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

2016 ◽  
Vol 90 (19) ◽  
pp. 8634-8643 ◽  
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.

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

2009 ◽  
Vol 84 (6) ◽  
pp. 3079-3093 ◽  
Author(s):  
Karen Tran ◽  
Jeffrey A. Mahr ◽  
Deborah H. Spector
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Proteasome Activity ◽  
Late Gene ◽  
Viral Dna ◽  
Rna Transcription ◽  
Viral Dna Replication ◽  
Early Protein

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.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Dongcheng Liu ◽  
Yan Wang ◽  
Yan Yuan
Keyword(s):  
Dna Replication ◽  
Noncoding Rna ◽  
Rna Binding ◽  
De Novo ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Noncoding Rnas ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation andde novoinfection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UVcross-linking andimmunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication duringde novoinfection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication duringde novoinfection.IMPORTANCEGenomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.

scholarly journals Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

2012 ◽  
Vol 86 (18) ◽  
pp. 9817-9827 ◽  
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.

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

2018 ◽  
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

scholarly journals Human cytomegalovirus RL13 protein interacts with host NUDT14 protein affecting viral DNA replication

2016 ◽  
Vol 13 (3) ◽  
pp. 2167-2174 ◽  
Author(s):  
GUILI WANG ◽  
GAOWEI REN ◽  
XIN CUI ◽  
ZHITAO LU ◽  
YANPING MA ◽  
...  
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Viral Dna ◽  
Viral Dna Replication

scholarly journals The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)

1999 ◽  
Vol 73 (12) ◽  
pp. 10458-10471 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Won-Jong Jang ◽  
Gary S. Hayward
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Promyelocytic Leukemia Protein ◽  
The Core ◽  
Replication Compartment

ABSTRACT During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation.

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