Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage

Abstract

The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease.IMPORTANCE Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.KEYWORDS human cytomegalovirus, terminase, pUL89 inhibitor, hydroxypyridonecarboxylic acid, metal chelation H uman cytomegalovirus (HCMV) infection is a significant cause of morbidity and mortality in immunocompromised patients, including transplant recipients and AIDS patients (reviewed in reference 1). In addition, HCMV infects 1% of all newborns and is a leading cause of brain damage and hearing loss (2). Currently, most of the U.S. Food and Drug Administration (FDA)-approved anti-HCMV drugs target the viral polymerase, including ganciclovir (GCV), valganciclovir, cidofovir, and foscarnet (reviewed in

Comparison of Cytomegalovirus Terminase Gene Mutations Selected after Exposure to Three Distinct Inhibitor Compounds

“…Colored residues 463, 534, and 651 in the nuclease domain are involved in metal ion coordination (12). molecules may effectively target the UL89 nuclease domain independently of the terminase inhibitors studied here (15,16).…”

Human herpesvirus portal proteins: Structure, function, and antiviral prospects

“…WAY‐150138 showed modest activity (13 μM) against HCMV (Table ), but the CMV portal was not experimentally confirmed to be the target. Although no compounds currently target pUL104, several compounds have been described that prevent encapsidation by inhibiting the viral terminase . Letermovir, a CMV‐specific terminase inhibitor in late stage development, showed no cross resistance with viral strains resistant to currently available drugs and few side effects in clinical trials .…”

“…The portal oligomer serves as a docking site for the encapsidation motor or terminase complex. Viral terminases possess ATPase and endonuclease activities and provide the necessary force to pack the increasingly rigid dsDNA into the capsid under pressure . DNA, though initially flexible, becomes semicrystalline as it fills the capsid .…”

Modern Ethiotropic Chemotherapy of Human Cytomegalovirus Infection: Clinical Effectiveness, Molecular Mechanism of Action, Drug Resistance, New Trends and Prospects. Part 1

“…В структуре рUL86 установлен домен, имеющий близкую структуру с РНКазаН-каталитическим сайтом обратной транскриптазы (ОТ) ВИЧ-1 и проявляющий РНКазаН/ интегразаподобную активность. РНКазный каталитический сайт ВИЧ-1 включает 4 аминокислотных остатка Asp443, Asp498, Glu478 и Asp549, координирующих 2 катиона Mg 2+ , а эндонуклеазный каталитический сайт pUL86 сформирован Asp463, Asp651, Glu534 и Ala465, которые координируют 2 катиона Mn 2+ [47,48]. Поэтому представляет очевидный интерес поиск структурных аналогов РТВ с более высокой антивирусной активностью.…”

“…3). Показано, что биомишенью для 10k является pUL86: в присутствии 10k ингибируется эндонуклеазная активность pUL86 и нарезание вирусной конкатемерной ДНК на единичные геномы [48].…”