scholarly journals Phosphorylation of β-d-Ribosylbenzimidazoles Is Not Required for Activity against Human Cytomegalovirus

2002 ◽  
Vol 46 (2) ◽  
pp. 478-486 ◽  
Author(s):  
Paula M. Krosky ◽  
Katherine Z. Borysko ◽  
M. Reza Nassiri ◽  
Rodrigo V. Devivar ◽  
Roger G. Ptak ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
High Performance ◽  
Selective Inhibitors ◽  
Viral Dna ◽  
Infected Cells ◽  
Hcmv Replication ◽  
Antiviral Nucleosides

ABSTRACT We have previously reported that 2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(β-d-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5′-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [3H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5′-monophosphate (BDCRB-5′-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5′-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5′-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5′-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5′-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5′-deoxy analog (5′-dTCRB) did not affect viral DNA synthesis, but 5′-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5′-dTCRB and the 5′-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5′-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5′ position for antiviral activity.

scholarly journals Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

1997 ◽  
Vol 41 (12) ◽  
pp. 2680-2685 ◽  
Author(s):  
D J Tenney ◽  
G Yamanaka ◽  
S M Voss ◽  
C W Cianci ◽  
A V Tuomari ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Potent Inhibitor ◽  
Viral Dna ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv Thymidine Kinase

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.

scholarly journals Wnt Modulating Agents Inhibit Human Cytomegalovirus Replication

2013 ◽  
Vol 57 (6) ◽  
pp. 2761-2767 ◽  
Author(s):  
Arun Kapoor ◽  
Ran He ◽  
Rajkumar Venkatadri ◽  
Michael Forman ◽  
Ravit Arav-Boger
Keyword(s):  
Stem Cell ◽  
Cancer Stem Cell ◽  
Human Cytomegalovirus ◽  
Wnt Pathway ◽  
Critical Role ◽  
Wnt Signaling Pathway ◽  
Negative Regulator ◽  
Infected Cells ◽  
Hcmv Replication

ABSTRACTInfection with human cytomegalovirus (HCMV) continues to be a threat for pregnant women and immunocompromised hosts. Although limited anti-HCMV therapies are available, development of new agents is desired. The Wnt signaling pathway plays a critical role in embryonic and cancer stem cell development and is targeted by gammaherpesviruses, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV). HCMV infects stem cells, including neural progenitor cells, during embryogenesis. To investigate the role of Wnt in HCMV replicationin vitro, we tested monensin, nigericin, and salinomycin, compounds that inhibit cancer stem cell growth by modulating the Wnt pathway. These compounds inhibited the replication of HCMV Towne and a clinical isolate. Inhibition occurred prior to DNA replication but persisted throughout the full replication cycle. There was a significant decrease in expression of IE2, UL44, and pp65 proteins. HCMV infection resulted in a significant and sustained decrease in expression of phosphorylated and total lipoprotein receptor-related protein 6 (pLRP6 and LRP6, respectively), Wnt 5a/b, and β-catenin and a modest decrease in Dvl2/3, while levels of the negative regulator axin 1 were increased. Nigericin decreased the expression of pLRP6, LRP6, axin 1, and Wnt 5a/b in noninfected and HCMV-infected cells. For all three compounds, a correlation was found between expression levels of Wnt 5a/b and axin 1 and HCMV inhibition. The decrease in Wnt 5a/b and axin 1 expression was more significant in HCMV-infected cells than noninfected cells. These data illustrate the complex effects of HCMV on the Wnt pathway and the fine balance between Wnt and HCMV, resulting in abrogation of HCMV replication. Additional studies are required to elucidate how HCMV targets Wnt for its benefit.

scholarly journals 4′-C-Methyl-2′-Deoxyadenosine and 4′-C-Ethyl-2′-Deoxyadenosine Inhibit HIV-1 Replication

2011 ◽  
Vol 55 (5) ◽  
pp. 2379-2389 ◽  
Author(s):  
B. Christie Vu ◽  
Paul L. Boyer ◽  
Maqbool A. Siddiqui ◽  
Victor E. Marquez ◽  
Stephen H. Hughes
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Cultured Cells ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Resistance Mutations ◽  
Viral Dna ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTIt is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3′-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4′-C-alkyl-deoxyadenosine (4′-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4′-C-methyl- and 4′-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4′-C-methyl- or 4′-C-ethyl-2′-deoxyadenosine.In vitroexperiments with purified HIV-1 RT showed that 4′-C-methyl-2′-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4′-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.

scholarly journals Relationship of avian retrovirus DNA synthesis to integration in vitro.

