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

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.

Combinatorial Loss of the Enzymatic Activities of Viral Uracil-DNA Glycosylase and Viral dUTPase Impairs Murine Gammaherpesvirus Pathogenesis and Leads to Increased Recombination-Based Deletion in the Viral Genome

“…In summary, the dUTPase activity of ORF54 promotes early replication during acute infection. As observed for other MHV68 mutants impaired for acute replication ( 10 , 30 – 33 , 36 ), ORF54.CM was compromised in latency establishment at 16 dpi, but reached levels comparable to those of the MR virus by 42 dpi. These phenotypes were not observed for a similar ORF54.CM mutant in BALB/c mice ( 21 ).…”

“…MHV68 ORF46.stop virus and ORF46.stop marker rescue (MR) virus on the H2BYFP BAC were previously described ( 10 ) and then made on the M3Luc BAC in this study. Recombinant MHV68 BACs with mutations in ORF46 and ORF54 were generated by en passant mutagenesis as previously described ( 36 ). The primers and gene blocks (gBlocks) used to generate mutant viruses are listed in Table S1 .…”

Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire

“…In many MHV68 pathogenesis studies, there is an apparent disconnect in the phenotypes at early and late-stage infection. For instance, in the absence of a particular host gene or viral factor, a substantial defect in the establishment of splenic latency at 14–18 dpi is often observed (some examples include [ 226 , 227 , 228 , 229 ]). More recently, latency in particular subsets has been analyzed for such phenotypes, and the GC was found to harbor less virus [ 172 , 177 ].…”

Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68

“…For example, MHV68 antisense noncoding transcripts may regulate the expression of sense strand ORF75 genes, which encode an essential tegument protein that is conserved among gammaherpesviruses (Tsai et al, 2015). MHV68 expresses 3 partially homologous genes from this locus, which appear to play important but distinct roles in different stages of MHV68 infection (Cheng et al, 2012;Van Skike et al, 2018), and it is plausible that antisense noncoding transcripts may regulate their differential expression.…”