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

Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity.

Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined. The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus. Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75%. The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry. On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered. The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors. Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells

Daclatasvir plasma level and resistance selection in HIV patients with hepatitis C virus cirrhosis treated with daclatasvir, sofosbuvir, and ribavirin

ObjectivesEffective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV–HCV co-infected patients.MethodsThis study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV–HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing.ResultsDCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA <12 IU/ml at week 12 and undetectable at week 16.ConclusionsSub-optimal DCV drug levels allow the selection of resistance-associated variants and fail to contribute to antiviral activity. No definite reason for the low DCV level was found. Quantifying the drug is suggested in difficult-to-treat patients

Human neural progenitor cell models to study the antiviral effects and neuroprotective potential of approved and investigational human cytomegalovirus inhibitors

: Human cytomegalovirus (HCMV) is the viral leading cause of congenital defects in newborns worldwide. Many aspects of congenital CMV (cCMV) infection, which currently lacks a specific treatment, as well as the main determinants of neuropathogenesis in the developing brain during HCMV infection are unclear. In this study, we modeled HCMV infection at different stages of neural development. Moreover, we evaluated the effects of both approved and investigational anti-HCMV drugs on viral replication and gene expression in two different neural progenitor cell lines, i.e., human embryonic stem cells-derived neural stem cells (NSCs) and fetus-derived neuroepithelial stem (NES) cells. Ganciclovir, letermovir, nitazoxanide, and the ozonide OZ418 reduced viral DNA synthesis and the production of infectious virus in both lines of neural progenitors. HCMV infection dysregulated the expression of genes that either are markers of neural progenitors, such as SOX2, NESTIN, PAX-6, or play a role in neurogenesis, such as Doublecortin. Treatment with antiviral drugs had different effects on HCMV-induced dysregulation of the genes under investigation. This study contributes to the understanding of the molecular mechanisms of cCMV neuropathogenesis and paves the way for further consideration of anti-HCMV drugs as candidate therapeutic agents for the amelioration of cCMV-associated neurological manifestations

Exploring a New Generation of Pyrimidine and Pyridine Derivatives as Anti-Influenza Agents Targeting the Polymerase PA–PB1 Subunits Interaction

The limited range of available flu treatments due to virus mutations and drug resistance have prompted the search for new therapies. RNA-dependent RNA polymerase (RdRp) is a heterotrimeric complex of three subunits, i.e., polymerase acidic protein (PA) and polymerase basic proteins 1 and 2 (PB1 and PB2). It is widely recognized as one of the most promising anti-flu targets because of its critical role in influenza infection and high amino acid conservation. In particular, the disruption of RdRp complex assembly through protein-protein interaction (PPI) inhibition has emerged as a valuable strategy for discovering a new therapy. Our group previously identified the 3-cyano-4,6-diphenyl-pyridine core as a privileged scaffold for developing PA-PB1 PPI inhibitors. Encouraged by these findings, we synthesized a small library of pyridine and pyrimidine derivatives decorated with a thio-N-(m-tolyl)acetamide side chain (compounds 2a-n) or several amino acid groups (compounds 3a-n) at the C2 position. Interestingly, derivative 2d, characterized by a pyrimidine core and a phenyl and 4-chloro phenyl ring at the C4 and C6 positions, respectively, showed an IC50 value of 90.1 mu M in PA-PB1 ELISA, an EC50 value of 2.8 mu M in PRA, and a favorable cytotoxic profile, emerging as a significant breakthrough in the pursuit of new PPI inhibitors. A molecular modeling study was also completed as part of this project, allowing us to clarify the biological profile of these compounds

A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer

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