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.
Bibliographical noteFunding Information:
NDVS was supported by the National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov/) under grant number T32AI007539. LTK was supported by an American Cancer Society (https://www.cancer.org/research/we-fund-cancer-research/apply-research-grant/extramural-grants-staff-contacts.html) Research Scholar Grant, RSG-1-160-01-MPC and National Institute of Allergy and Infectious Diseases AI097875. SAT was supported by the National Institutes of Health (https://grants.nih.gov/grants/oer.htm) R01AI108407 and P01CA214091. DGO and DWW were supported by the Gundersen Medical Foundation (http://www.gundersenhealth.org/foundation/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Hong Wang for technical assistance with library preparation for MiSeq, and Clint Paden, Alisa Yurovsky, and Laraib Malik for assistance with sequencing analysis. Dr. Britt Glaunsinger and Dr. Jason Upton for technical advice. Special thanks to Varvara Kirillov and Swati Gupta for technical support, Dr. Patrick Hearing for critical discussions, and members of the Krug, French, and Hearing laboratories for helpful discussions.