Human cytomegalovirus DNA replication: antiviral targets and drugs

Mercorelli, Beatrice; Sinigalia, Elisa; Loregian, Arianna; Palù, Giorgio

doi:10.1002/rmv.558

Cited by 65 publications

(44 citation statements)

References 225 publications

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“…Given the ability of quinolones to interact with protein-nucleic acid complexes (2,30), the possibility existed that WC5 might inhibit HCMV DNA replication either by affecting the activity of a component of the DNA replication complex other than the viral polymerase or of a protein(s) indirectly involved in virus genome replication (26,29) or by interfering with DNA maturation. To test this hypothesis, the effect of WC5 on viral DNA synthesis in HCMV-infected cells was quantitatively determined by qPCR at 72 h p.i.…”

Section: Resultsmentioning

confidence: 99%

The 6-Aminoquinolone WC5 Inhibits Human Cytomegalovirus Replication at an Early Stage by Interfering with the Transactivating Activity of Viral Immediate-Early 2 Protein

Loregian

Mercorelli

Muratore

et al. 2010

Antimicrob Agents Chemother

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WC5 is a 6-aminoquinolone that potently inhibits the replication of human cytomegalovirus (HCMV) but has no activity, or significantly less activity, against other herpesviruses. Here we investigated the nature of its specific anti-HCMV activity. Structure-activity relationship studies on a small series of analogues showed that WC5 possesses the most suitable pattern of substitutions around the quinolone scaffold to give potent and selective anti-HCMV activity. Studies performed to identify the possible target of WC5 indicated that it prevents viral DNA synthesis but does not significantly affect DNA polymerase activity. In yield reduction experiments with different multiplicities of infection, the anti-HCMV activity of WC5 appeared to be highly dependent on the viral inoculum, suggesting that WC5 may act at an initial stage of virus replication. Consistently, time-of-addition and time-of-removal studies demonstrated that WC5 affects a phase of the HCMV replicative cycle that precedes viral DNA synthesis. Experiments to monitor the effects of the compound on virus attachment and entry showed that it does not inhibit either process. Evaluation of viral mRNA and protein expression revealed that WC5 targets an event of the HCMV replicative cycle that follows the transcription and translation of immediate-early genes and precedes those of early and late genes. In cell-based assays to test the effects of WC5 on the transactivating activity of the HCMV immediate-early 2 (IE2) protein, WC5 markedly interfered with IE2-mediated transactivation of viral early promoters. Finally, WC5 combined with ganciclovir in checkerboard experiments exhibited highly synergistic activity. These findings suggest that WC5 deserves further investigation as a candidate anti-HCMV drug with a novel mechanism of action.

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Section: Resultsmentioning

confidence: 99%

The 6-Aminoquinolone WC5 Inhibits Human Cytomegalovirus Replication at an Early Stage by Interfering with the Transactivating Activity of Viral Immediate-Early 2 Protein

Loregian

Mercorelli

Muratore

et al. 2010

Antimicrob Agents Chemother

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“…HCMV is the most complex human-pathogenic herpesvirus and encodes approximately 180 proteins, of which 45 were categorized as absolutely essential and 35 were assumed to be critical for efficient viral replication in cell culture (14,33,48). Thus, in addition to the well-established antiviral target molecules comprising the viral protein kinase UL97 and the DNA polymerase UL54, HCMV encodes several more target proteins that deserve further evaluation and validation (32). In this report, we focused on the gene product encoded by the open reading frame UL84, which has previously been shown to be essential for the initiation of viral lytic DNA replication (34,35,37).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, increasing evidence suggests that atherosclerotic vascular disease manifestations, such as coronary restenosis or transplant atherosclerosis, are linked to HCMV infection (5). Despite considerable diagnostic and therapeutic progress in recent years, the clinical application of all presently licensed anti-HCMV drugs is limited due to several drawbacks, including toxicity and the emergence of drug-resistant virus strains after prolonged therapy (27,32). Consequently, new therapeutic strategies, as well as novel antiviral targets, are urgently required to improve the treatment options for life-threatening HCMV infections.…”

