scholarly journals DNA Polymerase Processivity Factor of Human Cytomegalovirus May Be a Key Molecule for Molecular Coupling of Viral DNA Replication to Transcription

10.5772/18144 ◽  
2011 ◽  
Author(s):  
Hiroki Isomura
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Processivity Factor

scholarly journals Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

2009 ◽  
Vol 83 (15) ◽  
pp. 7581-7589 ◽  
Author(s):  
Blair L. Strang ◽  
Elisa Sinigalia ◽  
Laurie A. Silva ◽  
Donald M. Coen ◽  
Arianna Loregian
Keyword(s):  
Dna Replication ◽  
Infected Cell ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Nuclear Antigen ◽  
Replication Proteins ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Processivity Factor ◽  
Replication Factors

ABSTRACT The central enzyme responsible for human cytomegalovirus (HCMV) DNA synthesis is a virally encoded DNA polymerase that includes a catalytic subunit, UL54, and a homodimeric accessory subunit, UL44, the presumptive HCMV DNA polymerase processivity factor. The structure of UL44 is similar to that of the eukaryotic processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous other proteins required for faithful DNA replication. We sought to determine whether, like PCNA, UL44 is capable of interacting with multiple DNA replication proteins and, if so, whether these proteins bind UL44 at the site corresponding to where multiple proteins bind to PCNA. Initially, several proteins, including the viral DNA replication factors UL84 and UL57, were identified by mass spectrometry in immunoprecipitates of UL44 from infected cell lysate. The association of UL44/UL84, but not UL44/UL57, was confirmed by reciprocal coimmunoprecipitation of these proteins from infected cell lysates and was resistant to nuclease treatment. Yeast two-hybrid analyses demonstrated that the substitution of residues in UL44 that prevent UL44 homodimerization or abrogate the binding of UL54 to UL44 do not abrogate the UL44/UL84 interaction. Reciprocal glutathione-S-transferase (GST) pulldown experiments using bacterially expressed UL44 and UL84 confirmed these results and, further, demonstrated that a UL54-derived peptide that competes with UL54 for UL44 binding does not prevent the association of UL84 with UL44. Taken together, our results strongly suggest that UL44 and UL84 interact directly using a region of UL44 different from the UL54 binding site. Thus, UL44 can bind interacting replication proteins using a mechanism different from that of PCNA.

scholarly journals Role of the Specific Interaction of UL112-113 p84 with UL44 DNA Polymerase Processivity Factor in Promoting DNA Replication of Human Cytomegalovirus

2010 ◽  
Vol 84 (17) ◽  
pp. 8409-8421 ◽  
Author(s):  
Young-Eui Kim ◽  
Jin-Hyun Ahn
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Specific Interaction ◽  
Terminal Region ◽  
Replication Proteins ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Carboxy Terminal Region ◽  
Processivity Factor ◽  
Carboxy Terminal

ABSTRACT The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.

scholarly journals Sumoylation of the Carboxy-Terminal of Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Attenuates Viral DNA Replication

2021 ◽  
Vol 12 ◽  
Author(s):  
Jun Chen ◽  
Guanlie Li ◽  
Haiqing He ◽  
Xin Li ◽  
Wenjing Niu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Sumoylation Site ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Infected Cells ◽  
Processivity Factor ◽  
Carboxy Terminal

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.

scholarly journals Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Tim Schommartz ◽  
Jiajia Tang ◽  
Rebekka Brost ◽  
Wolfram Brune
Keyword(s):  
Endothelial Cells ◽  
Dna Replication ◽  
Viral Replication ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Cell Nucleus ◽  
Human Fibroblasts ◽  
Gene Products ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The UL112-113 gene is one of the few alternatively spliced genes of human cytomegalovirus (HCMV). It codes for four phosphoproteins, p34, p43, p50, and p84, all of which are expressed with early kinetics and accumulate at sites of viral DNA replication within the host cell nucleus. Although these proteins are known to play important, possibly essential, roles in the viral replication cycle, little is known about the contribution of individual UL112-113 protein products. Here we used splice site mutagenesis, intron deletion and substitution, and nonsense mutagenesis to prevent the individual expression of each UL112-113 protein isoform and to investigate the importance of each isoform for viral replication. We show that HCMV mutants lacking p34 or p50 expression replicated to high titers in human fibroblasts and endothelial cells, indicating that these proteins are nonessential for viral replication, while mutant viruses carrying a stop mutation within the p84 coding sequence were severely growth impaired. Viral replication could not be detected upon the inactivation of p43 expression, indicating that this UL112-113 protein is essential for viral replication. We also analyzed the ability of UL112-113 proteins to recruit other viral proteins to intranuclear prereplication compartments. While UL112-113 expression was sufficient to recruit the UL44-encoded viral DNA polymerase processivity factor, it was not sufficient for the recruitment of the viral UL84 and UL117 proteins. Remarkably, both the p43 and p84 isoforms were required for the efficient recruitment of pUL44, which is consistent with their critical role in the viral life cycle. IMPORTANCE Human cytomegalovirus requires gene products from 11 genetic loci for the lytic replication of its genome. One of these loci, UL112-113, encodes four proteins with common N termini by alternative splicing. In this study, we inactivated the expression of each of the four UL112-113 proteins individually and determined their requirement for HCMV replication. We found that two of the UL112-113 gene products were dispensable for viral replication in human fibroblasts and endothelial cells. In contrast, viral replication was severely reduced or absent when one of the other two gene products was inactivated, indicating that they are of crucial importance for the viral replication cycle. We further showed that the latter two gene products are involved in the recruitment of pUL44, an essential cofactor of the viral DNA polymerase, to specific sites within the cell nucleus that are thought to serve as starting points for viral DNA replication.

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
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