Human Herpesvirus-8 ORF K8.1 Gene Encodes Immunogenic Glycoproteins Generated by Spliced Transcripts

Abstract

A cDNA library from phorbol ester-induced human herpesvirus-8 (HHV-8) carrying BCBL-1 cells was screened with an HIV+KS+ serum, and several cDNA clones encoding HHV-8 proteins were identified. Sequence analysis of two full-length cDNA clones show open reading frames (ORFs) encoded by spliced messages originating from the HHV-8 K8.1 gene. One cDNA encodes an ORF of 228 amino acids, designated K8. 1.A, with a cleavable signal sequence, a transmembrane domain, and four N-glycosylation sites. The splicing event generated the transmembrane domain in the ORF not seen in the genomic K8.1 ORF. Another cDNA encodes an ORF of 167 amino acids, designated K8.1.B, that shares similar amino and carboxyl termini with ORF K8.1.A but with an in-frame deletion. The primary translation products of ORF K8.1A (34 kDa) and K8.1B (20 kDa) in the in vitro-transcription-translation experiments shifted into glycosylated species of 43 and 32 kDa, respectively, in the presence of microsomal membranes. This suggested that the ORF K8.1A and K8.1B encode for glycoproteins. Riboprobes from the K8.1A cDNA insert hybridized with an HHV-8-specific 0.9-kb abundant transcript from BCBL-1 cells. Synthesis of this RNA was eliminated in the presence of a DNA synthesis inhibitor, suggesting that this RNA was a late gene transcript. Because ORFs K8.1A and K8.1B are unique for HHV-8, human sera were tested in Western blot reactions for antibodies against glutathione-S-transferase-ORF K8.1A fusion protein. All sera that were positive for HHV-8 antibodies in immunofluorescence assays with phorbol ester-induced BCBL-1 cells were also positive for anti-ORF K8.1A antibodies. This suggests that measurement of anti-ORF K8.1A antibodies would provide an HHV-8-specific serological assay. Further work is needed to define the biological role of the HHV-8 ORF K8.1A and K8.1B glycoproteins.

The role of Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 regulator of transcription activation (RTA) in control of gene expression

“…In addition, another species of ORF50 mRNA of about 3.4 kb was identified and was found to contain the ORF 50 K8 and K8.1 ORFs (Lukac et al, 1999). The K8.1 ORF encodes for a virion-associated glycoprotein that is involved in infection and in the binding to the cellular receptor (Chandran et al, 1998;Raab et al, 1998;Li et al, 1999).…”

Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein K8.1 Is Dispensable for Virus Entry

“…These include glycoprotein B (gB) (open reading frame [ORF] 8 [ORF8]), gH (ORF22), gM (ORF39), gL (ORF47) (36,48), and gN (ORF53) (27). In addition, the K1, K8.1, and vOX2 (K14) glycoproteins are unique to KSHV, with no counterparts in other herpesviruses (10,11,36,49). KSHV gB and glycoprotein K8.1A mediate the initial binding of virions to glycosaminoglycans, e.g., heparan sulfate on cell surfaces (2,4,7,57).…”

“…The K8.1A cDNA encodes a 228-amino-acid protein containing a signal sequence, transmembrane domain, and four N glycosylation sites. The K8.1B cDNA encodes a 167-amino-acid glycoprotein sharing similar amino and carboxy termini with K8.1A but containing an in-frame deletion (10,44). K8.1A is the predominant form detected within infected cells and virion envelopes (62).…”

Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein

“…51 gpK8.1A is a 228 amino acid late lytic glycoprotein [52][53][54] displayed on KSHV virions; 55,56 it binds to heparin sulfate on the target cell surface, 57,58 thereby facilitating virus entry and consequent spread of infection. Antibodies against gpK8.1A are important serological markers for KSHV disease.…”