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.

Human herpesvirus-8 K8.1 gene encodes for two immunogenic class I glycoproteins, K8.1A and B, originating from spliced messages [(1998) Virology 243, 208-217]. The 228-amino-acid-long K8.1A open reading frame (ORF) contains four N-glycosylation sites and the 167-amino-acid-long K8.1B ORF contains three N-glycosylation sites, sharing similar amino- and carboxyl-termini with ORF K8.1A but with an in-frame deletion [(1998) Virology 249, 140-149]. To characterize the K8.1A and B glycoproteins in the infected body cavity-based B cell lymphoma (BCBL-1) cells and in the virion envelopes, monoclonal antibodies (MAbs) recognizing only K8.1A protein or both K8.1A and B proteins were generated. These antibodies reacted with the infected cell membranes and virion envelopes. Stable COS-1 transformant cells expressed the K8.1A and B proteins independently on the plasma membranes. MAbs recognized multiple proteins with molecular weights ranging from 23 to 72 kDa from the BCBL-1 cells and COS-1 cells and the 72 to 68 kDa molecular-weight proteins from the virion particles. The K8.1A is the predominant protein affinity purified from the infected BCBL-1 cells. Digestion with glycosidases show that these proteins contain both N- and O-linked sugars, suggesting that the multiple proteins recognized by the MAbs represent the precursor and product forms of K8.1A and B proteins, and the 72 to 68 kDa molecular-weight proteins represent the virion particle-associated mature forms of these glycoproteins.

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.

Human herpesvirus 8 (HHV8) infects Kaposi’s sarcoma (KS) spindle cells in situ, as well as the lesional endothelial cells considered to be spindle cell precursors. The HHV8 genome contains several oncogenes, suggesting that infection of endothelial and spindle cells could induce cellular transformation and tumorigenesis and promote the formation of KS lesions. To investigate the potential of HHV8 infection of endothelial cells to contribute to the development of KS, we have developed an in vitro model utilizing dermal microvascular endothelial cells that support significant HHV8 infection. In contrast to existing in vitro systems used to study HHV8 pathogenesis, the majority of dermal endothelial cells are infected with HHV8 and the viral genome is maintained indefinitely. Infection is predominantly latent, with a small percentage of cells supporting lytic replication, and latency is responsive to lytic induction stimuli. Infected endothelial cells develop a spindle shape resembling that of KS lesional cells and show characteristics of a transformed phenotype, including loss of contact inhibition and acquisition of anchorage-independent growth. These results describe a relevant model system in which to study virus-host interactions in vitro and demonstrate the ability of HHV8 to induce phenotypic changes in infected endothelial cells that resemble characteristics of KS spindle cells in vivo. Thus, our results are consistent with a direct role for HHV8 in the pathogenesis of KS.

Autoantibodies against RNA polymerase I (RNAPI), DNA, La and ribosomal P proteins were detected in the urine of systemic lupus erythematosus (SLE) patients, many with normal protein excretion rates. In a number of cases, the antibodies were detectable in the urine but not the serum sample of the same patient. The presence and relative concentrations of the urinary autoantibodies correlated with disease activity. RNAPI antigens were detected in the urine of SLE patients by radioimmunoassay and immunoblotting using rabbit antisera prepared against the purified holoenzyme. Immunoaffinity purification of the rabbit anti-RNAPI with SLE urine proteins resulted in antibodies directed primarily against the largest RNAPI subunit (S1; 194 kDa). Antibodies prepared against recombinant fusion proteins representing the DNA binding regions of human RNAPI(S1) reacted with a 35 kDa SLE urinary protein, a putative fragment of RNAPI(S1). Ribosomal protein P0 was detected in SLE patients' urine by immunoblotting, using rabbit antiserum prepared against recombinant human P1 fusion protein. The relative quantities of urinary P0 correlated with disease status. Analysis of urinary autoantibodies and corresponding antigens in conjunction with analysis of serum autoantibodies may be of value for the purpose of monitoring disease activity.