Recruitment of the Complete hTREX Complex Is Required for Kaposi's Sarcoma–Associated Herpesvirus Intronless mRNA Nuclear Export and Virus Replication

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Abstract
A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma–associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production. Following gene expression in the nucleus, newly transcribed messenger RNA (mRNA) is exported to the cytoplasm, where it is translated into protein. In mammals the vast majority of mRNAs contain introns that must be removed by the spliceosome prior to nuclear export. In addition to excising introns, splicing is also essential for the recruitment of a several protein complexes to mRNA, one example being the human transcription/export complex, which is required for mRNA export. Herpesviruses, such as Kaposi's sarcoma–associated herpesvirus, replicate by hijacking components of the host cells biological machinery, including those proteins necessary for mRNA export. An intriguing caveat in herpesvirology is that herpesviruses, such as Kaposi's sarcoma–associated herpesvirus, produce some mRNAs that lack introns and do not undergo splicing. How then are these intronless mRNAs exported to the cytoplasm? The answer lies in a virus protein called ORF57 that is able to bind to the intronless mRNA and then export them to the cytoplasm. ORF57 achieves this function by mimicking splicing and recruiting the human transcription/export complex to the intronless viral mRNA, thus facilitating its export into the cytoplasm.

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