Characterisation of the interactions between the KSHV ORF57 protein and the human TREX complex

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncovirus associated with multiple malignancies, including Kaposi’s sarcoma (KS). Like all herpesviruses, KSHV can establish either a latent or a lytic infection in host cells. During latency the virus remains in a dormant state, with limited gene expression. After reactivation, KSHV enters the lytic life cycle, characterised by production of infectious virions and subsequent dissemination of the virus from its latent reservoir. Importantly, lytic replication is critical for KS tumourigenesis. KSHV replicates in the nucleus of the host cell and requires cellular mRNA export factors to efficiently export viral mRNAs from the nucleus, allowing their translation in the cytoplasm. But while mammalian mRNA export is linked to splicing, the majority of the KSHV genome encodes intronless mRNAs, prompting the virus to circumvent this step. KSHV therefore expresses ORF57, a multifunctional protein essential for lytic replication. ORF57 recruits members of the cellular human transcription/export (hTREX) complex to form an export competent viral ribonucleoprotein particle (vRNP), facilitating efficient nuclear export of intronless KSHV mRNAs. This study presents a novel mechanism for the specific disruption of the vRNP and subsequent inhibition of virus lytic replication. Results suggest an ATP-cycle dependent remodelling of hTREX, which affects the ability of ORF57 to interact and form an export competent vRNP. Specifically, inhibiting ATP hydrolysis by the hTREX component and ATPase UAP56 prevents ORF57 from recruiting the hTREX complex, while key components of the endogenous complex remain able to interact. Following this finding, UAP56 was targeted by small molecule inhibitors, using a structure-based drug design approach, to prevent ATP hydrolysis and thereby inhibiting vRNP formation. Strikingly, a hit compound identified was shown to disrupt formation of the vRNP, lytic protein expression and infectious virion production, while allowing endogenous hTREX formation within a therapeutic window, where no cytotoxicity was observed. Bearing in mind the conserved mechanism for herpesvirus intronless mRNA export, this study finally presents a series of compounds that are able to prevent both KSHV and HSV-1 lytic replication.