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confidence: 99%

Inhibition of Human Cytomegalovirus Replication via Peptide Aptamers Directed against the Nonconventional Nuclear Localization Signal of the Essential Viral Replication Factor pUL84

Kaiser

Lischka

Wagenknecht

et al. 2009

J Virol

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The UL84 open reading frame of human cytomegalovirus encodes an essential multifunctional regulatory protein that is thought to act in the nucleus as an initiator of lytic viral replication. Nuclear trafficking of pUL84 is facilitated by a complex nonconventional nuclear localization signal (NLS) that mediates its interaction with the cellular importin-␣/␤ pathway. Since binding of pUL84 to importin-␣ proteins mechanistically differs from that of cellular proteins containing a classical NLS, we assumed that specific interference with the nuclear import of pUL84 might be possible and that this could constitute a novel principle for antiviral therapy. In order to test this hypothesis, we employed peptide aptamer technology and isolated several peptide aptamers from a randomized peptide expression library that specifically bind with high affinity to the unconventional pUL84 NLS under intracellular conditions. Coimmunoprecipitation experiments confirmed these interactions in mammalian cells, and the antiviral potential of the identified peptide aptamers was determined using three independent experimental approaches. (i) Infection experiments with a recombinant human cytomegalovirus expressing green fluorescent protein demonstrated 50 to 60% decreased viral replication in primary human fibroblasts stably expressing pUL84-specific aptamers. (ii) A 50 to 70% reduction of viral plaque formation, as well as a 70 to 90% inhibition of virus release in the presence of pUL84-specific aptamers, was observed. (iii) Immunofluorescence analyses revealed a shift from an almost exclusively nuclear pUL84 staining pattern to a nucleocytoplasmic distribution upon coexpression of the identified molecules, indicating that interference with the nuclear import of pUL84 contributes to the observed antiviral activity of the identified pUL84-binding aptamer molecules.Human cytomegalovirus (HCMV) is a widely distributed opportunistic betaherpesvirus with a 30 to 100% seroprevalence in the human population, depending on the socioeconomic status and geographic location of the country (5). Following primary infection, HCMV establishes lifelong latency and periodically reactivates, rarely causing symptoms in healthy individuals. In contrast, the virus still represents a major cause of morbidity and mortality in immunosuppressed patients receiving organ transplants or suffering from AIDS and tumors (5). Furthermore, HCMV is the leading viral pathogen of congenitally infected newborns (2). Although 90% of the congenitally infected infants are initially asymptomatic, a considerable proportion develop sequelae later in life, such as progressive sensorineural hearing loss. This is due to ongoing viral replication, indicating the urgent need for adequate antiviral treatment of these children (1). In addition, increasing evidence suggests that atherosclerotic vascular disease manifestations, such as coronary restenosis or transplant atherosclerosis, are linked to HCMV infection (5). Despite considerable diagnostic and therapeutic progress in recent yea...

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“…Subsequently, HCMV replication sites are enlarged to form globular replication compartments that eventually fill the nucleus at late stage [19,29,30]. Viral proteins involved in HCMV DNA replication at least include UL44 (the DNA polymerase processivity factor), UL84 (likely a replication initiator interacting with the RNA/DNA hybrid within oriLyt) and the immediate-early 2 (IE2) protein [29,[31][32][33][34]. HCMV with IE2 gene deleted is not viable [35].…”

Section: Introductionmentioning

confidence: 99%

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

Lee

et al. 2011

Cell Res

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Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.

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Human cytomegalovirus DNA replication: antiviral targets and drugs

Cited by 65 publications

References 225 publications

The 6-Aminoquinolone WC5 Inhibits Human Cytomegalovirus Replication at an Early Stage by Interfering with the Transactivating Activity of Viral Immediate-Early 2 Protein

The 6-Aminoquinolone WC5 Inhibits Human Cytomegalovirus Replication at an Early Stage by Interfering with the Transactivating Activity of Viral Immediate-Early 2 Protein

Inhibition of Human Cytomegalovirus Replication via Peptide Aptamers Directed against the Nonconventional Nuclear Localization Signal of the Essential Viral Replication Factor pUL84

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

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