) to support infection [1,2,3]. The human herpesviruses Epstein-Barr virus (EBV/HHV-4) and Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) each encode a single viral FGARAT (vFGARAT), yet other gammaherpesviruses encode multiple vFGARATs [1]. The primate rhadinovirus herpesvirus saimiri (HVS) encodes two vFGARATs with distinct functions [4], and the murine gammaherpesviruses have invested ~10% of their genomes to encode three vFGARATs, orf75A, orf75B, and orf75C

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