A small molecule exerts selective antiviral activity by targeting the human cytomegalovirus nuclear egress complex.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV. Author summary: Human cytomegalovirus (HCMV) causes severe diseases in newborns and in adults with compromised immunity. With no available vaccine for this virus, and with the drawbacks of existing anti-HCMV drugs, there is a pressing need for new effective treatments. HCMV, like other herpesviruses, uses an unusual process called nuclear egress by which viral capsids exit the nucleus. The interaction of two HCMV proteins plays a crucial role in nuclear egress. To investigate the possibility that a small compound can inhibit production of infectious HCMV by specifically targeting this interaction, we screened ~200,000 chemicals and found two related compounds that specifically inhibit the interaction of the two proteins in biochemical assays and exert anti-HCMV activity in cells. Both the inhibition of the interaction and the antiviral activity of at least one of the compounds depend upon the compounds binding to a specific location on one of the viral proteins. Our results provide compelling evidence that targeting these proteins is a valid strategy for developing new anti-HCMV drugs. The compounds that we identified might serve as a starting point for development of such drugs and as tools for studying the virus. [ABSTRACT FROM AUTHOR]