Uracil-DNA Glycosylase of Murine Gammaherpesvirus 68 Binds Cognate Viral Replication Factors Independently of its Catalytic Residues

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 comprised of the cognate viral DNA polymerase, vPOL encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF encoded by ORF59. MHV68 vUNG colocalized 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. Last, 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.


INTRODUCTION 63
Herpesviruses are large, double-stranded DNA viruses that encode proteins necessary FLAG-vPOL with vUNG-C1 mAb led to pull-down of FLAG-vPOL with vUNG.CM (Fig.  220   5E). An interaction between vUNG.CM and vPPF-V5 was observed following IP of 221 vUNG.CM with anti-V5 (Fig. 5F). Taken together, the vUNG, vPOL and vPPF interact in 222 pairs and in a complex independent of the enzymatic activity of vUNG, active viral DNA 223 replication, and other viral proteins. 224 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  In the current study, we identified non-enzymatic functions of the vUNG encoded by the 242 model gammaherpesvirus pathogen MHV68. We report that vUNG readily complexes 243 with vPOL and vPPF in the context of infection, independently of key catalytic residues. 244 This indicates that the vUNG may provide scaffolding functions for the viral DNA 245 replication machinery. Our data offers one potential explanation for the failure of the 246 and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 polymerase and host factors in vitro (17). These data are consistent with a model 290 whereby vUNG excision of uracil residues triggers BER to prevent recruitment of error-291 prone translesion DNA polymerases that would otherwise place the genomes at risk for 292 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023. In the case of HCMV infection, the Y-family of translesion DNA 293 polymerases are largely deleterious to viral DNA synthesis while the polymerase ζ 294 complex stabilizes viral genome integrity (33). Accelerated native isolation of proteins on 295 nascent DNA (aniPOND) is a powerful tool that has been used to identify viral and 296 cellular proteins on nascent herpesvirus replication forks (32,34,35). Anipond analysis 297 of HSV-1 infected cells identified host factors involved in BER, mismatch repair, and 298 double-strand DNA break sensing along with viral replication factors on nascent viral 299 DNA (32). KSHV LANA was observed to bind human UNG2 suggesting a requirement 300 for uracil excision at origins of viral DNA replication (36). We previously reported that an 301 was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. studies that dissect residues of MHV68 vUNG required for biochemical functions from 336 protein interactions will enable the role of complex formation with vPOL and vPPF to be 337 tested in the MHV68 pathogenesis system. 338 and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ; https://doi.org/10.1101/2023.05.19.541466 doi: bioRxiv preprint

MATERIALS AND METHODS 339
Cell culture. Immortalized murine fibroblast cells, NIH 3T3 and NIH 3T12, and HEK-340

293T cells were obtained from ATCC (Manassa, Virginia, USA) and maintained in 341
Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% (NIH 3T3 and NIH 342 3T12) or 10% (HEK-293T) fetal bovine serum, 1% L-glutamine, 100 U/ml penicillin, and 343 100 mg/ml streptomycin at 37°C in 5% CO 2 . were designed as previously described (25) with an aspartic acid in place of the wild-360 type asparagine (D85N) in the water activating loop, and a histidine (H207L) in place of 361 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 designed with 20 bp homology to the flanking region of the desired insertion site (in this 379 case, the GFP ORF in the BAC). The flanking regions up-and downstream of the GFP 380 ORF were PCR-amplified with GFP_FLANK 1 and GFP_FLANK 2 forward and reverse 381 primers. The PCR products were gel-purified (Qiagen) and assembled using Gibson 382 Assembly (New England Biolabs). To generate the final targeting construct, a final PCR 383 was performed using GFP_FLANK1_FOR and GFP_FLANK2_REV. This PCR product 384 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105  Virus passage and titer determination were performed as previously described (43). 406 RFLP with BamHI and HindIII, coupled with whole genome BAC sequencing (Illumina,407 and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105  3T12 cells with YFP+ signal (Fig S1A). vUNG was detected by staining the cells with 427 anti-vUNG-C1 and secondary anti-mouse AF647 (Jackson-Immuno, USA) prior to 428 analysis. A minimum of two biological replicates were performed for each experimental 429 analysis. 430 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ; https://doi.org/10.1101/2023.05.19.541466 doi: bioRxiv preprint

