James R. Boyne

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

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 Kaposis 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.

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription/export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA.

The prototype γ-2 herpesvirus nucleocytoplasmic shuttling protein, ORF 57, transports viral RNA through the cellular mRNA export pathway

HVS (herpesvirus saimiri) is the prototype gamma-2 herpesvirus. This is a subfamily of herpesviruses gaining importance since the identification of the first human gamma-2 herpesvirus, Kaposis sarcoma-associated herpesvirus. The HVS ORF 57 (open reading frame 57) protein is a multifunctional transregulatory protein homologous with genes identified in all classes of herpesviruses. Recent work has demonstrated that ORF 57 has the ability to bind viral RNA, shuttles between the nucleus and cytoplasm and promotes the nuclear export of viral transcripts. In the present study, we show that ORF 57 shuttles between the nucleus and cytoplasm in a CRM-1 (chromosomal region maintenance 1)-independent manner. ORF 57 interacts with the mRNA export factor REF (RNA export factor) and two other components of the exon junction complex, Y14 and Magoh. The association of ORF 57 with REF stimulates recruitment of the cellular mRNA export factor TAP (Tip-associated protein), and HVS infection triggers the relocalization of REF and TAP from the nuclear speckles to several large clumps within the cell. Using a dominant-negative form of TAP and RNA interference to deplete TAP, we show that it is essential for bulk mRNA export in mammalian cells and is required for ORF 57-mediated viral RNA export. Furthermore, we show that the disruption of TAP reduces viral replication. These results indicate that HVS utilizes ORF 57 to recruit components of the exon junction complex and subsequently TAP to promote viral RNA export through the cellular mRNA export pathway.

Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs.

Kaposis sarcoma‐associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing‐dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export‐competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon‐junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre‐initiation complex and co‐sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre‐initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

An interaction between KSHV ORF57 and UIF provides mRNA-adaptor redundancy in herpesvirus intronless mRNA export.

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposis sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs.

Nucleolar disruption impairs Kaposi's sarcoma-associated herpesvirus ORF57-mediated nuclear export of intronless viral mRNAs

MINT‐7291715: ORF57 (uniprotkb:Q2HR75) and Aly (uniprotkb:Q86V81) colocalise (MI:0403) by fluorescence microscopy (MI:0416)

Viral nucleolar localisation signals determine dynamic trafficking within the nucleolus

Abstract Localisation of both viral and cellular proteins to the nucleolus is determined by a variety of factors including nucleolar localisation signals (NoLSs), but how these signals operate is not clearly understood. The nucleolar trafficking of wild type viral proteins and chimeric proteins, which contain altered NoLSs, were compared to investigate the role of NoLSs in dynamic nucleolar trafficking. Three viral proteins from diverse viruses were selected which localised to the nucleolus; the coronavirus infectious bronchitis virus nucleocapsid (N) protein, the herpesvirus saimiri ORF57 protein and the HIV-1 Rev protein. The chimeric proteins were N protein and ORF57 protein which had their own NoLS replaced with those from ORF57 and Rev proteins, respectively. By analysing the sub-cellular localisation and trafficking of these viral proteins and their chimeras within and between nucleoli using confocal microscopy and photo-bleaching we show that NoLSs are responsible for different nucleolar localisations and trafficking rates.

Herpesvirus Saimiri-Based Gene Delivery Vectors

Herpesviruses possess a number of characteristics which make them promising gene delivery vectors. These include their capacity to package large amounts of heterologous DNA and an ability to establish persistent, lifelong infections, where the viral genome remains as a circular non-integrated episome. Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus and is currently being developed as a potential gene delivery vector. In addition to the above properties, HVS-based vectors have the ability to infect a wide range of human cell lines and primary cultures with high efficiencies. Moreover, upon infection the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny cells upon division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained heterologous gene expression. As such, it offers the characteristics of an artificial chromosome combined with a highly efficient delivery system. This review aims to describe the assessment of HVS-based vectors in both in vitro and in vivo studies, highlighting new developments and possible applications for the treatment of genetic diseases.

Mutation of a C-Terminal Motif Affects Kaposi's Sarcoma-Associated Herpesvirus ORF57 RNA Binding, Nuclear Trafficking, and Multimerization

ABSTRACT The Kaposis sarcoma-associated herpesvirus (KSHV) ORF57 protein is essential for virus lytic replication. ORF57 regulates virus gene expression at multiple levels, enhancing transcription, stability, nuclear export, and translation of viral transcripts. To enhance the nuclear export of viral intronless transcripts, ORF57 (i) binds viral intronless mRNAs, (ii) shuttles between the nucleus, nucleolus, and the cytoplasm, and (iii) interacts with multiple cellular nuclear export proteins to access the TAP-mediated nuclear export pathway. We investigated the implications on the subcellular trafficking, cellular nuclear export factor recruitment, and ultimately nuclear mRNA export of an ORF57 protein unable to bind RNA. We observed that mutation of a carboxy-terminal RGG motif, which prevents RNA binding, affects the subcellular localization and nuclear trafficking of the ORF57 protein, suggesting that it forms subnuclear aggregates. Further analysis of the mutant shows that although it still retains the ability to interact with cellular nuclear export proteins, it is unable to export viral intronless mRNAs from the nucleus. Moreover, computational molecular modeling and biochemical studies suggest that, unlike the wild-type protein, this mutant is unable to self-associate. Therefore, these results suggest the mutation of a carboxy-terminal RGG motif affects ORF57 RNA binding, nuclear trafficking, and multimerization.

ORF57: Master regulator of KSHV mRNA biogenesis

Comment on: Boyne JR, et al. EMBO J 2010; 29:1851-64.