Divide et impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication.

Hanieh Ghassabian,Federico Falchi,Gualtiero Alvisi,Martina Timmoneri,Arianna Loregian,Beatrice Mercorelli,Giorgio Palù

doi:10.3390/v13050941

Hanieh Ghassabian, Federico Falchi + Show 5 more

Open Access

https://doi.org/10.3390/v13050941

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Abstract

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited HCMV AD169 strain in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

Highlights

The β-Herpesvirinae member human cytomegalovirus (HCMV) is a major human pathogen, causing severe and life-threatening infections in immunocompromised subjects [1] and in congenitally infected newborns [2]
The molecular mechanisms involved in herpesvirus DNA replication and its regulation have been widely studied as they provide important models for the study of eukaryotic DNA replication and because viral enzymes involved in the process represent targets for antiviral therapy
We aimed at identifying small molecules (SMs) able to interfere with ppUL44 selfinteraction

Summary

Introduction

The β-Herpesvirinae member human cytomegalovirus (HCMV) is a major human pathogen, causing severe and life-threatening infections in immunocompromised subjects [1] and in congenitally infected newborns [2]. HCMV DNA polymerase holoenzyme is a multi-functional enzyme that plays a key role during viral infection ensuring replication of the viral genome, and consists of the catalytic subunit pUL54 and the processivity factor ppUL44, which physically and functionally interact thought specific residues [4,5,6]. The most widely antiviral agents used to fight HCMV infections target pUL54 and are either nucleoside or pyrophosphate analogues such as ganciclovir (GCV) or foscarnet (PAA), respectively [7]. Long-term administration of these antiviral agents frequently leads to the selection of viral isolates with reduced drug susceptibility, due to mutations of either pUL54 or of pUL97, the viral kinase phosphorylating GCV [8,9]. There is a recognized need for novel anti-HCMV compounds that target other viral functions [14]

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