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

Abstract

Misincorporation of uracil or spontaneous cytidine deamination is a common mutagenic insult to DNA. Herpesviruses encode a viral uracil-DNA glycosylase (vUNG) and a viral dUTPase (vDUT), each with enzymatic and nonenzymatic functions. However, the coordinated roles of these enzymatic activities in gammaherpesvirus pathogenesis and viral genomic stability have not been defined. In addition, potential compensation by the host UNG has not been examined in vivo The genetic tractability of the murine gammaherpesvirus 68 (MHV68) system enabled us to delineate the contribution of host and viral factors that prevent uracilated DNA. Recombinant MHV68 lacking vUNG (ORF46.stop) was not further impaired for acute replication in the lungs of UNG^-/- mice compared to wild-type (WT) mice, indicating host UNG does not compensate for the absence of vUNG. Next, we investigated the separate and combinatorial consequences of mutating the catalytic residues of the vUNG (ORF46.CM) and vDUT (ORF54.CM). ORF46.CM was not impaired for replication, while ORF54.CM had a slight transient defect in replication in the lungs. However, disabling both vUNG and vDUT led to a significant defect in acute expansion in the lungs, followed by impaired establishment of latency in the splenic reservoir. Upon serial passage of the ORF46.CM/ORF54.CM mutant in either fibroblasts or the lungs of mice, we noted rapid loss of the nonessential yellow fluorescent protein (YFP) reporter gene from the viral genome, due to recombination at repetitive elements. Taken together, our data indicate that the vUNG and vDUT coordinate to promote viral genomic stability and enable viral expansion prior to colonization of latent reservoirs.IMPORTANCE Unrepaired uracils in DNA can lead to mutations and compromise genomic stability. Herpesviruses have hijacked host processes of DNA repair and nucleotide metabolism by encoding a viral UNG that excises uracils and a viral dUTPase that initiates conversion of dUTP to dTTP. To better understand the impact of these processes on gammaherpesvirus pathogenesis, we examined the separate and collaborative roles of vUNG and vDUT upon MHV68 infection of mice. Simultaneous disruption of the enzymatic activities of both vUNG and vDUT led to a severe defect in acute replication and establishment of latency, while also revealing a novel, combinatorial function in promoting viral genomic stability. We propose that herpesviruses require these enzymatic processes to protect the viral genome from damage, possibly triggered by misincorporated uracil. This reveals a novel point of therapeutic intervention to potentially block viral replication and reduce the fitness of multiple herpesviruses.

Keywords: DNA replication; dUTPase; gammaherpesvirus; genomic stability; herpesviruses; latency; lytic replication; uracil-DNA glycosylase; viral pathogenesis; virus-host interactions.

FIG 1

The function of the viral UNG encoded by ORF46 is not compensated by host UNG during pathogenesis. (A) The UNGase assay was performed with lysates prepared from UNG^−/− or WT MEFs. UNGase activity was measured in lysates by the generation of a 9-mer cleavage product upon incubation with a 19-mer oligonucleotide containing a single uracil. (B) UNGase assay on lung and spleen tissue from UNG^−/− or WT mice. (C) Time course of ORF46 expression and UNGase activity. UNG^−/− MEFs were infected with recombinant MHV68 with a stop codon disruption in ORF46 (46.stop [stop lanes]) or with the marker rescue virus of ORF46.stop (46.MR [MR lanes]). The indicated viral and host proteins were detected by immunoblotting. (D) Multistep growth curve in primary UNG^−/− and WT MEFs with the indicated viruses (MOI of 0.01). (E) Acute replication in the lungs of UNG^−/− or WT C57BL/6 mice infected by the intranasal route with 1,000 PFU of the indicated viruses (M3Luc) at 9 dpi. Each symbol represents the titer per milliliter of lung homogenate in an individual mouse. The line indicates the geometric mean titer. The dashed line depicts the limit of detection at 50 PFU/ml. (F and G) UNG^−/− mice or WT C57BL/6 mice were infected by the intraperitoneal route with 1,000 PFU of the indicated viruses. (F) Reactivation frequency of splenocytes 16 dpi was examined by limiting dilution assay. Data were generated from two independent experiments with 3 to 6 mice per group (G) Frequency of MHV68 genome-positive splenocytes 42 dpi was determined by limiting dilution PCR. Data were generated from four replicates per condition. 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. For panel E, significance was determined by two-way unpaired t test on infected animals: *, P < 0.05; ***, P < 0.001.

