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 activi...

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Autores principales:	Qiwen Dong, Kyle R. Smith, Darby G. Oldenburg, Maxwell Shapiro, William R. Schutt, Laraib Malik, Joshua B. Plummer, Yunxiang Mu, Thomas MacCarthy, Douglas W. White, Kevin M. McBride, Laurie T. Krug
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	DNA replication dUTPase gammaherpesvirus genomic stability herpesviruses latency Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/e0bb07380d1b4f38bdcc807e729ffc60
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spelling	oai:doaj.org-article:e0bb07380d1b4f38bdcc807e729ffc602021-11-15T15:58:20ZCombinatorial 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 Genome10.1128/mBio.01831-182150-7511https://doaj.org/article/e0bb07380d1b4f38bdcc807e729ffc602018-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01831-18https://doaj.org/toc/2150-7511ABSTRACT 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.Qiwen DongKyle R. SmithDarby G. OldenburgMaxwell ShapiroWilliam R. SchuttLaraib MalikJoshua B. PlummerYunxiang MuThomas MacCarthyDouglas W. WhiteKevin M. McBrideLaurie T. KrugAmerican Society for MicrobiologyarticleDNA replicationdUTPasegammaherpesvirusgenomic stabilityherpesviruseslatencyMicrobiologyQR1-502ENmBio, Vol 9, Iss 5 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA replication dUTPase gammaherpesvirus genomic stability herpesviruses latency Microbiology QR1-502
spellingShingle	DNA replication dUTPase gammaherpesvirus genomic stability herpesviruses latency Microbiology QR1-502 Qiwen Dong Kyle R. Smith Darby G. Oldenburg Maxwell Shapiro William R. Schutt Laraib Malik Joshua B. Plummer Yunxiang Mu Thomas MacCarthy Douglas W. White Kevin M. McBride Laurie T. Krug 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
description	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.
format	article
author	Qiwen Dong Kyle R. Smith Darby G. Oldenburg Maxwell Shapiro William R. Schutt Laraib Malik Joshua B. Plummer Yunxiang Mu Thomas MacCarthy Douglas W. White Kevin M. McBride Laurie T. Krug
author_facet	Qiwen Dong Kyle R. Smith Darby G. Oldenburg Maxwell Shapiro William R. Schutt Laraib Malik Joshua B. Plummer Yunxiang Mu Thomas MacCarthy Douglas W. White Kevin M. McBride Laurie T. Krug
author_sort	Qiwen Dong
title	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
title_short	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
title_full	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
title_fullStr	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
title_full_unstemmed	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
title_sort	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
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/e0bb07380d1b4f38bdcc807e729ffc60
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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

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