Synthesis of Cytomegalovirus DNA Is an Antiviral Target Late in Virus Growth

Tyms, A. S.; Davis, Jennifer; Clarke, John R.; Jeffries, D. J.

doi:10.1099/0022-1317-68-6-1563

Cited by 13 publications

(8 citation statements)

References 41 publications

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“…This increase may be the result of input viral DNA binding to PI or of progression from G 0 /G 1 toward the G 1 /S border. The presence of 200 g of phosphoroacetic acid (PFA) per ml, a specific inhibitor of viral but not cellular DNA replication (46,47), abolished the increase in overall DNA content ( Fig. 1G, H, and I), consistent with our interpretation that accumulating progeny viral DNA contributed substantially to the DNA content at late times.…”

supporting

confidence: 86%

Human cytomegalovirus infection inhibits G1/S transition

Dittmer

Mocarski

1997

J Virol

189

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Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G 1 /S (hydroxyurea and mimosine) and G 2 /M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G 0) cells, the cell cycle did not progress beyond the G 1 /S border even after serum stimulation. Proteins which normally indicate G 1 /S transition (proliferating cell nuclear antigen [PCNA]) or G 2 /M transition (cyclin B 1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B 1 was induced in infected cells which exhibited a G 1 /S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.

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supporting

confidence: 86%

Human cytomegalovirus infection inhibits G1/S transition

Dittmer

Mocarski

1997

J Virol

189

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“…To verify this hypothesis, HFFs were infected with HCMV and treated with PFA, a selective inhibitor of viral DNA polymerase (63). As shown in Fig.…”

Section: The Journal Of Immunologymentioning

confidence: 99%

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

Pignoloni

Fionda

Dell’Oste

et al. 2016

The Journal of Immunology

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Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I–related chain (MIC) A/B and UL16-binding proteins (ULBP)1–6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell–mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3–related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.

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“…SA-␤-Gal induction in HCMV-infected cells treated with a viral DNA replication inhibitor was therefore assessed. Serum-starved HELF cells were infected with HCMV (MOI, 5) and maintained in the presence of the selective HCMV polymerase inhibitor PFA (58), which inhibits the expression of delayed early and late genes such as the pp65 gene. As expected, immunohistochemical staining for pp65 was negative in PFAtreated cells at 72 h p.i., whereas positive cells were observed in untreated cultures (Fig.…”

Section: Hcmv Induces Senescence In Primary Human Fibroblastsmentioning

confidence: 99%

Cell Cycle Arrest by Human Cytomegalovirus 86-kDa IE2 Protein Resembles Premature Senescence

Noris

Zannetti

Demurtas

et al. 2002

J Virol

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Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display ␤-galactosidase (␤-Gal) activity at neutral pH (senescence-associated ␤-Gal [SA-␤-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Human cytomegalovirus (HCMV), a member of the betaherpesvirus family, is widespread among humans, where it establishes a persistent infection that causes disease and mortality in immunocompromised individuals (44). HCMV remains the major viral cause of birth defects in humans, with an incidence of about 1% of live births. Among these congenital infections, the incidence of severe neurological complications is estimated to be at least 10%. Additionally, epidemiological data and pathological studies suggest a link between HCMV infection and atherosclerosis.Following infection of permissive cells, i.e., human embryo fibroblasts (HELF), HCMV gene expression occurs in three temporal phases, designated immediate-early (IE), early, and late (41). The most abundant IE transcripts arise from the major IE region in the unique long region of the genome: differential-splicing events give rise to the 72-kDa IE protein (IE1) and the 86-kDa IE gene product (IE2), plus other isomers that regulate genes that are expressed during the S phase of the cell cycle. Studies from many laboratories have shown that these proteins act as transcriptional regulators of both virus and host cellular genes, such as DNA polymerase ␣, dihydrofolate reductase, thymidylate synthase, and ribonucleotide reductase (for a review see references 18 and 41).Replication of HCMV, which encodes its own replication apparatus, appears to be favored in the absence of host cellular DNA synthesis (17). Fibroblasts experimentally infected in vitro by HCMV are halted in both the G 1 -S and G 2 -M compartments, depending on the cell cycle phase where infection occurs (5,15,28,35). This cell cycle control is mediated by several viral proteins, such as IE2 and UL69, using mechanisms that have been determined (9,24,30,43,60). HCMV demonstrates a rather unique cell cycle arrest, preceded by a virally mediated mitogenic response of infected cells, with the transient induction of c-fos, c-jun, and c-myc (3). It induces the expression of proliferation markers such as proliferating cellular nuclear antigen (PCNA) and topoisomerase II (15,26) and cellular enzymes involved in DNA synthesis (22,31,33,38), but cellular activation does not seem to lead to cell division (1, 30). However, elevated levels of both cyclin E and B and their associated kinase activities are induced following infection (4,40,47), suggesting that HCMV has evolved mechanisms that support viral DNA synthesis but disallow cellular DNA replication b...

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Synthesis of Cytomegalovirus DNA Is an Antiviral Target Late in Virus Growth

Cited by 13 publications

References 41 publications

Human cytomegalovirus infection inhibits G1/S transition

Human cytomegalovirus infection inhibits G1/S transition

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

Cell Cycle Arrest by Human Cytomegalovirus 86-kDa IE2 Protein Resembles Premature Senescence

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