1991 ◽  
Vol 11 (3) ◽  
pp. 1419-1430 ◽  
Author(s):  
Y M Lee ◽  
J M Coffin
Keyword(s):  
Polyethylene Glycol ◽  
Dna Synthesis ◽  
Strand Transfer ◽  
Donor Molecule ◽  
Viral Dna ◽  
Target Dna ◽  
Infected Cells ◽  
Relationship Of ◽  
Transfer Mechanisms

An in vitro integration system derived from avian leukosis virus-infected cells supports both intra- and intermolecular integration of the viral DNA. In the absence of polyethylene glycol, intramolecular integration of viral DNA molecules into themselves (autointegration) was preferred. In the presence of polyethylene glycol, integration into an exogenously supplied DNA target was greatly promoted. Analysis of integration intermediates revealed that the strand transfer mechanisms of both reactions were identical to those of retroviruses and some transposons: each 3' end of the donor molecule is joined to a 5' end of the cleaved target DNA. The immediate integration precursor appears to be linear viral DNA with the 3' ends shortened by 2 nucleotides. Finally, in the avian system, most cytoplasmic viral DNA appears to be incomplete and further DNA synthesis is required for integration in vitro.

scholarly journals Strigolactone Analogs Are Promising Antiviral Agents for the Treatment of Human Cytomegalovirus Infection

2020 ◽  
Vol 8 (5) ◽  
pp. 703
Author(s):  
Matteo Biolatti ◽  
Marco Blangetti ◽  
Giulia D’Arrigo ◽  
Francesca Spyrakis ◽  
Paola Cappello ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Human Cytomegalovirus ◽  
Antiviral Agents ◽  
Nucleoside Analogs ◽  
Developed Countries ◽  
Promising Alternative ◽  
Late Protein ◽  
Potential Applications ◽  
Infected Cells

The human cytomegalovirus (HCMV) is a widespread pathogen and is associated with severe diseases in immunocompromised individuals. Moreover, HCMV infection is the most frequent cause of congenital malformation in developed countries. Although nucleoside analogs have been successfully employed against HCMV, their use is hampered by the occurrence of serious side effects. There is thus an urgent clinical need for less toxic, but highly effective, antiviral drugs. Strigolactones (SLs) are a novel class of plant hormones with a multifaceted activity. While their role in plant-related fields has been extensively explored, their effects on human cells and their potential applications in medicine are far from being fully exploited. In particular, their antiviral activity has never been investigated. In the present study, a panel of SL analogs has been assessed for antiviral activity against HCMV. We demonstrate that TH-EGO and EDOT-EGO significantly inhibit HCMV replication in vitro, impairing late protein expression. Moreover, we show that the SL-dependent induction of apoptosis in HCMV-infected cells is a contributing mechanism to SL antiviral properties. Overall, our results indicate that SLs may be a promising alternative to nucleoside analogs for the treatment of HCMV infections.

Famciclovir and Penciclovir. The Mode of Action of Famciclovir Including Its Conversion to Penciclovir

1993 ◽  
Vol 4 (2) ◽  
pp. 67-84 ◽  
Author(s):  
R. A. Vere Hodge
Keyword(s):  
Antiviral Activity ◽  
Dna Synthesis ◽  
Cell Cultures ◽  
Oral Absorption ◽  
Dna Polymerases ◽  
Clinical Situation ◽  
Intestinal Wall ◽  
Human Tissues ◽  
Viral Dna ◽  
Infected Cells

Famciclovir is the oral form of penciclovir, a highly selective antiherpesvirus agent. Both famciclovir and penciclovir are being evaluated in clinical studies. This review covers the conversion of famciclovir to penciclovir, the formation and stability of penciclovir-triphosphate, its effect on herpesvirus DNA polymerases and the inhibition of viral DNA synthesis in cell cultures. The conversion of famciclovir into penciclovir is described, emphasising work with human tissues and including the elucidation of the structures of its chiral monoacetylated derivatives. To reflect its metabolism during oral absorption, famciclovir was incubated in duodenal contents, extracts of intestinal wall and liver, and in blood. The major metabolic route was by de-acetylation followed by oxidation of the purine to form penciclovir. This work with human tissues was predictive of the major metabolic pathway in humans. Penciclovir is phosphorylated much more efficiently than acyclovir in herpesvirus-infected cells yet the host cell kinases phosphorylate the two compounds to a small but comparable extent. This highly preferential metabolism in herpesvirus infected cells is a major factor in its selective antiviral activity. The ( S) enantiomer of penciclovir-triphosphate is the major metabolite and it has good stability in HSV-1, HSV-2, and VZV infected cells. At the high concentrations found in infected cells, penciclovir-triphosphate was an effective inhibitor of viral DNA polymerases and of viral DNA synthesis. The efficient entrapment of the active moiety can account for the markedly better antiviral activity of penciclovir than acyclovir when infected cell cultures were treated for a short time. Such assays reflect more closely the clinical situation after oral dosing than do standard assays in which the test compounds are present continuously. The role of these studies in compound selection and development is discussed.

