scholarly journals Human cytomegalovirus RNA2.7 regulates host cell cycle and facilitates viral DNA replication by inhibiting RNA polymerase II phosphorylation

2018 ◽  
Author(s):  
Yujing Huang ◽  
Jing Zhang ◽  
Xin Guo ◽  
Qing Wang ◽  
Zhongyang Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Cytomegalovirus ◽  
Cell Cycle Control ◽  
S Phase ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Pol Ii

AbstractHuman cytomegalovirus (HCMV) is a ubiquitous pathogen belongs to the beta herpesvirus family. RNA2.7 is a viral long non-coding RNA accounting for more than 20% of total viral transcripts at early time of infection. By construction of RNA2.7 deleted mutant and genome transcriptomic analysis, RNA2.7 is demonstrated to repress host cellular RNA polymerase II (Pol II)-dependent transcription through inhibiting the phosphorylation of RNA polymerase II (Pol II). Co-immunoprecipitation, RNA immunoprecipitation and RNA electrophoretic mobility shift assay are followed to investigate its mechnism. A 145nt-in-length fragment in RNA2.7 is identified to bind to Pol II and block the interaction between Pol II and phosphorylated cyckin-dependent kinase 9 (phospho-CDK9). By inhibiting Pol II phosphorylation, RNA2.7 decreases the transcription and expression levels of chromatin licensing and DNA replication factor 1 (Cdt1) and cell division cycle gene 6 (Cdc6). Through above way, RNA2.7 prevents the entry of cells into S phase and facilitates viral DNA replication. Our results discover the functions of HCMV RNA2.7 in regulation of Pol II phosphorylation and cell cycle control during infection.Author summaryHuman cytomegalovirus (HCMV) RNA2.7 is a viral lncRNA that is most abundant during infection. Here we show that a 145nt-in-length fragment in RNA2.7 binds to RNA polymerase II (Pol II) and blocks the interaction between Pol II and phosphorylated cyckin-dependent kinase 9 (phospho-CDK9). By inhibiting Pol II phosphorylation, RNA2.7 decreases the transcription and expression levels of chromatin licensing and DNA replication factor 1 (Cdt1) and cell division cycle gene 6 (Cdc6), and blocks host cells entering into S phase. RNA2.7 is confirmed to facilitate viral DNA replication through decreasing Cdt1 and Cdc6. Therefore, our results discover the functions of HCMV RNA2.7 in regulation of Pol II phosphorylation and cell cycle control during infection.

scholarly journals Human Parvovirus B19 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication

2013 ◽  
Vol 87 (23) ◽  
pp. 12766-12775 ◽  
Author(s):  
Yong Luo ◽  
Steve Kleiboeker ◽  
Xuefeng Deng ◽  
Jianming Qiu
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Content ◽  
Parvovirus B19 ◽  
S Phase ◽  
Human Parvovirus B19 ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cycle Arrest ◽  
Cellular Dna

Human parvovirus B19 (B19V) infection has a unique tropism to human erythroid progenitor cells (EPCs) in human bone marrow and the fetal liver. It has been reported that both B19V infection and expression of the large nonstructural protein NS1 arrested EPCs at a cell cycle status with a 4 N DNA content, which was previously claimed to be “G2/M arrest.” However, a B19V mutant infectious DNA (M20mTAD2) replicated well in B19V-semipermissive UT7/Epo-S1 cells but did not induce G2/M arrest (S. Lou, Y. Luo, F. Cheng, Q. Huang, W. Shen, S. Kleiboeker, J. F. Tisdale, Z. Liu, and J. Qiu, J. Virol.86:10748–10758, 2012). To further characterize cell cycle arrest during B19V infection of EPCs, we analyzed the cell cycle change using 5-bromo-2′-deoxyuridine (BrdU) pulse-labeling and DAPI (4′,6-diamidino-2-phenylindole) staining, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content. We found that although both B19V NS1 transduction and infection immediately arrested cells at a status of 4 N DNA content, B19V-infected 4 N cells still incorporated BrdU, indicating active DNA synthesis. Notably, the BrdU incorporation was caused neither by viral DNA replication nor by cellular DNA repair that could be initiated by B19V infection-induced cellular DNA damage. Moreover, several S phase regulators were abundantly expressed and colocalized within the B19V replication centers. More importantly, replication of the B19V wild-type infectious DNA, as well as the M20mTAD2mutant, arrested cells at S phase. Taken together, our results confirmed that B19V infection triggers late S phase arrest, which presumably provides cellular S phase factors for viral DNA replication.

