Binding Parameters and Thermodynamics of the Interaction of the Human Cytomegalovirus DNA Polymerase Accessory Protein, UL44, with DNA: Implications for the Processivity Mechanism

The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12 bp, with apparent dissociation constants in the nanomolar range for DNAs > 18 bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, $4 \pm 1$ monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity

Caratterizzazione funzionale e ricerca di partners proteici cellulari della subunità accessoria, UL44, della DNA polimerasi del citomegalovirus umano

The human cytomegalovirus (HCMV) DNA polymerase consists of a catalytic subunit, UL54, and an accessory protein, UL44, that is thought to act as a processivity factor. UL44 has been shown to bind double-stranded DNA, to specifically interact with UL54 and to stimulate long-chain DNA synthesis by UL54. The crystal structure of UL44 has recently been solved and has revealed that UL44 possesses a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and the eukaryotic sliding clamp PCNA. It has also been shown that UL44 forms a homodimer. Similarly to UL42, the putative DNA-binding face of UL44 contains multiple basic residues that could interact electrostatically with the phosphate backbone of DNA. Residues important for the dimerization of UL44 have been identified: L86, L87 and F121. Indeed, the F121A and L86A/L87A mutants, which behave like monomers, are impaired in the ability to bind double-strand DNA. In this work we performed some experiments to better understand the role of UL44 dimerization in vivo. We demonstrated that, as already shown for truncated UL44?C290, full-length UL44 can form dimers both in solution and in a cellular context. Moreover, while UL44 dimerization has been shown to be important for binding to DNA, it appears to be not required for binding to UL54 and for stimulation of DNA synthesis by UL54 in vitro. We also investigated the effect of the L86A/L87A and F121A mutations of UL44 on viral DNA replication through an in vivo assay. The homodimerization of UL44 turned out to be essential for the replication of the viral DNA in a cellular context. This observation suggests that the disruption of UL44 dimerization could represent a good strategy for the development of new antiviral compounds. How UL44 binds to DNA and the role of DNA binding in processivity function have not been yet elucidated. To begin to understand these mechanism, we characterized the interaction of UL44 with DNA by means of filter-binding assays and electrophoretic mobility shift assays (EMSA). We found that, similar to HSV-1 UL42, UL44 binds directly to DNA with nanomolar affinity in a manner that does not require ATP hydrolysis or accessory proteins. UL44 binds DNA as a dimer in a sequence-non specific manner and displays higher affinity for ds DNA compared to ss DNA. Affinity of UL44 for ds DNA decreases with increasing ionic strength and this effect is mediated by ion release, suggesting that DNA binding entails electrostatic interactions between the negatively charged phosphates on DNA backbone and the positive charge of basic residues on the “back” face and disordered loops of UL44. Finally, several observations suggest that UL44 could have other function(s) besides that of DNA processivity factor. Hence, we performed a two-hybrid system screening in order to identify cellular protein partners of UL44 using a cellular cDNA library. We were able to identify 7 cellular proteins which could represent UL44 functional partners

Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12 bp, with apparent dissociation constants in the nanomolar range for DNAs >18 bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, 4 +/- 1 monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity

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

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

Sulfated Derivatives of Escherichia coli K5 Capsular Polysaccharide Are Potent Inhibitors of Human Cytomegalovirus▿

To date, there are few drugs licensed for the treatment of human cytomegalovirus (HCMV) infections, most of which target the viral DNA polymerase and suffer from many drawbacks. Thus, there is still a strong need for new anti-HCMV compounds with novel mechanisms of action. In this study, we investigated the anti-HCMV activity of chemically sulfated derivatives of Escherichia coli K5 capsular polysaccharide. These compounds are structurally related to cellular heparan sulfate and have been previously shown to be effective against some enveloped and nonenveloped viruses. We demonstrated that two derivatives, i.e., K5-N,OS(H) and K5-N,OS(L), are able to prevent cell infection by different strains of HCMV at concentrations in the nanomolar range while having no significant cytotoxicity. Studies performed to elucidate the mechanism of action of their anti-HCMV activity revealed that these compounds do not interact with either the host cell or the viral particle but need a virus-cell interaction to exert antiviral effects. Furthermore, these K5 derivatives were able to inhibit the attachment step of HCMV infection, as well as the viral cell-to-cell spread. Since the mode of inhibition of these compounds appears to differ from that of the available anti-HCMV drugs, sulfated K5 derivatives could represent the basis for the development of a novel class of potent anti-HCMV compounds. Interestingly, our studies highlight that small variations of the K5 derivatives structure can modulate the selectivity and potency of their activities against different viruses, including viruses belonging to the same family

A 6-Aminoquinolone Compound, WC5, with Potent and Selective Anti-Human Cytomegalovirus Activity

We identified a 6-aminoquinolone compound, WC5, that inhibits human cytomegalovirus (HCMV) replication with a selectivity index of approximately 500. WC5 also showed activity against drug-resistant HCMV strains. In contrast, it did not significantly affect the replication of human herpesvirus 6 and 8 and was approximately 10-fold less active against murine cytomegalovirus. Thus, WC5 may represent a lead for the development of new, potent, and selective anti-HCMV compounds

Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells▿

The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy

Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells▿

The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy

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

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