Identification and analysis of the K5 gene of Kaposi's sarcoma-associated herpesvirus.
Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes approximately 80 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 immediate-early gene product; determination of its specific biological function requires further investigation.
Haque
M
M
Department of Microbiology, Osaka University Medical School C1, 2-2 Yamadaoka, Suita 565-0871, Japan.
Chen
J
J
Ueda
K
K
Mori
Y
Y
Nakano
K
K
Hirata
Y
Y
Kanamori
S
S
Uchiyama
Y
Y
Inagi
R
R
Okuno
T
T
Yamanishi
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D019556:COS Cells; D002460:Cell Line; D004279:DNA, Viral; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004279:DNA, Viral; D017874:Immediate-Early Proteins; C405867:K5 protein, Human herpesvirus 8; D014764:Viral Proteins