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

<div><p>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 <i>orf75A</i> 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.</p></div

Accelerated gene expression coupled with replication defect upon 75A.stop infection of primary cells.

<p><b>(A)</b> Single-step growth curve in primary BMDM at an MOI of 5 with 75A.stop1.2 and 75A.stop1MR. <b>(B)</b> Timecourse analysis of gene products upon a single-step infection of BMDMs. <b>(C)</b> Multi-step growth curve in primary MEFs at an MOI of 0.05 with 75A.stop1.2 and 75A.stop1MR. <b>(D)</b> Timecourse analysis of gene products upon a low MOI infection of MEFs. <b>(E)</b> Schematic of the ORF75 locus and the multicistronic transcripts with strand-specific probes to indicated ORFs. <b>(F)</b> Northern blot analysis of NIH 3T12 fibroblast cells infected with indicated viruses at an MOI of 5. Membrane was hybridized with strand-specific ³²P-labled cDNA of ORF75A, ORF75B, ORF75C, M3, or 18S. Longer exposures with ORF75C probe detects the <i>orf75A/B/C</i> transcript. <b>(G)</b> Viral DNA quantification of input genomes at 2 hpi and subsequent replication at 24 hpi. For A, C, and E, bars represent the mean of three independent biological replicates +/- SD.</p

Input of equivalent genomes reveals role for ORF75A in driving productive replication in cell culture and in mice.

<p><b>(A)</b> Multi-step growth curve in NIH 3T12 cells infected with equivalent PFU or equivalent genomes. <b>(B-D)</b> Expression of the YFP reporter gene in NIH 3T12 fibroblasts <b>(B)</b>, primary fibroblasts (MEFs) <b>(C)</b>, and primary bone marrow-derived macrophage cells (BMDM) <b>(D)</b> upon infection with indicated viruses at an equivalent PFU or with equivalent genomes. <b>(E)</b> Acute replication in the lungs of mice 7 dpi. Mice were infected with indicated viruses at 1000 PFU or adjusted to infect at equivalent FFU. * p ≤ 0.05, ** p ≤ 0.005 ***, p ≤ 0.0005, and **** p ≤ 0.00005; significance determined by two-way ANOVA for A and one-way ANOVA for B-E.</p

Frequencies of cells harboring viral genomes in C57BL/6 mice.

<p>Frequencies of cells harboring viral genomes in C57BL/6 mice.</p

Divergence of the gammaherpesvirus vFGARAT family.

<p><b>(A)</b> Phylogenetic tree of the vFGARATs of the indicated γ-hepesviruses (Genebank ID located in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006843#ppat.1006843.s007" target="_blank">S1 Table</a>). <b>(B)</b> Phylogenetic alignment using BLOSUM62 with previously characterized domains of the vFGARATs. <b>(C)</b> Pairwise alignment showing degrees of identity (lower left corner of table) or similarity (upper right corner of table) between the respective N-terminal or C-terminal portions of the indicated vFGARATs. The division point for each vFGARAT (red vertical line in B) was based on dotplot analysis for each indicated vFGARATS compared to MHV68 ORF75C.</p

Intraperitoneal administration of MHV68 reveals a reactivation defect in the absence of ORF75A.

<p>C57BL/6 mice were infected at 1000 PFU by the intraperitoneal route with the indicated viruses. <b>(A)</b> Splenomegaly at 18 dpi with indicated viruses. Each symbol represents an individual mouse. Line indicates geometric mean titer. <b>(B)</b> Frequency of splenocytes harboring genomes 18 dpi. <b>(C)</b> Frequency of splenocytes spontaneously reactivating from latency 18 dpi. <b>(D)</b> Reactivation efficiency of splenocytes 18 dpi. <b>(E)</b> Frequency of PECs harboring genomes 18 dpi. <b>(F)</b> Frequency of PECs spontaneously reactivating from latency 18 dpi. For the limiting dilution analyses, curve fit lines were determined by nonlinear regression analysis. Using Poisson analysis, the intersection of the nonlinear regression curves with the dashed line at 63.2% was used to determine the frequency of cells that were either positive for the viral genome or reactivating virus. Data is generated from 5 independent experiments with 4–5 mice per group for splenocyte data and 2 independent experiments with 4 mice per group for PEC data. ** p ≤ 0.005, *** p ≤ 0.0005, and **** p ≤ 0.00005; for A significance determined by two-way unpaired t-test; for B-F significance determined by two-way paired t-test.</p

Frequencies of cell populations reactivating viral genomes in C57BL/6 mice.

<p>Frequencies of cell populations reactivating viral genomes in C57BL/6 mice.</p

Input of equivalent genomes ameliorates the high particle to PFU phenotype of 75A.stop.