scholarly journals Nucleolin Associates with the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 and Is Necessary for Efficient Viral Replication

2009 ◽  
Vol 84 (4) ◽  
pp. 1771-1784 ◽  
Author(s):  
Blair L. Strang ◽  
Steeve Boulant ◽  
Donald M. Coen
Keyword(s):  
Infected Cell ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Western Blotting ◽  
Human Cytomegalovirus ◽  
Major Protein ◽  
Cell Nuclei ◽  
Viral Dna ◽  
Infected Cells ◽  
Processivity Factor

ABSTRACT In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.

Relationship of avian retrovirus DNA synthesis to integration in vitro

1991 ◽  
Vol 11 (3) ◽  
pp. 1419-1430
Author(s):  
Y M Lee ◽  
J M Coffin
Keyword(s):  
Polyethylene Glycol ◽  
Dna Synthesis ◽  
Strand Transfer ◽  
Donor Molecule ◽  
Viral Dna ◽  
Target Dna ◽  
Infected Cells ◽  
Relationship Of ◽  
Transfer Mechanisms

An in vitro integration system derived from avian leukosis virus-infected cells supports both intra- and intermolecular integration of the viral DNA. In the absence of polyethylene glycol, intramolecular integration of viral DNA molecules into themselves (autointegration) was preferred. In the presence of polyethylene glycol, integration into an exogenously supplied DNA target was greatly promoted. Analysis of integration intermediates revealed that the strand transfer mechanisms of both reactions were identical to those of retroviruses and some transposons: each 3' end of the donor molecule is joined to a 5' end of the cleaved target DNA. The immediate integration precursor appears to be linear viral DNA with the 3' ends shortened by 2 nucleotides. Finally, in the avian system, most cytoplasmic viral DNA appears to be incomplete and further DNA synthesis is required for integration in vitro.

scholarly journals Host Cell Nucleolin Is Required To Maintain the Architecture of Human Cytomegalovirus Replication Compartments

mBio ◽  
2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Blair L. Strang ◽  
Steeve Boulant ◽  
Tomas Kirchhausen ◽  
Donald M. Coen
Keyword(s):  
Viral Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Cellular Protein ◽  
Major Protein ◽  
Viral Dna ◽  
Infected Cells ◽  
Interfering Rna ◽  
Replication Compartment

ABSTRACTDrastic reorganization of the nucleus is a hallmark of herpesvirus replication. This reorganization includes the formation of viral replication compartments, the subnuclear structures in which the viral DNA genome is replicated. The architecture of replication compartments is poorly understood. However, recent work with human cytomegalovirus (HCMV) showed that the viral DNA polymerase subunit UL44 concentrates and viral DNA synthesis occurs at the periphery of these compartments. Any cellular factors involved in replication compartment architecture are largely unknown. Previously, we found that nucleolin, a major protein component of nucleoli, associates with HCMV UL44 in infected cells and is required for efficient viral DNA synthesis. Here, we show that nucleolin binds to purified UL44. Confocal immunofluorescence analysis demonstrated colocalization of nucleolin with UL44 at the periphery of replication compartments. Pharmacological inhibition of viral DNA synthesis prevented the formation of replication compartments but did not abrogate association of UL44 and nucleolin. Thus, association of UL44 and nucleolin is unlikely to be a nonspecific effect related to development of replication compartments. No detectable colocalization of 5-ethynyl-2′-deoxyuridine (EdU)-labeled viral DNA with nucleolin was observed, suggesting that nucleolin is not directly involved in viral DNA synthesis. Small interfering RNA (siRNA)-mediated knockdown of nucleolin caused improper localization of UL44 and a defect in EdU incorporation into viral DNA. We propose a model in which nucleolin anchors UL44 at the periphery of replication compartments to maintain their architecture and promote viral DNA synthesis.IMPORTANCEHuman cytomegalovirus (HCMV) is an important human pathogen. HCMV infection causes considerable rearrangement of the structure of the nucleus, largely due to the formation of viral replication compartments within the nucleus. Within these compartments, the virus replicates its DNA genome. We previously demonstrated that nucleolin is required for efficient viral DNA synthesis and now find that the nucleolar protein nucleolin interacts with a subunit of the viral DNA polymerase, UL44, specifically at the periphery of replication compartments. Moreover, we find that nucleolin is required to properly localize UL44 at this region. Nucleolin is, therefore, involved in the organization of proteins within replication compartments. This, to our knowledge, is the first report identifying a cellular protein required for maintaining replication compartment architecture.

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.

Sign in / Sign up

Export Citation Format

Share Document