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

The Antifungal Drug Isavuconazole Inhibits the Replication of Human Cytomegalovirus (HCMV) and Acts Synergistically with Anti-HCMV Drugs

We recently reported that some clinically approved antifungal drugs are potent inhibitors of human cytomegalovirus (HCMV). Here, we report the broad-spectrum activity against HCMV of isavuconazole (ICZ), a new extended-spectrum triazolic antifungal drug. ICZ inhibited the replication of clinical isolates of HCMV as well as strains resistant to the currently available DNA polymerase inhibitors. The antiviral activity of ICZ against HCMV could be linked to the inhibition of human cytochrome P450 51 (hCYP51), an enzyme whose activity we previously demonstrated to be required for productive HCMV infection. Moreover, time-of-addition studies indicated that ICZ might have additional inhibitory effects during the first phase of HCMV replication. Importantly, ICZ showed synergistic antiviral activity in vitro when administered in combination with different approved anti-HCMV drugs at clinically relevant doses. Together, these results pave the way to possible future clinical studies aimed at evaluating the repurposing potential of ICZ in the treatment of HCMV-associated diseases

Human Cytomegalovirus Inhibitor AL18 Also Possesses Activity against Influenza A and B Viruses

AL18, an inhibitor of human cytomegalovirus DNA polymerase, was serendipitously found to also block the interaction between the PB1 and PA polymerase subunits of influenza A virus. Furthermore, AL18 effectively inhibited influenza A virus polymerase activity and the overall replication of influenza A and B viruses. A molecular model to explain the binding of AL18 to both cytomegalovirus and influenza targets is proposed. Thus, AL18 represents an interesting lead for the development of new antivirals

A Point Mutation in a Herpesvirus Polymerase Determines Neuropathogenicity

Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission

Caratterizzazione di nuovi composti antivirali contro il citomegalovirus umano e studio del meccanismo di neuropatogenocità nel herpesvirus equino di tipo 1

