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. 2023 Oct 24;8(5):e0027823.
doi: 10.1128/msphere.00278-23. Epub 2023 Sep 25.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues

Kyle R Smith  1   2 , Somnath Paul  3 , Qiwen Dong  4 , Orchi Anannya  5 , Darby G Oldenburg  6 , J Craig Forrest  7 , Kevin M McBride  3 , Laurie T Krug  1   2
Affiliations

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues

Kyle R Smith et al. mSphere. .

Abstract

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Keywords: gammaherpesvirus; lytic replication; uracil-DNA glycosylase; viral DNA polymerase; viral DNA polymerase processivity factor; viral replication compartment; virus-host interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
The MHV68 vUNG interacts with the vPOL and vPPF upon de novo lytic infection. NIH 3T12 cells were infected with MHV68 H2b-YFP at an MOI of 3.0. (A) Cells were fixed and stained for flow cytometry at the indicated hpi. Plots indicate the gating strategy used to identify YFP+ infected cells on the y-axis and cells expressing vUNG detected by the vUNG-C1 mAb conjugated to AF647+ on the x-axis. (B) Line graph of percentage of cells with YFP or vUNG from flow data collected in (A). (C) Cell viability of mock and infected cells was measured by exclusion of propidium iodide by flow cytometry. Symbols represent three biological replicates ±SEM. Statistical significance was evaluated by a two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01. (D) Immunoprecipitation of the vUNG using the vUNG-C1 mAb from infected NIH 3T12 cells at 36 hpi. (E) Enriched peptides identified by mass spectrometry with a Log2 fold change following IP of vUNG compared to control IgG. The orange spheres denote MHV68-specific peptides detected, while the black spheres denote host/murine peptides.
FIG 2
FIG 2
Characterization and immunofluorescence imaging of recombinant MHV68 expressing GFP-tagged vPOL and vPPF. (A) Single-step growth curve to compare replication of wild-type MHV68 (WT) or recombinant MHV68 expressing GFP fusions to vPOL (viral DNA polymerase encoded by ORF9) or vPPF (viral DNA polymerase processivity factor encoded by ORF59) in NIH 3T12 fibroblasts, MOI 3.0. Symbols represent three biological replicates ±SD. Statistical significance was evaluated by two-way ANOVA with Tukey’s multiple comparisons test, and differences were noted between ORF9-GFP and WT at 12 and 36 hpi and between ORF9-GFP and ORF59-GFP at 12 hpi. *, P < 0.05; **, P < 0.01 (B) Timecourse of viral protein expression by immunoblot under conditions described in A. (C) Immunofluorescence timecourse of viral protein co-localization in NIH 3T3 cells infected with the indicated viruses, MOI 3.0. vUNG was detected with vUNG pAb, followed by secondary AF568; vPOL and vPPF fusion proteins were detected via GFP expression without antibodies; DNA was stained with DAPI.
FIG 3
FIG 3
vUNG co-localizes with the viral DNA polymerase and viral DNA polymerase processivity factor in infected fibroblasts. (A and B) NIH 3T3 cells were infected with recombinant MHV68 virus expressing vPOL-GFP (A) or vPPF-GFP (B) at an MOI of 3.0 and treated with 300 μg/mL PAA at 1 hpi where indicated. Cells were fixed and stained for immunofluorescence imaging at the indicated hpi. vUNG was detected with vUNG-C1 mAb conjugated to AF647; vPOL and vPPF fusion proteins were detected via GFP expression without antibodies; DNA was stained with DAPI. Nikon NIS-Elements line analysis of the indicated cells at 17 hpi is located below each panel (A and B).
FIG 4
FIG 4
vUNG interacts in complex with vPOL and vPPF. At 48 h post-transfection with the indicated expression constructs, protein lysates were DNAse treated and sonicated prior to IP and immunoblot analysis. (A) IP with anti-vUNG C1 mAb or anti-V5 mAb (B) in lysates from co-transfections with FLAG-vPOL, vPPF-V5, and vUNG-MYC. (C and D) IP with anti-FLAG mAb (C) or anti-V5 mAb (D) in lysates from co-transfections with FLAG-vPOL and vPPF-V5. (E) IP with anti-FLAG mAb in lysates from co-transfections with vUNG-MYC and FLAG-vPOL. (F) IP with anti-V5 mAb in lysates from co-transfections with vUNG-FLAG and vPPF-V5.
FIG 5
FIG 5
vUNG complexes with vPOL and vPPF independent of vUNG enzymatic activity. (A and B) NIH 3T3 cells were infected with recombinant MHV68 ORF46CM.MR virus expressing a wild-type vUNG (A) or with MHV68 ORF46.CM expressing the vUNG CM (B) at an MOI of 3.0. vUNG was detected with the vUNG-AF647 mAb; vPPF was detected with the vPPF pAb, followed by secondary AF555; DNA was stained with DAPI. Nikon NIS-Elements line analysis of the indicated cells at 17 hpi is located below each panel (A and B). (C–F) HEK-293T cells were transfected with the indicated expression constructs. At 48 h post-transfection, protein lysates were DNAse treated and sonicated prior to immunoprecipitation and immunoblot analysis. IP with anti-vUNG C1 mAb (C) or anti-V5 mAb (D) in lysates from co-transfections with FLAG-vPOL, vPPF-V5, and vUNG.CM-MYC. (E) IP with anti-vUNG mAb in lysates from co-transfections with vUNG.CM-MYC and FLAG-vPOL. (F) IP with anti-V5 mAb in lysates from co-transfections with vUNG.CM-FLAG and vPPF-V5. (G) NIH 3T12 cells were infected with the indicated viruses for 36 h, and protein lysates were subjected to SDS-PAGE. Tubulin was used as a loading control. (H) Co-IP of NIH 3T12-infected cell lysates at 36 hpi with anti-vUNG-C1 or IgG control. vUNG was detected with the vUNG pAb, and vPPF was detected with the anti-vPPF pAb. The data represent three biological replicates.
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