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Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication.

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.

SUBMITTER: Boyne JR

PROVIDER: S-EPMC2569588 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication.

Boyne James R JR Colgan Kevin J KJ Whitehouse Adrian A

PLoS pathogens 20081031 10

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 ...[more]

PMID: 18974867

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human gammaherpesvirus and the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). During reactivation, viral genes are expressed in a temporal manner. These lytic genes encode transactivators, core replication proteins, or structural proteins. During reactivation, other viral factors that are required for lytic replication are expressed. The most abundant viral transcript is the long noncoding RNA (lncRNA) known as polyadenylated nuclear (PAN) RNA. lncRNAs have diverse functions, including the regulation of gene expression and the immune response. PAN possesses two main cis-acting elements, the Mta response element (MRE) and the expression and nuclear retention element (ENE). While PAN has been demonstrated to be required for efficient viral replication, the function of these elements within PAN remains unclear. Our goal was to determine if the ENE of PAN is required in the context of infection. A KSHV bacmid containing a deletion of the 79-nucleotide (nt) ENE in PAN was generated to assess the effects of the ENE during viral replication. Our studies demonstrated that the ENE is not required for viral DNA synthesis, lytic gene expression, or the production of infectious virus. Although the ENE is not required for viral replication, we found that the ENE functions to retain PAN in the nucleus, and the absence of the ENE results in an increased accumulation of PAN in the cytoplasm. Furthermore, open reading frame 59 (ORF59), LANA, ORF57, H1.4, and H2A still retain the ability to bind to PAN in the absence of the ENE. Together, our data highlight how the ENE affects the nuclear retention of PAN but ultimately does not play an essential role during lytic replication. Our data suggest that PAN may have other functional domains apart from the ENE. IMPORTANCE KSHV is an oncogenic herpesvirus that establishes latency and exhibits episodes of reactivation. KSHV disease pathologies are most often associated with the lytic replication of the virus. PAN RNA is the most abundant viral transcript during the reactivation of KSHV and is required for viral replication. Deletion and knockdown of PAN resulted in defects in viral replication and reduced virion production in the absence of PAN RNA. To better understand how the cis elements within PAN may contribute to its function, we investigated if the ENE of PAN was necessary for viral replication. Although the ENE had previously been extensively studied with both biochemical and in vitro approaches, this is the first study to demonstrate the role of the ENE in the context of infection and that the ENE of PAN is not required for the lytic replication of KSHV.

Dataset Information

Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication.

Publications

Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication.

Similar Datasets

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