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Functional analysis of Kaposi's sarcoma-associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation.

ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contributes to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NF?B pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. Here we show that a 252-nt fragment directly upstream of vFLIP, within a coding region, directs translation. We have established its RNA structure and demonstrate that IRES activity requires the presence of eIF4A and an intact eIF4G. Furthermore, and unusually for an IRES, eIF4E is part of the complex assembled onto the vFLIP IRES to direct translation. These molecular interactions define a new paradigm for IRES-mediated translation.

SUBMITTER: Othman Z

PROVIDER: S-EPMC4201831 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Functional analysis of Kaposi's sarcoma-associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation.

Othman Zulkefley Z Sulaiman Mariam K MK Willcocks Margaret M MM Ulryck Nathalie N Blackbourn David J DJ Sargueil Bruno B Roberts Lisa O LO Locker Nicolas N

RNA (New York, N.Y.) 20140922 11

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contributes to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFκB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level ...[more]

PMID: 25246653

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Project description:UnlabelledKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countries-where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap since it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment protocol followed by Illumina deep-sequencing, we generated and analyzed 16 unique Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting highly confident polymorphism analysis. Multiple alignment of the 16 newly sequenced KSHV genomes showed low level variability across the entire central conserved region. This variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic sequences and those derived from Western countries. Importantly, the phylogenetic segregation of Zambian from Western sequences occurred irrespective of inclusion of the highly variable genes K1 and K15. We also show that four genes within the more conserved region of the KSHV genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole KSHV genome should be taken into consideration for accurate viral characterization.ImportanceOur results represent the largest number of KSHV whole-genomic sequences published to date and the first time that multiple genomes have been sequenced from sub-Saharan Africa, a geographic area where KS is highly endemic. Based on our new sequence data, it is apparent that whole-genome KSHV diversity is greater than previously appreciated and differential phylogenetic clustering exists between viral genomes of Zambia and Western countries. Furthermore, individual genes may be insufficient for KSHV genetic characterization. Continued investigation of the KSHV genetic landscape is necessary in order to effectively understand the role of viral evolution and sequence diversity on KSHV gene functions and pathogenesis.

Project description:UnlabelledThe human oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a set of ∼20 viral microRNAs (miRNAs). miR-K10a stands out among these miRNAs because its entire stem-loop precursor overlaps the coding sequence for the Kaposin (Kap) A/C proteins. The ectopic expression of KapA has been reported to lead to transformation of rodent fibroblasts. However, these experiments inadvertently also introduced miR-K10a, which raises the question whether the transforming activity of the locus could in fact be due to miR-K10a expression. To answer this question, we have uncoupled miR-K10a and KapA expression. Our experiments revealed that miR-K10a alone transformed cells with an efficiency similar to that when it was coexpressed with KapA. Maintenance of the transformed phenotype was conditional upon continued miR-K10a but not KapA protein expression, consistent with its dependence on miRNA-mediated changes in gene expression. Importantly, miR-K10a taps into an evolutionarily conserved network of miR-142-3p targets, several of which are expressed in 3T3 cells and are also known inhibitors of cellular transformation. In summary, our studies of miR-K10a serve as an example of an unsuspected function of an mRNA whose precursor is embedded within a coding transcript. In addition, our identification of conserved miR-K10a targets that limit transformation will point the way to a better understanding of the role of this miRNA in KSHV-associated tumors.ImportanceKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus. The viral Kaposin locus has known oncogenic potential, which has previously been attributed to the encoded KapA protein. Here we show that the virally encoded miR-K10a miRNA, whose precursor overlaps the KapA-coding region, may account for the oncogenic properties of this locus. Our data suggest that miR-K10a mimics the cellular miRNA miR-142-3p and thereby represses several known inhibitors of oncogenic transformation. Our work demonstrates that functional properties attributed to a coding region may in fact be carried out by an embedded noncoding element and sheds light on the functions of viral miR-K10a.

Project description:Studies on "hit-and-run" effects by viral proteins are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled identification of both static and dynamic protein-protein interactions. In this study, we applied a PL method by generating recombinant Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV, a gammaherpesvirus, uniquely encodes four interferon regulatory factors (IRF-1 to -4) that suppress host interferon responses, and we examined KSHV IRF-1 and IRF-4 neighbor proteins to identify cellular proteins involved in innate immune regulation. PL identified 213 and 70 proteins as neighboring proteins of viral IRF-1 (vIRF-1) and vIRF-4 during viral reactivation, and 47 proteins were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological roles such as mRNA processing and transcriptional regulation by TP53. Innate immune regulation by these commonly interacting 44 cellular proteins was examined with small interfering RNAs (siRNAs), and the splicing factor 3B family proteins were found to be associated with interferon transcription and to act as suppressors of KSHV reactivation. We propose that recombinant mini-TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication and that selective splicing factors have a function in the regulation of innate immune responses.IMPORTANCE Viral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to the use of affinity precipitation approaches, the majority of studies have focused on how the virus takes advantage of the newly formed protein interactions for its own replication. Proximity labeling (PL), however, can also highlight transient and negative effects-those interactions which lead to dissociation from the existing protein complex. Here, we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

Dataset Information

Functional analysis of Kaposi's sarcoma-associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation.

Publications

Functional analysis of Kaposi's sarcoma-associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation.

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