Brian R. Jackson

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.

Merkel cell polyomavirus small T antigen targets the NEMO adaptor protein to disrupt inflammatory signaling.

ABSTRACT Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKβ-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aβ, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell.

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.

A novel mechanism inducing genome instability in Kaposi's sarcoma-associated herpesvirus infected cells.

Kaposis sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies. Like other herpesviruses, KSHV has a biphasic life cycle and both the lytic and latent phases are required for tumorigenesis. Evidence suggests that KSHV lytic replication can cause genome instability in KSHV-infected cells, although no mechanism has thus far been described. A surprising link has recently been suggested between mRNA export, genome instability and cancer development. Notably, aberrations in the cellular transcription and export complex (hTREX) proteins have been identified in high-grade tumours and these defects contribute to genome instability. We have previously shown that the lytically expressed KSHV ORF57 protein interacts with the complete hTREX complex; therefore, we investigated the possible intriguing link between ORF57, hTREX and KSHV-induced genome instability. Herein, we show that lytically active KSHV infected cells induce a DNA damage response and, importantly, we demonstrate directly that this is due to DNA strand breaks. Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage. Moreover, we describe a novel mechanism showing that the genetic instability observed is a consequence of R-loop formation. Importantly, the link between hTREX sequestration and DNA damage may be a common feature in herpesvirus infection, as a similar phenotype was observed with the herpes simplex virus 1 (HSV-1) ICP27 protein. Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX.

Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

NEDDylation Is Essential for Kaposi's Sarcoma-Associated Herpesvirus Latency and Lytic Reactivation and Represents a Novel Anti-KSHV Target

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposis sarcoma (KS) and primary effusion lymphoma (PEL), which are aggressive malignancies associated with immunocompromised patients. For many non-viral malignancies, therapeutically targeting the ubiquitin proteasome system (UPS) has been successful. Likewise, laboratory studies have demonstrated that inhibition of the UPS might provide a promising avenue for the treatment of KSHV-associated diseases. The largest class of E3 ubiquitin ligases are the cullin-RING ligases (CRLs) that are activated by an additional ubiquitin-like protein, NEDD8. We show that pharmacological inhibition of NEDDylation (using the small molecule inhibitor MLN4924) is cytotoxic to PEL cells by inhibiting NF-κB. We also show that CRL4B is a novel regulator of latency as its inhibition reactivated lytic gene expression. Furthermore, we uncovered a requirement for NEDDylation during the reactivation of the KSHV lytic cycle. Intriguingly, inhibition prevented viral DNA replication but not lytic cycle-associated gene expression, highlighting a novel mechanism that uncouples these two features of KSHV biology. Mechanistically, we show that MLN4924 treatment precluded the recruitment of the viral pre-replication complex to the origin of lytic DNA replication (OriLyt). These new findings have revealed novel mechanisms that regulate KSHV latency and reactivation. Moreover, they demonstrate that inhibition of NEDDylation represents a novel approach for the treatment of KSHV-associated malignancies.

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.

The Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Its Multiple Roles in mRNA Biogenesis

Post-transcriptional events which regulate mRNA biogenesis are fundamental to the control of gene expression. A nascent mRNA is therefore steered through multimeric RNA–protein complexes that mediate its capping, splicing, polyadenylation, nuclear export, and ultimately its translation. Kaposi’s sarcoma-associated herpesvirus (KSHV) mRNA transport and accumulation protein, or ORF57, is a functionally conserved protein found in all herpesviruses which plays a pivotal role in enhancing viral gene expression at a post-transcriptional level. As such, ORF57 has been implicated in multiple steps of RNA biogenesis, including augmenting viral splicing, protecting viral RNAs from degradation to enhancing viral mRNA nuclear export and translation. In this review, we highlight the multiple roles of KSHV ORF57 in regulating the post-transcriptional events which are fundamental to the control of virus gene expression.

ORF57: Master regulator of KSHV mRNA biogenesis

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

Targeting the ATP-dependent formation of herpesvirus ribonucleoprotein particle assembly as an antiviral approach

Human herpesviruses are responsible for a range of debilitating acute and recurrent diseases, including a number of malignancies. Current treatments are limited to targeting the herpesvirus DNA polymerases, but with emerging viral resistance and little efficacy against the oncogenic herpesviruses, there is an urgent need for new antiviral strategies. Here, we describe a mechanism to inhibit the replication of the oncogenic herpesvirus Kaposis sarcoma-associated herpesvirus (KSHV), by targeting the ATP-dependent formation of viral ribonucleoprotein particles (vRNPs). We demonstrate that small-molecule inhibitors which selectively inhibit the ATPase activity of the cellular human transcription/export complex (hTREX) protein UAP56 result in effective inhibition of vRNP formation, viral lytic replication and infectious virion production. Strikingly, as all human herpesviruses use conserved mRNA processing pathways involving hTREX components, we demonstrate the feasibility of this approach for pan-herpesvirus inhibition.