Co-Immunoprecipitation and mass spectrometry following infection. NIH 3T12 431
cells were infected at an MOI of 3.0 and harvested at 36 hpi for co-IP/MS analysis. Cell 432 pellets were washed with cold phosphate-buffered saline (PBS), followed by incubation 433 with non-denaturing lysis buffer (20mM Tris HCl pH 8, 137mM NaCl, 10% glycerol, 1% 434 NP-40, and 2mM EDTA) containing 50 μg/mL protease inhibitors (phenylmethylsulfonyl 435 chloride), and 1 μg/mL aprotinin for 1 h at 4° C with constant agitation. The lysates were 436 clarified by centrifugation, and the protein concentration of the supernatant was 437 calculated as follows. First, for each peptide spectral match (PSMs), the false discovery 452 rate (FDR) was set at 0.1% followed by a minimum cutoff of 10 unique peptides for any 453 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ; https://doi.org/10.1101/2023.05.19.541466 doi: bioRxiv preprint protein were considered for downstream analysis (Table S2). Further, with Scaffold 5.0, 454 protein identifications were accepted if they could be established at greater than 99.0% 455 probability of being protein and not a decoy. Second, non-specific binding of host 456 protein from NIH 3T12 cells was addressed by discarding antigen IDs that were not 457 enriched at least 3-fold in the infected sample when compared with the mock IP (Table  458 S3). Finally, the Log 2 fold change (FC) was calculated by comparing αvUNG and IgG 459 isotype control (Table S4). prior to SDS PAGE in 4-12% gradient gels (Invitrogen) with MOPs buffer (Invitrogen). 471 Following electrophoresis, the gels were transferred to nitrocellulose membranes 472 (Invitrogen) with an iBlot 2 dry blotting system (Thermo Scientific). Primary antibodies 473 include vPPF, which was detected using an affinity-purified rabbit polyclonal anti-ORF59 474 antibody that was generated (GenScript, Piscataway, NJ, USA) against the peptides 475 GKKTRGGNKASDSGT and KRPPPKKDREPTTKRPKL, corresponding to amino acids 476 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 210-224 and 372-388, respectively, of the predicted vPPF protein of MHV68 477 (Genbank:AAF19323) (47). vUNG was detected using anti-vUNG polyclonal sera 478 (vUNG pAb) (25). GFP was detected with mouse anti-GFP mAb (Invitrogen, Clone 479 GF28R, CA# MA5-15256), mouse sera harvested from MHV68-infected C57BL/6 mice 480 28 dpi was used to detect viral antigen. β-actin was detected with rabbit mAb (Cell was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 protein A (lane 3) from input fraction (lane 1). As such, during experimental detection of 500 immunoprecipitated vUNG using mouse sera harvested from MHV68-infected C57BL/6 501 mice, the bands detected around 25-27KDa is vUNG and not the light chain. 502 503 For transfected lysates, protein was quantified via BCA assay (Thermo Fisher) and 504 input and co-IP samples were subjected to SDS PAGE in 4-12% gradient gels 505 (Invitrogen) with MOPs buffer (Invitrogen). Following electrophoresis, the gels were 506 transferred to nitrocellulose membranes (Invitrogen) with an iBlot 2 dry blotting system 507 (Thermo Scientific). Primary antibodies used were vUNG pAb (25) infection at an MOI of 3. Cells were fixed in 4% paraformaldehyde in PBS for 10 min 520 and permeabilized in 0.2% Triton X-100 in PBS for 5 min. Cells were blocked in 10% 521 normal goat serum (Thermo Scientific, Waltham, MA) in PBS for 1 h, followed by 522 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 incubation with primary antibodies diluted in blocking buffer for 1 h at room temperature 523 with rocking, and then two PBS washes. Primary antibodies include anti-vPPF pAb, 524 vUNG pAb, and anti-vUNG conjugated to Alexa-Fluor 647 (29). vPPF or vUNG was 525 detected by incubation with the respective secondary antibodies, goat anti-mouse IgG 526 conjugated to Alexa Fluor 568 (Invitrogen, CA# A11061) or goat anti-rabbit IgG Alexa 527 Flour 555 (Invitrogen, CA# A27039) diluted in 10% blocking buffer for 1 h at room temp 528 with rocking. Cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Thermo 529 Fisher) nuclear stain diluted in PBS for 5 min with rocking. Coverslips were mounted 530 using Prolong Gold antifade reagent (Invitrogen). Confocal microscopy was conducted 531 using a spinning disk confocal with a Nikon T2i Eclipse Microscope (Nikon, Minato City, 532 Tokyo, Japan). Z-stack images consisting of ten 2.5 μm optical sections were generated 533 using Nikon NIS-Elements software and processed using ImageJ software (NIH,534 Bethesda, MD, USA). Line graphs of distance and intensity were generated using Nikon 535 NIS-Elements software. pulses on ice using a Branson Sonifier 450 (duty cycle 10%, output control 2) and 545 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 cleared by 14,000 x g centrifugation for 5 min at 4°C. Lysates were treated with 0.5 U 546 DNase I per 100 uL lysate (Invitrogen, Waltham, MA, USA) for 15 min. For the analysis 547 of vPPF interaction with vUNG, protein was lysed in M-PER buffer supplemented with 548 EDTA-free protease inhibitor and 0.1 M NaCl and 0.1% Triton X-100. Lysates were 549 treated with 10 U DNaseI per 100 uL lysate (Invitrogen) for 60 minutes at 37°C prior to 550 IP steps. 551 552 For IP, lysates were incubated with primary antibodies (1:100) for 1 h with rotation at 553 4°C, and then rotated overnight at 4°C with 1.5 mg protein G (Dynabead, Invitrogen). 554 Complexes were precipitated by centrifugation and washed three times in PBS 555 supplemented 0.02% Tween 20 and resuspended in 100 uL PBS prior to elution with 40 556 uL of 2 parts 0.1M glycine pH 2.8 and 1 part NuPage sample buffer and reducing agent 557 (Invitrogen). Samples were heated at 70°C for 10 min prior to magnetic removal of 558 Dynabeads and storage at -20°C. 559 560 Statistics. All data were analyzed using Prism 9 software (GraphPad, La Jolla, CA). 561 Statistical significance was determined using two-way Anova with Tukey's multiple 562 comparisons test. 563 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023

ACKNOWLEDGEMENTS 564
We thank Dr. Michael Kruhlak of the CCR Confocal Microscopy Core facility and Dr. Jan 565

Wizniewski of the NCI Experimental Immunology Branch Microscopy and Digital 566
Imaging Facility for their assistance with confocal microscopy and image analysis. and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105  two-way Anova with Tukey's multiple comparisons test and differences were noted 601 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 between ORF9-GFP and WT at 12 and 36 hpi, and between ORF9-GFP and ORF59-602 was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 vUNG-FLAG and vPPF-V5. replicates. 645 646 and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted May 19, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023