Identification and Analysis of the K5 Gene of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes ϳ80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5 and 3 ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediateearly gene product; determination of its specific biological function requires further investigation.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

“…The very rapid disappearance of H-2D b in the presence of ␥HV-68 K3 was reminiscent of the effects of the US2 and US11 proteins of cytomegalovirus, both of which export HLA antigens from the endoplasmic reticulum to be degraded in the cytosol (32,33). Interestingly, the KSHV K5 protein is known to be expressed in the endoplasmic reticulum (34), where MHC class I͞␤ 2 -microglobulin͞peptide complexes are first assembled. Precisely how ␥HV-68 K3 and its homologs work has not yet been determined.…”

Autoregulation of MARCH1 Expression by Dimerization and Autoubiquitination

“…Interestingly, MARCH9 is capable of autoubiquitination and dimerizes with a RING-less splice variant, confirming the possible RING-independent dimer formation of ubiquitin ligases (25). Alternatively, the viral MIR1 and MIR2 also homodimerize but most likely via their transmembrane domains (26)(27)(28). In this study, we investigated the possible role of autoubiquitination in the posttranslational regulation of MARCH1 expression.…”

Split genes and their expression in Kaposi's sarcoma‐associated herpesvirus

“…As discussed above, not all exons in a split KSHV gene have coding potential. Some are leader exons preceding a coding exon, as can be found in the ORF 73/72/K13 [43][44][45][46] and K5 transcripts [48]. Others are terminal exons that make the transcripts accessible to a proximal pA signal for RNA processing.…”