FIG 2

MHV68 lacking the enzymatic activity of vUNG has no replication defect in cell culture or in mouse lungs. (A) Schematic of the MHV68 vUNG catalytic mutant virus (ORF46.CM) with the luciferase reporter gene under the control of the M3 lytic promoter (M3Luc). Alignment of the conserved enzymatic motifs (black amino acids) and the location of the two mutations in ORF46.CM (blue amino acids) are presented below. (B) Immunoblot validation of mutant ORF46 expression in infected UNG^−/− MEFs. (C) Lack of UNGase activity in lysates of UNG^−/− cells infected with ORF46.CM. (D) Multistep growth curve in primary WT MEFs with the indicated viruses (MOI of 0.01). (E to G) WT C57BL/6 mice were infected by the intranasal route with 1,000 PFU of the indicated viruses. (E) Representative pictures of mock-infected or MHV68-infected mice upon in vivo imaging of chemiluminescence in the thorax at 5 and 9 dpi. The scale bar shows photons s⁻¹cm⁻²sr⁻¹. (F) Each symbol represents the total flux radiance within the region of interest for an individual mouse. The dashed line depicts the limit of detection at 5 × 10⁴ photons s⁻¹. (G) Acute replication in lungs imaged in panel F determined by plaque assay. Each symbol represents the titer per milliliter of lung homogenate in an individual mouse. The line indicates the geometric mean titer. The dashed line depicts the limit of detection at 50 PFU/ml. For panels F and G, significance was determined by two-way unpaired t test on infected animals: *, P < 0.05; ***, P < 0.001.

FIG 3

MHV68 lacking the enzymatic activity of vUNG has no defect in the establishment of latency or reactivation from latency in mice. WT C57BL/6 mice were infected by either the intranasal (IN [A to C]) or intraperitoneal (IP [D to F]) route with 1,000 PFU of the indicated viruses. (A and D) Weights of spleens from uninfected, naive mice or infected mice at 16 dpi. (B and E) Frequency of splenocytes harboring latent genomes at 16 dpi. (C and F) Frequency of splenocytes capable of reactivation from latency upon explant at 16 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. For panels A to C, data are generated from three independent experiments with 3 to 6 mice per group. For panels D to H, data are generated from two experiments with 3 to 6 mice per group. Error bars indicate standard errors of the mean (SEM). For panel A, ** indicates P < 0.01 by two-way unpaired t test.

FIG 4

MHV68 lacking the enzymatic activity of the vDUT encoded by ORF54 leads to a transient decrease in acute replication and a reduction in splenic latency at 16 dpi. (A) Schematic of the MHV68 vDUT catalytic mutant virus (ORF54.CM) with the H2BYFP reporter gene located between ORF27 and ORF29b. Alignment of the conserved enzymatic motifs (black amino acids) and the locations of the two mutations in ORF54.CM (red amino acids) are below. WT C57BL/6 mice were infected by either the intranasal (IN [B to E]) or intraperitoneal (IP [F to H]) route with 1,000 PFU of the indicated viruses. (B) Acute replication in the lungs at 5, 7, and 9 dpi. Each symbol represents the titer per milliliter of lung homogenate in an individual mouse. The line indicates the geometric mean titer. The dashed line depicts the limit of detection at 50 PFU/ml. (C and F) Weights of spleens from uninfected, naive mice or infected mice at 16 dpi. (D and G) Frequency of splenocytes harboring latent genomes at 16 dpi. (E and H) Frequency of splenocytes capable of reactivation from latency upon explant at 16 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. For panels C to H, data were generated from three independent experiments, with 3 to 6 mice per group. Error bars indicate SEM. For panels B and C, significance was determined by two-way unpaired t test on infected animals: *, P < 0.05; **, P < 0.01.

