Proteasome Subunits Relocalize during Human Cytomegalovirus Infection, and Proteasome Activity Is Necessary for Efficient Viral Gene Transcription

ABSTRACT We have continued studies to further understand the role of the ubiquitin-proteasome system (UPS) in human cytomegalovirus (HCMV) infection. With specific inhibitors of the proteasome, we show that ongoing proteasome activity is necessary for facilitating the various stages of the infection. Immediate-early protein 2 expression is modestly reduced with addition of proteasome inhibitors at the onset of infection; however, both early and late gene expression are significantly delayed, even if the inhibitor is removed at 12 h postinfection. Adding the inhibitor at later times during the infection blocks the further accumulation of viral early and late gene products, the severity of which is dependent on when the proteasome is inhibited. This can be attributed primarily to a block in viral RNA transcription, although DNA synthesis is also partially inhibited. Proteasome activity and expression increase as the infection progresses, and this coincides with the relocalization of active proteasomes to the periphery of the viral DNA replication center, where there is active RNA transcription. Interestingly, one 19S subunit, Rpn2, is specifically recruited into the viral DNA replication center. The relocalization of the subunits requires viral DNA replication, but their maintenance around or within the replication center is not dependent on continued viral DNA synthesis or the proteolytic activity of the proteasome. These studies highlight the importance of the UPS at all stages of the HCMV infection and support further studies into this pathway as a potential antiviral target.

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Human Cytomegalovirus Can Procure Deoxyribonucleotides for Viral DNA Replication in the Absence of Retinoblastoma Protein Phosphorylation

Journal of Virology ◽

10.1128/jvi.00731-16 ◽

2016 ◽

Vol 90 (19) ◽

pp. 8634-8643 ◽

Cited By ~ 2

Author(s):

Chad V. Kuny ◽

Robert F. Kalejta

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Human Cytomegalovirus ◽

De Novo ◽

Dna Virus ◽

Viral Dna ◽

Viral Oncoproteins ◽

Viral Dna Replication ◽

Rb Phosphorylation

ABSTRACTViral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or throughde novosynthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing thede novoproduction of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that bothde novoand salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner.IMPORTANCETransforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally mimics these viral oncoproteins through phosphorylation of Rb, fails to induce the production of nonlimiting amounts of dNTPs. This finding challenges the paradigm of the role of Rb inactivation during DNA virus infection and uncovers the existence of an alternative mechanism by which UL97 contributes to HCMV DNA synthesis. The ineffectiveness of the UL97 inhibitor maribavir in clinical trials might be better explained with a fuller understanding of the role of UL97 during infection. Furthermore, as the nucleoside analog ganciclovir is the current drug of choice for treating HCMV, knowing the provenance of the dNTPs incorporated into viral DNA may help inform antiviral therapeutic regimens.

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ORF18 Is a Transfactor That Is Essential for Late Gene Transcription of a Gammaherpesvirus

Journal of Virology ◽

10.1128/jvi.00246-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9730-9740 ◽

Cited By ~ 44

Author(s):

Vaithilingaraja Arumugaswami ◽

Ting-Ting Wu ◽

DeeAnn Martinez-Guzman ◽

Qingmei Jia ◽

Hongyu Deng ◽

...

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Dna Synthesis ◽

Lytic Replication ◽

Early Gene ◽

Late Gene ◽

Viral Dna ◽

Viral Dna Replication ◽

Murine Gammaherpesvirus ◽

Late Genes

ABSTRACT Lytic replication of the tumor-associated human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus has important implications in pathogenesis and tumorigenesis. Herpesvirus lytic genes have been temporally classified as exhibiting immediate-early (IE), early, and late expression kinetics. Though the regulation of IE and early gene expression has been studied extensively, very little is known regarding the regulation of late gene expression. Late genes, which primarily encode virion structural proteins, require viral DNA replication for their expression. We have identified a murine gammaherpesvirus 68 (MHV-68) early lytic gene, ORF18, essential for viral replication. ORF18 is conserved in both beta- and gammaherpesviruses. By generating an MHV-68 ORF18-null virus, we characterized the stage of the virus lytic cascade that requires the function of ORF18. Gene expression profiling and quantitation of viral DNA synthesis of the ORF18-null virus revealed that the expression of early genes and viral DNA replication were not affected; however, the transcription of late genes was abolished. Hence, we have identified a gammaherpesvirus-encoded factor essential for the expression of late genes independently of viral DNA synthesis.

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Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

Journal of Virology ◽

10.1128/jvi.00581-12 ◽

2012 ◽

Vol 86 (18) ◽

pp. 9817-9827 ◽

Cited By ~ 19

Author(s):

Alexandra Nitzsche ◽

Charlotte Steinhäußer ◽

Katrin Mücke ◽

Christina Paulus ◽

Michael Nevels

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Histone Modification ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Viral Genome ◽

Histone H3 ◽

Viral Dna ◽

The Impact ◽

Preferential Incorporation

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using “click chemistry” revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.

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Human cytomegalovirus UL141 protein interacts with CELF5 and affects viral DNA replication

Molecular Medicine Reports ◽

10.3892/mmr.2018.8419 ◽

2018 ◽

Cited By ~ 2

Author(s):

Fei Zou ◽

Zhi‑Tao Lu ◽

Shuang Wang ◽

Si Wu ◽

Ying‑Ying Wu ◽

...

Keyword(s):

Dna Replication ◽

Human Cytomegalovirus ◽

Viral Dna ◽

Viral Dna Replication

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Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

Microbiology and Immunology ◽

10.1111/j.1348-0421.1994.tb01785.x ◽

1994 ◽

Vol 38 (4) ◽

pp. 321-325 ◽

Cited By ~ 24

Author(s):

Kappei Kobayashi ◽

Eriko Ohgitani ◽

Yasuyuki Tanaka ◽

Masakazu Kita ◽

Jiro Imanishi

Keyword(s):

Protein Synthesis ◽

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Heat Shock ◽

Heat Shock Protein ◽

Viral Dna ◽

Viral Dna Replication ◽

Early Protein ◽

Simplex Virus

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Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

Journal of Virology ◽

10.1128/jvi.00115-10 ◽

2010 ◽

Vol 84 (12) ◽

pp. 6153-6162 ◽

Cited By ~ 13

Author(s):

Mei Yu ◽

Eric B. Carstens

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Viral Genome ◽

Virus Production ◽

Autographa Californica ◽

Late Gene ◽

Late Gene Expression ◽

Viral Dna ◽

Viral Dna Replication ◽

Budded Virus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.

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Analysis of an Autographa californica Multicapsid Nucleopolyhedrovirus lef-6-Null Virus: LEF-6 Is Not Essential for Viral Replication but Appears To Accelerate Late Gene Transcription

Journal of Virology ◽

10.1128/jvi.76.11.5503-5514.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5503-5514 ◽

Cited By ~ 37

Author(s):

Guangyun Lin ◽

Gary W. Blissard

Keyword(s):

Dna Replication ◽

Viral Replication ◽

Gene Transcription ◽

Cellular Localization ◽

Autographa Californica ◽

Late Gene ◽

Infection Cycle ◽

Viral Dna ◽

Viral Dna Replication ◽

Late Transcription

ABSTRACT The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli. Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAclef6KO) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two “repair” AcMNPV BACmids (vAclef6KO-REP-P and vAclef6KO-REP-ie1P) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAclef6KO BACmid. Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus. The lef-6-null BACmid (vAclef6KO) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription. Therefore, lef-6 appears to accelerate the infection cycle of AcMNPV.

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