{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T19:30:41Z","timestamp":1768505441674,"version":"3.49.0"},"posted":{"date-parts":[[2023,5,19]]},"group-title":"Microbiology","reference-count":47,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,5,19]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  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\n                  <jats:italic>ORF46<\/jats:italic>\n                  (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency\n                  <jats:italic>in vivo.<\/jats:italic>\n                  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\n                  <jats:italic>ORF9<\/jats:italic>\n                  , and the viral DNA polymerase processivity factor, vPPF encoded by\n                  <jats:italic>ORF59<\/jats:italic>\n                  . 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.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    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\n                    <jats:italic>in vivo<\/jats:italic>\n                    . In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus to form 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.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2023.05.19.541466","type":"posted-content","created":{"date-parts":[[2023,5,22]],"date-time":"2023-05-22T13:55:11Z","timestamp":1684763711000},"source":"Crossref","is-referenced-by-count":0,"title":["Uracil-DNA Glycosylase of Murine Gammaherpesvirus 68 Binds Cognate Viral Replication Factors Independently of its Catalytic Residues"],"prefix":"10.64898","author":[{"given":"Kyle R.","family":"Smith","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Somnath","family":"Paul","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiwen","family":"Dong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Orchi","family":"Anannya","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Darby G.","family":"Oldenburg","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J. Craig","family":"Forrest","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9646-152X","authenticated-orcid":false,"given":"Kevin M.","family":"McBride","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9648-522X","authenticated-orcid":false,"given":"Laurie T.","family":"Krug","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023052302400483000_2023.05.19.541466v1.1","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a013011"},{"key":"2023052302400483000_2023.05.19.541466v1.2","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1146\/annurev-virology-011921-082615","article-title":"Conquering the Host: Determinants of Pathogenesis Learned from Murine Gammaherpesvirus 68","volume":"8","year":"2021","journal-title":"Annual Review of Virology"},{"key":"2023052302400483000_2023.05.19.541466v1.3","doi-asserted-by":"crossref","first-page":"4514","DOI":"10.1128\/jvi.68.7.4514-4524.1994","article-title":"Mutations in accessory DNA replicating functions alter the relative mutation frequency of herpes simplex virus type 1 strains in cultured murine cells","volume":"68","year":"1994","journal-title":"J Virol"},{"key":"2023052302400483000_2023.05.19.541466v1.4","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.70.5.3018-3025.1996"},{"key":"2023052302400483000_2023.05.19.541466v1.5","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.75.16.7592-7601.2001"},{"key":"2023052302400483000_2023.05.19.541466v1.6","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a012583"},{"key":"2023052302400483000_2023.05.19.541466v1.7","doi-asserted-by":"publisher","DOI":"10.1021\/ar800114w"},{"key":"2023052302400483000_2023.05.19.541466v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.dnarep.2006.10.014"},{"key":"2023052302400483000_2023.05.19.541466v1.9","doi-asserted-by":"crossref","unstructured":"Cheng AZ , Moraes SN , Shaban NM , Fanunza E , Bierle CJ , Southern PJ , Bresnahan WA , Rice SA , Harris RS . 2021. APOBECs and Herpesviruses. Viruses 13.","DOI":"10.3390\/v13030390"},{"key":"2023052302400483000_2023.05.19.541466v1.10","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00290-11"},{"key":"2023052302400483000_2023.05.19.541466v1.11","doi-asserted-by":"crossref","first-page":"2015","DOI":"10.3390\/v13102015","article-title":"HSV-1 DNA Replication\u2014Coordinated Regulation by Viral and Cellular Factors","volume":"13","year":"2021","journal-title":"Viruses"},{"key":"2023052302400483000_2023.05.19.541466v1.12","doi-asserted-by":"crossref","first-page":"e1011252","DOI":"10.1371\/journal.ppat.1011252","article-title":"Human DNA tumor viruses evade uracil-mediated antiviral immunity","volume":"19","year":"2023","journal-title":"PLoS pathogens"},{"key":"2023052302400483000_2023.05.19.541466v1.13","first-page":"78","article-title":"Epstein-Barr virus BORF2 inhibits cellular APOBEC3B to preserve viral genome integrity","volume":"4","year":"2019","journal-title":"Nat