scholarly journals S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression

1998 ◽  
Vol 336 (3) ◽  
pp. 619-624 ◽  
Author(s):  
Maya CESARI ◽  
Laurent HÉLIOT ◽  
Catherine MEPLAN ◽  
Michel PABION ◽  
Saadi KHOCHBIN
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Regulation Of Gene Expression ◽  
S Phase ◽  
Chromatin Assembly

Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phase of the cell cycle. Thus treatment of cells with an inhibitor of translation, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cycle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase-dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and show that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, which is again S-phase-dependent. The modest effect of CX in inducing the activity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observe transcriptional activation. We therefore suggest that the uncoupled DNA replication and histone synthesis that occur after CX treatment induces a general modification of chromatin structure, and propose that this general disorganization of chromatin structure is responsible for a widespread activation of RNA polymerase II-mediated gene transcription.

scholarly journals Cytomegalovirus late transcription factor target sequence diversity orchestrates viral early to late transcription

2021 ◽  
Vol 17 (8) ◽  
pp. e1009796
Author(s):  
Ming Li ◽  
Qiaolin Hu ◽  
Geoffrey Collins ◽  
Mrutyunjaya Parida ◽  
Christopher B. Ball ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Core Promoter ◽  
Target Sequence ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Pol Ii ◽  
Viral Promoters ◽  
Late Transcription ◽  
Target Sequences

Beta- and gammaherpesviruses late transcription factors (LTFs) target viral promoters containing a TATT sequence to drive transcription after viral DNA replication has begun. Human cytomegalovirus (HCMV), a betaherpesvirus, uses the UL87 LTF to bind both TATT and host RNA polymerase II (Pol II), whereas the UL79 LTF has been suggested to drive productive elongation. Here we apply integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, and promoter function assays) to uncover the contribution of diversity in LTF target sequences in determining degree and scope to which LTFs drive viral transcription. We characterize the DNA sequence patterns in LTF-responsive and -unresponsive promoter populations, determine where and when Pol II initiates transcription, identify sites of LTF binding genome-wide, and quantify change in nascent transcripts from individual promoters in relation to core promoter sequences, LTF loss, stage of infection, and viral DNA replication. We find that HCMV UL79 and UL87 LTFs function concordantly to initiate transcription from over half of all active viral promoters in late infection, while not appreciably affecting host transcription. Both LTFs act on and bind to viral early-late and late kinetic-class promoters. Over one-third of these core promoters lack the TATT and instead have a TATAT, TGTT, or YRYT. The TATT and non-TATT motifs are part of a sequence block with a sequence code that correlates with promoter transcription level. LTF occupancy of a TATATA palindrome shared by back-to-back promoters is linked to bidirectional transcription. We conclude that diversity in LTF target sequences shapes the LTF-transformative program that drives the viral early-to-late transcription switch.

scholarly journals Transient DNA Binding Induces RNA Polymerase II Compartmentalization During Herpesviral Infection Distinct From Phase Separation

2018 ◽  
Author(s):  
David T McSwiggen ◽  
Anders S Hansen ◽  
Hervé Marie-Nelly ◽  
Sheila Teves ◽  
Alec B Heckert ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Simplex Virus

SummaryDuring lytic infection, Herpes Simplex Virus 1 generates replication compartments (RCs) in host nuclei that efficiently recruit protein factors, including host RNA Polymerase II (Pol II). Pol II and other cellular factors form hubs in uninfected cells that are proposed to phase separate via multivalent protein-protein interactions mediated by their intrinsically disordered regions. Using a battery of live cell microscopic techniques, we show that although RCs superficially exhibit many characteristics of phase separation, the recruitment of Pol II instead derives from nonspecific interactions with the viral DNA. We find that the viral genome remains nucleosome-free, profoundly affecting the way Pol II explores RCs by causing it to repetitively visit nearby binding sites, thereby creating local Pol II accumulations. This mechanism, distinct from phase separation, allows viral DNA to outcompete host DNA for cellular proteins. Our work provides new insights into the strategies used to create local molecular hubs in cells.

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 Adenovirus E1B55K Region Is Required To Enhance Cyclin E Expression for Efficient Viral DNA Replication

2008 ◽  
Vol 82 (7) ◽  
pp. 3415-3427 ◽  
Author(s):  
Xinyu Zheng ◽  
Xiao-Mei Rao ◽  
Jorge G. Gomez-Gutierrez ◽  
Hongying Hao ◽  
Kelly M. McMasters ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cancer Cells ◽  
Cyclin E ◽  
Serum Starvation ◽  
Viral Dna ◽  
Human Lung Fibroblast ◽  
Viral Dna Replication ◽  
Efficient Replication ◽  
Interfering Rna

ABSTRACT Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G0 phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G0 phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

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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