<p><b>(A)</b> Immunoblot analysis of purified virions from three independent virus stock preparations. <b>(B)</b> Quantification of the particle to PFU ratio. Particles were enumerated by qPCR of encapsidated genomes. Error bars represent SEM of four independent ORF75.stop1.2 virus stocks and their respective marker rescue viruses generated in parallel. Significance determined by the one-way paired t-test. <b>(C)</b> Quantification of the infectious particle to PFU ratio. Infectious particles were enumerated by qPCR of cell-associated viral genomes 2 hpi upon 75A.stop1 or 75A.stop1MR infection of primary MEFs. Ratios of 75A.stop1 viruses are relative to the 75A.stop1MR fraction; significance determined by one-way ANOVA. <b>(D)</b> Timecourse analysis of immediate early promoter activity during lytic infection. NIH 3T12 cells were transfected with a RTA responsive immediate-early promoter expressing luciferase, and subsequently infected with equivalent PFU (MOI of 5) or equivalent genomes. <b>(E)</b> Timecourse analysis of NF-κB activation during lytic infection. NIH 3T12 cells were transfected with a NF-κB responsive promoter expressing luciferase, and subsequently infected as described in (D). <b>(F)</b> Time course analysis of TNFα release in BMDMs infected as described in (D). Dashed line indicates the limit of detection for the assay. Samples that are below the limit of detection (B.L.D.) are indicated. <b>(G)</b> Timecourse analysis of LDH release as described in (F). Error bars indicate SD with three independent biological replicates. * p ≤ 0.05 ** p ≤ 0.005, and **** p ≤ 0.00005; significance determined by one-way ANOVA compared to respective 75A.stop1MR at matched timepoints (D-G).</p

Generation and characterization of 75A.stop and ORF75B.stop viruses.

<p><b>(A)</b> Schematic of the 75A.stop mutations in MHV68. Restriction sites used to verify stop codon insertions are underlined. <b>(B)</b> Restriction fragment length polymorphism (RFLP) analysis of 75A.stop mutants after BamHI digestion. <b>(C)</b> Single-step growth curve of 75A.stop mutants and parental H2B-YFP viruses in NIH 3T3 fibroblasts (MOI 5). <b>(D)</b> Schematic of the ORF75B mutations in MHV68. <b>(E)</b> RFLP analysis of ORF75B.stop mutants after EcoRI digestion. <b>(F)</b> Single-step growth of ORF75B.stop mutants and marker rescue virus in NIH 3T3 fibroblasts (MOI 5). Error bars represent +/- SD of triplicate infections. C57B/6 mice were inoculated with 1000 PFU via the intranasal route with the indicated viruses. Lung homogenates from mice were titered by plaque assay. <b>(G)</b> Timecourse analysis of acute replication on the lungs. <b>(H)</b> Acute replication in the lungs 7 dpi. Each symbol represents an individual mouse. Line indicates geometric mean titer. The dashed line depicts the limit of detection at 50 PFU/ml of lung homogenate (log₁₀ of 1.7). * p ≤ 0.05; ** p ≤ 0.005; **** p ≤ 0.0005; for G-H, significance determined by one-way ANOVA analysis.</p

MHV68 ORF75A is essential for efficient seeding of intermediate reservoirs and the establishment of latency in the spleen at early, but not late times of chronic infection after intranasal inoculation.

<p>C57BL/6 mice were infected at 1000 PFU by the intranasal route with the indicated viruses. <b>(A)</b> Frequency of cells in the mediastinal lymph node harboring viral genomes 9 dpi. Data is a compilation of 75A.stop1.1 (n = 1) with 75A.stop1.2 (n = 4) and of H2BYFP (n = 1) with 75A.stop1MR (n = 4). <b>(B)</b> Frequency of peripheral blood mononuclear cells harboring genomes 9 dpi. Data is generated from 3 independent experiments, 5 mice per group. <b>(C)</b> Splenomegaly 16 dpi with indicated viruses. Each symbol represents an individual mouse. Line indicates geometric mean titer. ORF75A mutants are blue and WT control viruses are black. <b>(D)</b> Frequency of splenocytes harboring genomes 16 dpi. <b>(E)</b> Frequency of splenocytes undergoing reactivation from latency upon explant 16 dpi. <b>(F)</b> Frequency of splenocytes harboring genomes at six weeks post-infection. For the limiting dilution analyses, curve fit lines were determined by nonlinear regression analysis. Using Poisson analysis, the intersection of the nonlinear regression curves with the dashed line at 63.2% was used to determine the frequency of cells that were either positive for the viral genome or reactivating virus. <b>(C-F)</b> Data is generated from two independent experiments for 75A.stop2 and 75a.dbl.stop viruses, and three independent experiments for 75A.stop1.2 and 75A.stop1MR viruses. Each experiment contained three to six mice per group for 16 dpi. Data for 42–49 dpi is generated from two experiments with four to five mice per group. Error bars indicate SEM. * p ≤ 0.05, *** p ≤ 0.0005, and **** p ≤ 0.00005; for A, B, D-F, significance determined by two-way paired t-test; for C, significance determined by two-way unpaired t-test.</p