Microbiol"},{"key":"2023052302400483000_2023.05.19.541466v1.14","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M207107200"},{"key":"2023052302400483000_2023.05.19.541466v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrfmmm.2009.10.017"},{"key":"2023052302400483000_2023.05.19.541466v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/sj.onc.1205996"},{"key":"2023052302400483000_2023.05.19.541466v1.17","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109.010413"},{"key":"2023052302400483000_2023.05.19.541466v1.18","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.03111-14"},{"key":"2023052302400483000_2023.05.19.541466v1.19","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1998.9428"},{"key":"2023052302400483000_2023.05.19.541466v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2009.01.013"},{"key":"2023052302400483000_2023.05.19.541466v1.21","doi-asserted-by":"publisher","DOI":"10.1099\/0022-1317-70-2-449"},{"key":"2023052302400483000_2023.05.19.541466v1.22","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.68.8.4963-4972.1994"},{"key":"2023052302400483000_2023.05.19.541466v1.23","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-2-55"},{"key":"2023052302400483000_2023.05.19.541466v1.24","doi-asserted-by":"crossref","unstructured":"Prichard MN , Duke GM , Mocarski ES . 1996. Human Cytomegalovirus Uracil DNA Glycosylase Is Required for the Normal Temporal Regulation of both DNA Synthesis and Viral Replication. Journal of Virology J Virol:3018\u201325.","DOI":"10.1128\/jvi.70.5.3018-3025.1996"},{"key":"2023052302400483000_2023.05.19.541466v1.25","doi-asserted-by":"crossref","unstructured":"Dong Q , Smith KR , Oldenburg DG , Shapiro M , Schutt WR , Malik L , Plummer JB , Mu Y , MacCarthy T , White DW , McBride KM , Krug LT . 2018. 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. mBio 9.","DOI":"10.1128\/mBio.01831-18"},{"key":"2023052302400483000_2023.05.19.541466v1.26","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00950-14"},{"key":"2023052302400483000_2023.05.19.541466v1.27","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.131235"},{"key":"2023052302400483000_2023.05.19.541466v1.28","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.022160-0"},{"key":"2023052302400483000_2023.05.19.541466v1.29","doi-asserted-by":"publisher","DOI":"10.1101\/2023.05.17.541089:2023.05.17.541089"},{"key":"2023052302400483000_2023.05.19.541466v1.30","doi-asserted-by":"crossref","first-page":"110469","DOI":"10.1016\/j.celrep.2022.110469","article-title":"Human cytomegalovirus forms phase-separated compartments at viral genomes to facilitate viral replication","volume":"38","year":"2022","journal-title":"Cell Reports"},{"key":"2023052302400483000_2023.05.19.541466v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.05.028"},{"key":"2023052302400483000_2023.05.19.541466v1.32","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004939"},{"key":"2023052302400483000_2023.05.19.541466v1.33","doi-asserted-by":"crossref","first-page":"e2203203119","DOI":"10.1073\/pnas.2203203119","article-title":"Host translesion polymerases are required for viral genome integrity","volume":"119","year":"2022","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2023052302400483000_2023.05.19.541466v1.34","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1006166"},{"key":"2023052302400483000_2023.05.19.541466v1.35","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.virol.2021.11.004","article-title":"Identification of cellular proteins associated with human cytomegalovirus (HCMV) DNA replication suggests novel cellular and viral interactions","volume":"566","year":"2022","journal-title":"Virology"},{"key":"2023052302400483000_2023.05.19.541466v1.36","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01334-06"},{"key":"2023052302400483000_2023.05.19.541466v1.37","doi-asserted-by":"crossref","first-page":"e1010236","DOI":"10.1371\/journal.ppat.1010236","article-title":"A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains","volume":"18","year":"2022","journal-title":"PLoS Pathog"},{"key":"2023052302400483000_2023.05.19.541466v1.38","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003748"},{"key":"2023052302400483000_2023.05.19.541466v1.39","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01152-08"},{"key":"2023052302400483000_2023.05.19.541466v1.40","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0033230"},{"key":"2023052302400483000_2023.05.19.541466v1.41","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60761-652-8_30"},{"key":"2023052302400483000_2023.05.19.541466v1.42","doi-asserted-by":"publisher","DOI":"10.2144\/000112096"},{"key":"2023052302400483000_2023.05.19.541466v1.43","first-page":"e00027","article-title":"IKK\u03b1-Mediated Noncanonical NF-\u03baB Signaling Is Required To Support Murine Gammaherpesvirus 68 Latency In Vivo","volume":"96","year":"2022","journal-title":"Journal of Virology"},{"key":"2023052302400483000_2023.05.19.541466v1.44","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac247"},{"key":"2023052302400483000_2023.05.19.541466v1.45","doi-asserted-by":"publisher","DOI":"10.1002\/rcm.1198"},{"key":"2023052302400483000_2023.05.19.541466v1.46","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.200900437"},{"key":"2023052302400483000_2023.05.19.541466v1.47","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2005.07.014"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2023.05.19.541466","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T04:40:37Z","timestamp":1768452037000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2023.05.19.541466"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,19]]},"references-count":47,"URL":"https:\/\/doi.org\/10.1101\/2023.05.19.541466","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1128\/msphere.00278-23","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2023,5,19]]},"subtype":"preprint"}}