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Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 3 inhibits gamma interferon and major histocompatibility complex class II expression.

ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) carries four genes with homology to human interferon regulatory factors (IRFs). One of these IRFs, the viral interferon regulatory factor 3 (vIRF-3), is expressed in latently infected primary effusion lymphoma (PEL) cells and required for their continuous proliferation. Moreover, vIRF-3 is known to be involved in modulation of the type I interferon (IFN) response. We now show that vIRF-3 also interferes with the type II interferon system and antigen presentation to the adaptive immune system. Starting with an analysis of the transcriptome, we show that vIRF-3 inhibits expression of major histocompatibility complex class II (MHC II) molecules: small interfering RNA (siRNA)-mediated knockdown of vIRF-3 in KSHV-infected PEL cell lines resulted in increased MHC II levels; overexpression of vIRF-3 in KSHV-negative B cells leads to downmodulation of MHC II. This regulation could be traced back to inhibition of class II transactivator (CIITA) transcription by vIRF-3. Reporter assays revealed that the gamma interferon (IFN-?)-sensitive CIITA promoters PIV and PIII were inhibited by vIRF-3. Consistently, IFN-? levels increased upon vIRF-3 knockdown in PEL cells. IFN-? regulation by vIRF-3 was confirmed in reporter assays as well as by upregulation of typical IFN-? target genes upon knockdown of vIRF-3 in PEL cells. In summary, we conclude that vIRF-3 contributes to the viral immunoevasion by downregulation of IFN-? and CIITA and thus MHC II expression.

SUBMITTER: Schmidt K

PROVIDER: S-EPMC3126280 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 3 inhibits gamma interferon and major histocompatibility complex class II expression.

Schmidt Katharina K Wies Effi E Neipel Frank F

Journal of virology 20110223 9

Kaposi's sarcoma-associated herpesvirus (KSHV) carries four genes with homology to human interferon regulatory factors (IRFs). One of these IRFs, the viral interferon regulatory factor 3 (vIRF-3), is expressed in latently infected primary effusion lymphoma (PEL) cells and required for their continuous proliferation. Moreover, vIRF-3 is known to be involved in modulation of the type I interferon (IFN) response. We now show that vIRF-3 also interferes with the type II interferon system and antigen ...[more]

PMID: 21345951

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Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor (vIRF) that functions as a repressor for interferon-mediated signal transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5' rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box, but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cell's interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate beta-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its transactivation by ORF 50.

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: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

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 3 inhibits gamma interferon and major histocompatibility complex class II expression.

Publications

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 3 inhibits gamma interferon and major histocompatibility complex class II expression.

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