Kaposi's Sarcoma-Associated Herpesvirus ORF57 Functions as a Viral Splicing Factor and Promotes Expression of Intron-Containing Viral Lytic Genes in Spliceosome-Mediated RNA Splicing

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 facilitates the expression of both intronless viral ORF59 genes and intron-containing viral K8 and K8.1 genes (V. Majerciak, N. Pripuzova, J. P. McCoy, S. J. Gao, and Z. M. Zheng, J. Virol. 81:1062-1071, 2007). In this study, we showed that disruption of ORF57 in a KSHV genome led to increased accumulation of ORF50 and K8 pre-mRNAs and reduced expression of ORF50 and K-bZIP proteins but had no effect on latency-associated nuclear antigen (LANA). Cotransfection of ORF57 and K8␤ cDNA, which retains a suboptimal intron of K8 pre-mRNA due to alternative splicing, promoted RNA splicing of K8␤ and production of K8␣ (K-bZIP). Although Epstein-Barr virus EB2, a closely related homolog of ORF57, had a similar activity in the cotransfection assays, herpes simplex virus type 1 ICP27 was inactive. This enhancement of RNA splicing by ORF57 correlates with the intact N-terminal nuclear localization signal motifs of ORF57 and takes place in the absence of other viral proteins. In activated KSHV-infected B cells, KSHV ORF57 partially colocalizes with splicing factors in nuclear speckles and assembles into spliceosomal complexes in association with low-abundance viral ORF50 and K8 pre-mRNAs and essential splicing components. The association of ORF57 with snRNAs occurs by ORF57-Sm protein interaction. We also found that ORF57 binds K8␤ pre-mRNAs in vitro in the presence of nuclear extracts. Collectively our data indicate that KSHV ORF57 functions as a novel splicing factor in the spliceosome-mediated splicing of viral RNA transcripts.Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is a human gammaherpesvirus that is closely related to Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) (7,41,45). KSHV infection is associated with all forms of Kaposi's sarcoma, primary effusion lymphoma or body cavity-based B-cell lymphoma, and multicentric Castleman disease (20,53,55,62). Latent KSHV infection in Kaposi's sarcoma tissues and B-cell lines features the restricted expression of only five viral genes (13, 74). The lytic KSHV infection produces progeny virus from infected cells and can be induced by chemicals such as tetradecanoyl phorbol acetate (43), butyrate (39), or valproic acid (25) or by hypoxia (12) in primary effusion lymphoma-derived B cells with latent KSHV infection. Chemical induction in latently infected cells initiates the expression of a viral transactivator, ORF50, which is essential for the switch from KSHV latency to the lytic phase (32, 57).KSHV ORF57 (mRNA transcript accumulation [MTA]), which is transactivated by ORF50 (31, 63), encodes a viral early nuclear protein of 455 amino acid (aa) residues (15, 24) that is homologous to herpes simplex virus (HSV) ICP27 (IE63), EBV EB2 (SM), and HVS ORF57. KSHV ORF57 promotes the expression of ORF56 and ORF59 genes at the posttranscriptional level (24,34,35), but how ORF57 functions is poorly understood. Previous reports indicated that HSV ICP27 mediates viral intronless RNA export (...

Caspase-7 Cleavage of Kaposi Sarcoma-associated Herpesvirus ORF57 Confers a Cellular Function against Viral Lytic Gene Expression

“…8B) from K8␤ transcripts (17). The reduction of K8␣ production from K8␤ transcripts indicates that the truncated mutant is deficient in promoting viral RNA splicing (13). When coexpressed with full-length ORF57, the truncated mutant in a small amount was suppressive for the full-length ORF57 activity in promotion of K8␤ RNA splicing (supplemental Fig.…”

“…Cells and KSHV Lytic Induction-KSHV latently infected primary effusion lymphoma cell lines BCBL-1 (EBV Ϫ ) and JSC-1 (EBV an ORF57 knock-out KSHV genome) (11) were cultivated as described (13). To induce ORF57 expression and virus lytic replication, KSHV-infected cells were treated with 0.6 mM valproate (VA), 0.3 mM butyrate, or 20 ng/ml of 12-O-tetradecanoylphorbol-13-acetate.…”

Identification of a response element in a herpesvirus saimiri mRNA recognized by the ORF57 protein

“…In addition, some spliced HVS RNAs also contain the core response element. This was initially surprising given the pivotal role of ORF57 in nuclear export of intronless HVS RNAs; however, recent analysis suggests that KSHV and EBV ORF57 homologues also function as splicing factors (Majerciak et al, 2008;Verma & Swaminathan, 2008). If HVS ORF57 has a similar role, this would also require a core response element within spliced HVS RNAs.…”

Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication

“…Moreover, BRLF2 protein interaction networks include a cluster of RNA splicing factors, many of which overlap RNA-processing proteins present in mLANA interaction networks described in this study (76). Perhaps ORF52 tethers ORF57 to mLANA through RNA-processing proteins, which are also engaged by ORF57 (62,76,77). Our inability to validate other "specific" interactions such as with ORF57 and M3 is consistent with the notion that such proteins engage mLANA by tethering through other proteins or viral DNA present only within infected cells.…”