Several members of the Herpesviride family are pathogens for both humans and animals. Both the human cytomegalovirus (HCMV) and the equine herpesvirus-1 (EHV-1) belong to this family and are subject of this study. HCMV is a pathogen of major importance, since it infects the major part of the human population all over the world. Although it causes infections that are asymptomatic or benign in the immunocompetent subjects, HCMV infection causes serious diseases (such as gastroenteritis, pulmonitis, retinitis) that can result even fatal in immuncompromised individuals, such as transplant recipients, AIDS patients, and immunodepressed. Indeed, HCMV is also a major cause of congenital defects in the newborn children. To date, there are a few drugs licenced for the treatment of HCMV infections, which are ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS), but none of them has been approved for the treatment of congenital infections. All these drugs are targeted at the viral DNA polymerase and they suffer from many drawbacks, such as poor bioavailability, long-term toxicity, and low effectiveness. Furthermore, the outcome of HCMV strains resistant to these drugs has become a serious problem in the management of transplant recipients, which become even worse because often the strains resistant to one drug often are cross-resistant to the others. For these reasons many efforts have been recently directed to the identification of new, less toxic, more effective anti-HCMV compounds with a mechanism of action different from that of the currently available drugs. One of the aims of this study was to identify new molecules with antiviral activity, directed in particular against HCMV, with a novel mechanism of action. Therefore, three different classes of molecules have been under investigation: some 6-aminoquinolone (6-AQ) derivatives, a compound (AL18), belonging to the AL compounds class (a series of molecule of different nature identified by HTS as inhibitors of the interaction between the two subunits of the HCMV-encoded DNA polymerase), and some sulfated derivatives of the K5 polysaccharide from E. coli. The 6-AQs and the K5 derivatives were assayed for the anti-HCMV activity. Then the most promising molecules selected for further characterisation of their antiviral properties were assayed: for the activity against other herpesviruses, to identify the step in the replication cycle that was affected, and finally to elucidate their mechanism of action. AL18 was assayed for its activty against herpesviruses other than HCMV and for its specificity. These studies led to the identification of a 6-AQ, WC5, with specific anti-HCMV activity and of two sulfated derivatives of K5 polysaccharide from E. coli, K5-N,OS (H) and (L), that act by blocking HCMV infectivity. Furthermore, we demonstrated that AL18 is a specific anti-HCMV agent that represents the ideal candidate for the development of new drugs targeted at the viral DNA polymerase, but with a mechanism of action that differs from that of the currently available anti-HCMV drugs. The second part of this study regards EHV-1, which is a major pathogen for horses, causing diseases ranging from respiratory infections to induction of abortion, up to neuropathologies that can result in paralysis or be fatal for the animal. Recently, we characterised the DNA polymerase complex of EHV-1 and analysed the effects of some polymorfisms identified in field isolates on its enzymatic activity. Infact, since the range of EHV-1-induced pathologies is so broad, it has been hypothesised that different strains may exist with different neuropathogenic potential. To test this hypothesis, the genomic sequences of several EHV-1 strains have been compared in order to identify genetic markers associated with the neuropathogenic potential. It has been demonstrated that only a single aminoacid variation (D/N752) in the catalytic subunit of the viral DNA polymerase is associated with the neuropathogenic potential and that this change is able to reduce the virulence of the respective virus strain and to increase the sensitivity to a polymerase inhibitor, aphidicolin (AFC), that acts by competing with dNTPs substrates. Therefore, we performed some biochemical assays with the D/N752 polymerase variants to determine the affinity costants (KM) for dNTPs and the inhibition costants (KI) for AFC, to highlight biochemical differences that could explain the differences in the neuropathogenic potential showed in vivo by the two viruses. From these studies we observed that the non-neuropathogenic N752 variant has lower affinity for two of the dNTPs substrates than the neuropathogenic D752 variant does. Furthermore, the N752 variant is more sensitive to AFC than the D752 variant. These biochemical observations support the hypothesis that the two polymerase variants have a different biochemical "behaviour" with respect of the dNTPs substrates and contribute to the formulation of a model that may eplain the differences in the neuropathogenic potential showed by the different viral strains.La famiglia Herpesviridae comprende molti virus, patogeni sia per l'uomo che per gli animali. A questa famiglia appartengono anche il citomegalovirus umano (HCMV) e l'herpesvirus equino di tipo 1 (EHV-1), che sono oggetto di questo studio. HCMV è un patogeno di enorme importanza, dal momento che esso infetta la maggior parte della popolazione mondiale. Sebbene esso causi patologie asintomatiche o benigne nei soggetti immunocompetenti, l'infezione da HCMV è causa di patologie molto gravi (come gastroenteriti, polmoniti, retiniti, ecc.) che possono risultare anche fatali nei pazienti immunocompromessi, come trapiantati, affetti da AIDS e immunodepressi. Inoltre, HCMV è anche la prima causa di malformazioni e difetti congeniti nei neonati. Attualmente esistono alcuni farmaci approvati per il trattamento delle infezioni da HCMV, tra cui ganciclovir (GCV), cidofovir (CDV) e foscarnet (FOS), anche se nessuno di questi è stato approvato per il trattamento dell'infezione congenita. Tali farmaci hanno tutti come bersaglio comune la DNA polimerasi virale e hanno svantaggi non trascurabili, come la scarsa biodisponibilità, la tossicità elevata a lungo termine e la bassa efficacia. Inoltre, la comparsa di ceppi di HCMV resistenti a questi farmaci sta diventando un gravissimo problema nel management dei pazienti trapiantati, aggravato dal fatto che spesso i ceppi resistenti a un farmaco sono cross-resistenti anche agli altri. Per questi motivi, negli ultimi anni sono stati fatti grandi sforzi nella ricerca di nuovi composti anti-HCMV, meno tossici, più efficaci e soprattutto con un meccanismo d'azione diverso da quello dei farmaci di cui si dispone attualmente. Uno degli obbiettivi di questo studio è stato quello di identificare e caratterizzare nuove molecole con attività antivirale, in particolare dirette contro HCMV e dotate di un meccanismo d'azione diverso da quello dei farmaci commercializzati. Per questo scopo sono stati presi in considerazione tre tipi diversi di molecole: alcuni derivati 6-amminochinolonici (6-AQ), alcuni derivati solfatati del polisaccaride K5 di E. coli e un composto (AL18) della serie AL (insieme di molecole di diversa natura chimica identificate mediante HTS come inibitori dell'nterazione tra le subunità  della DNA polimerasi di HCMV). Per i 6-AQ e i derivati di K5 è stata saggiata l'attività contro HCMV e, una volta identificate le molecole più promettenti per le loro proprietà antivirali, sono stati effettuati diversi studi per caratterizzare ulteriormente la loro attività contro HCMV e altri herpesvirus, per definire lo stadio del ciclo replicativo virale in cui essi agiscono e per chiarire a livello molecolare il loro meccanismo d'azione. Invece, per quanto riguarda AL18, sono stati effettuati studi riguardanti la specificità contro HCMV rispetto ad altri herpesvirus. Questi studi hanno portato all'identificazione di un 6-AQ, WC5, con potente attività antivirale specifica per HCMV e di due derivati solfatati del polisaccaride K5 di E. coli, K5-N,OS (H) e (L), che agiscono bloccando l'nfettività di HCMV. Inoltre si è potuto dimostrare che il composto AL18 è specifico per HCMV e che rappresenta quindi un candidato ideale per lo sviluppo di nuovi farmaci sempre diretti contro la DNA polimerasi di HCMV, ma dotati di un meccanismo d'azione diverso da quello dei farmaci attualmente utilizzati in terapia. La seconda parte del progetto riguarda EHV-1, che è un patogeno molto importante per i cavalli, essendo in grado di causare patologie che vanno dalle infezioni respiratorie, all'induzione di aborto e fino a neuropatologie che possono portare anche alla paralisi e alla morte dell'animale. Recentemente il nostro gruppo di ricerca ha caratterizzato il complesso della DNA polimerasi di EHV-1 e l'effetto che alcuni polimorfismi riscontrati in isolati virali hanno sulla sua attività enzimatica. Infatti, data la varietà di patologie indotte da EHV-1, è stato ipotizzato che esistessero diversi ceppi con un diverso potenziale neuropatogenico a causa di un diverso corredo genetico e per questo sono state comparate le sequenze genomiche di numerosi ceppi di EHV-1, con lo scopo di identificare potenziali marcatori genetici associabili al potenziale neuropatogenico. E' stato dimostrato che l'unica variazione amminoacidica (D/N752) associata al potenziale neuropatogenico si trova nella subunità  catalitica della DNA polimerasi virale ed è in grado di provocare una attenuazione generale nella virulenza del ceppo da cui viene espressa e una maggiore sensibilità della polimerasi virale a un inibitore che agisce per competizione con i substrati nucleotidici, l'afidicolina (AFC). Per questo motivo sono stati effettuati dei saggi biochimici con le due varianti polimerasiche D/N752 per determinare le costanti di affinità (KM) per i substrati nucleotidici e di inibizione (KI) per AFC, con lo scopo di evidenziare delle eventuali differenze dal punto di vista biochimico in grado di contribuire alla delucidazione del meccanismo responsabile del diverso grado di neuropatogenicità  dimostrato in vivo dai rispettivi virus. Da questi studi è emerso che la variante N752 non-neuropatogenica ha un'affinità minore per due dei substrati nucleotidici rispetto alla variante D752 neuropatogenica. Inoltre, la variante N752 ha maggiore affinità per l'inibitore AFC rispetto alla variante D752. Questi dati biochimici supportano l'ipotesi di un diverso "comportamento" biochimico delle due varianti proteiche della DNA polimerasi virale nei confronti dei loro substrati dNTPs e contribuiscono alla formulazione di un modello per spiegare le differenze nel potenziale neuropatogenico dei diversi ceppi virali

