Identification of the DNA Sequence Interacting with Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 1

The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a ⌬K3 ⌬K5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1␣-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFP high and pK8.1-driven blue fluorescent proteinpositive (BFP ؉ ) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages. IMPORTANCEAlthough the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.

Section: Discussionmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have Stage-Specific Immune Evasion Roles during Lytic Replication

Brulois

Tóth

Wong

et al. 2014

“…The N terminus of vIRF4 is predicted to fold into a helical structure resembling the winged helix DBD of cellular IRFs, and even though it lacks some of the critical tyrosine residues, substitutions in the putative DNA recognition helix (helix 3) interfere with its transcription function. It is worth emphasizing that the equivalent region of vIRF1 has also diverged from the IRF pattern and yet binds to palindromic DNA sequences identified through an in vitro binding site selection assay (37). As might be expected, the consensus vIRF1-binding sequence (5=-GCGTCnnGACGC-3=, where n is any nucleotide) shares no obvious similarity to cellular IFN-stimulated response elements (ISREs) (5=-GAAAnnGAAAC T-3=) (13), providing a strong hint that the vIRFs may have evolved to recognize their own unique response elements.…”

Section: Discussionmentioning

confidence: 99%

Cooperation between Viral Interferon Regulatory Factor 4 and RTA To Activate a Subset of Kaposi's Sarcoma-Associated Herpesvirus Lytic Promoters

Persson

O’Brien

et al. 2012

The four Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded interferon (IFN) regulatory factor homologues (vIRF1 to vIRF4) are used to counter innate immune defenses and suppress p53. The vIRF genes are arranged in tandem but differ in function and expression. In KSHV-infected effusion lymphoma lines, K10.5/vIRF3 and K11/vIRF2 mRNAs are readily detected during latency, whereas K9/vIRF1 and K10/vIRF4 mRNAs are upregulated during reactivation. Here we show that the K10/vIRF4 promoter responds to the lytic switch protein RTA in KSHV-infected cells but is essentially unresponsive in uninfected cells. Coexpression of RTA with vIRF4 is sufficient to restore regulation, a property not shared by other vIRFs. The K9/vIRF1 promoter behaves similarly, and production of infectious virus is enhanced by the presence of vIRF4. Synergy requires the DNAbinding domain (DBD) and C-terminal IRF homology regions of vIRF4. Mutations of arginine residues within the putative DNA recognition helix of vIRF4 or the invariant cysteines of the adjacent CxxC motif abolish cooperation with RTA, in the latter case by preventing self-association. The oligomerization and transactivation functions of RTA are also essential for synergy. The K10/ vIRF4 promoter contains two transcription start sites (TSSs), and a 105-bp fragment containing the proximal promoter is responsive to vIRF4/RTA. Binding of a cellular factor(s) to this fragment is altered when both viral proteins are present, suggesting a possible mechanism for transcriptional synergy. Reliance on coregulators encoded by either the host or viral genome provides an elegant strategy for expanding the regulatory potential of a master regulator, such as RTA.

“…Indeed, B cell hyperplasia is thought to be driven by vIL-6, a functional homologue of its cellular counterpart (25,(36)(37)(38). Previous studies showed that KSHV vIRF-1 can bind to the KSHV vIL-6 promoter and drive its transcription in latently infected PEL cells in vitro (39). Therefore, if vIL-6 expression is even partially controlled by a vIRF(s) during RRV infection, this may be contributing to the decreased B cell hyperplasia following vIRF-ko RRV infection.…”

Section: Discussionmentioning

confidence: 99%

Viral Interferon Regulatory Factors Are Critical for Delay of the Host Immune Response against Rhesus Macaque Rhadinovirus Infection

Robinson

O’Connor

et al. 2012

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a gamma-2 gammaherpesvirus that was identified in 1994 to be the cause of AIDS-related KS (11), one of the most common malignancies within human immunodeficiency virus (HIV)-infected individuals. KSHV establishes a persistent infection within B cells (2,14) and is also associated with the malignant B cell disorders, multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL) (9, 48). Patients who develop MCD and PEL often have a poor outcome, especially if these B cell disorders develop before the introduction of highly active antiretroviral therapy (HAART) (7). Moreover, recent studies have documented an increase in KS within individuals on long-term therapy, despite maintenance of low HIV loads and high CD4 T cell numbers (31). These findings now suggest that the development of KSHV-associated diseases is dependent on other unidentified factors and not simply just immune suppression.Studying host-pathogen interactions during de novo KSHV infection and disease pathogenesis has been hindered by the scarcity of animal models that recapitulate the human disease (10, 16, 42). Rhesus macaque (RM) rhadinovirus (RRV) is a closely related gammaherpesvirus (1, 13, 15, 46) that naturally infects RMs and establishes latency within B cells (4). Moreover, RRV infection of RMs induces an acute hyperproliferation of B cells (17, 54) that often develops into diseases that resemble non-Hodgkin's lymphoma and MCD in immune-compromised animals (37). The striking similarities between KSHV-and RRV-associated pathologies (4,17,37,54), along with the nearly colinear genomic organization (1, 46), make RRV infection of RMs an ideal model for studying KSHV disease. Moreover, recent generation and characterization of a bacterial artificial chromosome (BAC) clone of RRV isolate 17577 (wild-type BAC-derived [WT BAC ] RRV 17577 ) (17) allow specific genes to be targeted for deletion to effectively address their roles during infection.KSHV and RRV encode a number of viral homologues of cellular genes involved in immune signaling, apoptosis, and cellular growth and differentiation. Accordingly, these viral homologues play critical roles in subverting the immune response (3). In particular, KSHV and RRV both encode a cluster of viral interferon ( (5, 6, 18-20, 24, 29, 52, 55). Furthermore, KSHV vIRF-1 displayed tumorigenic potential in NIH 3T3 cells and nude mice (20), and KSHV vIRF-1, -3, and -4 independently disrupt p53 function, inhibiting p53-induced apoptosis and/or cell cycle control (28,34,43,47). Additional antiapoptotic functions have also been attributed to KSHV vIRF-1 and vIRF-3. For example, KSHV vIRF-1 binds and sequesters the proapoptotic protein Bim, reducing levels of apoptosis (12), and RNA interference knockdown of KSHV vIRF-3 results in increased activity of effector caspases in KSHV-infected PEL cells (53). Collectively, these data illustrate the functional breadth and diversity of pathways/cellular functions that are targeted by the vIRFs, but these da...