FIG 5

Loss of the enzymatic activity of both vUNG and vDUT does not impact MHV68 replication in cell culture, but reduces viral replication in the lung and seeding of the latency reservoir in the spleen after intranasal inoculation of mice. (A) Schematic of the combinatorial vUNG and vDUT catalytic mutant virus (46.CM/54.CM) with the H2BYFP reporter gene located between ORF27 and ORF29b. (B) Multistep growth curve in primary WT MEFs with the indicated viruses (MOI of 0.01). WT C57BL/6 mice were infected by either the intranasal (IN [C to F]) or intraperitoneal (IP [G to I]) route with 1,000 PFU of the indicated viruses. (C) Acute replication in the lungs at 5 and 9 dpi. Each symbol represents the titer per milliliter of lung homogenate in an individual mouse. The line indicates the geometric mean titer. The dashed line depicts the limit of detection at 50 PFU/ml. (D and G) Weights of spleens from uninfected, naive mice or infected mice at 16 dpi. (E and H) Frequency of splenocytes harboring latent genomes at 16 dpi. (F and I) Frequency of splenocytes capable of reactivation from latency upon explant at 16 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. For panels D to F, data are generated from five independent experiments with 3 to 6 mice per group. For panels G to I, data are generated from two independent experiments with 3 to 6 mice per group. Error bars indicate SEM. For panels C and D, significance was determined by two-way unpaired t test on infected animals: *, P < 0.05; ****, P < 0.0001.

FIG 6

Loss of enzymatic activities of both MHV68 vUNG and vDUT leads to loss of YFP reporter gene in cell culture. (A) Schematic of serial passage experiment in MEFs. For passage 1, UNG^−/− or WT MEFs were infected at an MOI of 0.01 with the recombinant MHV68 lacking the enzymatic activity of both vUNG and vDUT (46.CM/54.CM) or the MR virus. Passage 1 viruses were harvested when 50% CPE was observed, and titers were quantified by plaque assay. Passage 1 viruses were then normalized to an MOI of 0.01 for the second passage of infection in the same cell type. (B and C) The percentage of YFP-positive plaques was quantified by fluorescence microscopy at 7 to 8 dpi. Samples were harvested following one or two passages in WT MEFs (B) or UNG^−/− MEFs (C). **, P < 0.01, and ***, P < 0.001, by two-way unpaired t test.

FIG 7

Loss of enzymatic activities of both MHV68 vUNG and vDUT leads to loss of YFP reporter gene in vivo. (A) Schematic of serial passage experiment in mouse lungs. For passage 1, UNG^−/− mice were infected by the intranasal route with 1,000 PFU with the recombinant MHV68 lacking the enzymatic activity of both vUNG and vDUT (46.CM/54.CM) or the MR virus. Titers of viruses recovered from passage 1 UNG^−/− animals 7 dpi were quantified by plaque assay. Passage 1 viruses were then normalized to 100 PFU for the second passage of infection in the UNG^−/− mice. WT mice were infected by the intranasal route with 1,000 PFU with the recombinant MHV68 lacking the enzymatic activity of vUNG (ORF46.CM), vDUT(ORF54.CM), or both vUNG and vDUT (46.CM/54.CM) and their MR viruses. Lung homogenates were prepared 9 dpi for plaque assay. (B) Passage 2 viruses in lung homogenates from UNG^−/− mice were determined by plaque assay at 9 dpi. The line indicates the geometric mean titer. Each symbol represents an individual mouse. The dashed line depicts the limit of detection at 50 PFU/ml of lung homogenate. (C) The percentage of YFP-positive plaques was quantified by fluorescence microscopy at 7 to 8 dpi for lung homogenate from infected UNG^−/− mice. Each symbol represents one mouse. (D) The increase in the percentage of YFP loss from passage 1 to passage 2. (E) WT C57BL/6 mice were infected by the intranasal route with 1,000 PFU of the indicated viruses. The percentage of YFP-positive plaques was quantified by fluorescence microscopy at 7 to 8 dpi. Each symbol represents one mouse. For panels B to D, significance was determined by two-way unpaired t test, and for panel E, significance was determined by one-way ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n.s., not significant.

FIG 8

Loss of enzymatic activities of both MHV68 vUNG and vDUT results in higher YFP loss due to recombination. (A) Schematic of H2BYFP reporter gene position between two repeated XL9 elements in the MHV68 genome. The locations of primers for PCR and restriction enzyme sites and probe for Southern blotting are indicated; the expected sizes of PCR amplimers and restriction fragments are shown to the right. (B) PCR products from isolated YFP-positive and YFP-negative plaques are consistent with the presence or loss of the YFP reporter gene. ORF9 amplification serves as the template input control. Multiple YFP-positive and YFP-negative plaques (n > 20 for each) were screened with the representative gel shown here. (C) The Southern blot probe for XL9 demonstrates a shift in EcoRV/ScaI restriction fragment size in DNA isolated from YFP-positive and YFP-negative plaques, consistent with recombination between XL9 repeats and loss of the H2BYFP reporter gene.