Early inhibitors of human cytomegalovirus: State-of-art and therapeutic perspectives.

Human cytomegalovirus (HCMV) infection is associated with severe morbidity and mortality in immunocompromised individuals, mainly transplant recipients and AIDS patients, and is the most frequent cause of congenital malformations in newborn children. To date, few drugs are licensed for the treatment of HCMV infections, most of which target the viral DNA polymerase and suffer from many drawbacks, including long-term toxicity, low potency, and poor bioavailability. In addition, the emergence of drug-resistant viral strains is becoming an increasing problem for disease management. Finally, none of the current anti-HCMV drugs have been approved for the treatment of congenital infections. For all these reasons, there is still a strong need for new anti-HCMV drugs with novel mechanisms of action. The first events of the virus replication cycle, including attachment, entry, immediate-early gene expression, and immediate-early functions-in particular that of Immediate-Early 2 protein-represent attractive targets for the development of novel antiviral compounds. Such inhibitors would block not only the expression of viral immediate-early proteins, which play a key role in the pathogenesis of HCMV infection, but also the host immunomodulation and the changes to cell physiology induced by the first events of virus infection. This review describes the current knowledge on the initial phases of HCMV replication, their validation as potential novel antiviral targets, and the development of compounds that block such processes

Targeted Disruption of E6/p53 Binding Exerts Broad Activity and Synergism with Paclitaxel and Topotecan against HPV-Transformed Cancer Cells

High-risk human papillomaviruses (HR-HPV) are the etiological agents of almost all cervical cancer cases and a high percentage of head-and-neck malignancies. Although HPV vaccination can reduce cancer incidence, its coverage significantly differs among countries, and, therefore, in the next decades HPV-related tumors will not likely be eradicated worldwide. Thus, the need of specific treatments persists, since no anti-HPV drug is yet available. We recently discovered a small molecule (Cpd12) able to inhibit the E6-mediated degradation of p53 through the disruption of E6/p53 binding in HPV16- and HPV18-positive cervical cancer cells. By employing several biochemical and cellular assays, here we show that Cpd12 is also active against cervical cancer cells transformed by other HR-HPV strains, such as HPV68 and HPV45, and against a HPV16-transformed head-and-neck cancer cell line, suggesting the possibility to employ Cpd12 as a targeted drug against a broad range of HPV-induced cancers. In these cancer cell lines, the antitumoral mechanism of action of Cpd12 involves p53-dependent cell cycle arrest, a senescent response, and inhibition of cancer cell migration. Finally, we show that Cpd12 can strongly synergize with taxanes and topoisomerase inhibitors, encouraging the evaluation of Cpd12 in preclinical studies for the targeted treatment of HPV-related carcinomas