Rolf Renne

Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi's sarcoma-associated herpesvirus.

Over 85% of patients with Kaposis sarcoma (KS) are seropositive for antibodies to the latency-associated nuclear antigen (LANA) expressed in B cell lines infected with Kaposis sarcoma-associated herpesvirus (KSHV). The presence of antibodies to LANA strongly correlates with the risk of developing the disease. However, the identity of the protein(s) comprising LANA and the corresponding gene(s) has remained unclear. To identify potential latent gene candidates for LANA, we probed total RNA extracted from BCBL-1 cells (a B cell line latently infected with KSHV) using lambda clones that span the KSHV genome. One region encoding latent transcripts spanned KSHV open reading frames (orfs) 71 (K13), 72 (v-cyclin), and 73. Among these, however, only orf 73, when expressed in heterologous mammalian cell systems, reacted with KSHV antibody-positive human sera, resulting in a punctate nuclear staining pattern reminiscent of LANA in BCBL-1 cells. Furthermore, extracts from cells expressing the orf 73 protein product specifically blocked the binding of KS patient antibodies to LANA. Finally, seroreactivity with recombinant orf 73 protein exactly paralleled reactivity with classical LANA as expressed in BCBL-1 cells, both in KS patients and in other groups. Together, these data support the identification of KSHV orf 73 as the gene encoding the dominant immunogenic component of LANA.

Modulation of Cellular and Viral Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposis sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)—and from NF-κB-dependent reporter genes—was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection.

Experimental transmission of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) to SIV-positive and SIV-negative rhesus macaques

Abstract:  Kaposis sarcoma‐associated herpesvirus (KSHV) is a γ‐herpesvirus associated with Kaposis sarcoma (KS) and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castlemans disease. Studies on the biology and pathogenesis of KSHV have been limited by lack of efficient cell culture systems and lack of a suitable animal model for KS. Here we report on the experimental inoculation of SIV‐positive and SIV‐negative rhesus macaques with KSHV‐infected PEL cells or KSHV preparations derived from PEL cells. Low levels of viral DNA could be detected in cultivated peripheral blood mononuclear cell of all animals, as well as in the bone marrow of one monkey that died from SAIDS. However, we were not able to detect KSHV‐specific antibodies or transcripts, nor did we observe any symptoms clearly related to KSHV infection (e.g. KS or lympho‐proliferative disease). Hence, although KSHV replicates in rhesus macaques at very low levels, this non‐human primate host is unlikely to provide a useful animal model for disease.

Human herpesvirus 8 glycoprotein K8.1: expression, post-translational modification and localization analyzed by monoclonal antibody.

BACKGROUND The genome of human herpesvirus 8 (HHV-8) contains at least 84 open reading frames, including the highly immunogenic K8.1. Other studies have determined that K8.1 gene generates at least two spliced transcripts in the HHV-8 infected BCBL-1 cells, termed as glycoprotein (gp)K8.1A and gpK8.1B. OBJECTIVE To analyze the expression, post-translational modification and localization of HHV-8 gpK8.1 by monoclonal antibody (mAb). STUDY DESIGN Mabs to HHV-8 produced by conventional hybridization and several clones identified. A mAb was used by various immunological assays to analyze HHV-8 K8.1 proteins in BCBL-1- and Sf9 insect cells. RESULTS MAb clone 19B4 identified a 0.75-kb insert from the lambdaZAP cDNA expression library of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced BCBL-1 cells. Sequence analysis revealed that the cDNA insert corresponds to the published spliced ORF K8.1 mRNA of HHV-8. By immunofluorescence assay, the mAb stained the cell membrane, cytoplasm and perinuclear region of TPA induced BCBL-1 cells and showed no cross-reactivity with other herpesviruses. By immunoblotting assay, mAb 19B4 reacted with two species polypeptides giving a diffuse band with rMW from 42 to 64 kDa (gpK8.1A) and two closely migrating polypeptides of rMW 35/37 kDa (gpK8.1B). Both species were labeled by [14C]glucosamine, indicating that they are glycosylated and only gpK8.1A was detected in the virions. Expression of the full length K8.1 derived from cDNA in baculovirus system confirmed that these two glycoproteins are encoded by K8.1 gene. Enzymatic deglycosylation with endoF/peptide-N-glycosidase F, led to the reduction of rMW of both polypeptides whereas deglycosylation with O-glycosidase led only the reduction of rMW of K8.1A. CONCLUSION The mAb 19B4 reacts specifically with BCBL-1 and Sf9 cells infected with recombinant baculovirus containing HHV-8 K8.1 gene. In several assays the mAb reacts with gpK8.1A and gpK8.1B. Only the mature spliced gpK8.1A is incorporated into virion. Enzymatic deglacosylation determined that gpK8.1A is N- and O-glycosylated, whereas gpK8.1B may lack O-glycosylation.