Elizabeth Grogan

Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50/Rta Protein Activates the Entire Viral Lytic Cycle in the HH-B2 Primary Effusion Lymphoma Cell Line

ABSTRACT Rta, the gene product of Kaposis sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.

Antibodies to Butyrate-Inducible Antigens of Kaposi's Sarcoma–Associated Herpesvirus in Patients with HIV-1 Infection

BACKGROUND The recent identification in patients with Kaposis sarcoma of DNA sequences with homology to gammaherpesviruses has led to the hypothesis that a newly identified virus, Kaposis sarcoma-associated herpeslike virus (KSHV), has a role in the pathogenesis of Kaposis sarcoma. We developed serologic markers for KSHV infection. METHODS KSHV antigens were prepared from a cell line (BC-1) that contains the genomes of both KSHV and the Epstein-Barr virus (EBV). We used immunoblot and immunofluorescence assays to examine serum samples from 102 patients with human immunodeficiency virus type 1 (HIV-1) infection for antibodies to KSHV-associated proteins and to distinguish these antibodies from antibodies to EBV antigens. A positive serologic response was defined by the recognition of an antigenic polypeptide, p40, in n-butyrate-treated BC-1 cells and by the absence of p40 recognition in untreated BC-1 cells or EBV-infected, KSHV-negative cells. The detection by the immunofluorescence assay of 10 to 20 times more antigen-positive cells in n-butyrate-treated BC-1 cells than in untreated cells was considered a positive response. RESULTS Antibodies to the p40 antigen expressed by chemically treated BC-1 cells were identified in 32 of 48 HIV-1-infected patients with Kaposis sarcoma (67 percent), as compared with only 7 of 54 HIV-1-infected patients without Kaposis sarcoma (13 percent). These results were confirmed by an immunofluorescence assay. The positive predictive value of the serologic tests for Kaposis sarcoma was 82 percent, and the negative predictive value 75 percent. CONCLUSIONS The presence of antibodies to a KSHV antigenic peptide correlates with the presence of Kaposis sarcoma in a high-risk population and provides further evidence of an etiologic role for KSHV.

Antibody responses to two Epstein-barr virus nuclear antigens defined by gene transfer

By transfecting small fragments of Epstein-Barr virus (EBV) DNA into cells, we defined two nuclear antigens, termed M and K, and examined serum from 258 subjects for antibodies against these antigens. We hoped to learn whether such single-antigen systems would clarify the association of EBV with various diseases. Although reactivity to M antigen was found in only 14 per cent of healthy EBV-seropositive subjects, 90 per cent of Chinese and North African patients with nasopharyngeal carcinoma had antibody to M. Nearly all persons (96 per cent) who were EBV seropositive, as judged by their serologic reaction to a nuclear antigen encoded by the complete virus (EBNA), had a reaction to K antigen. However, serum samples from three patients with chronic active EBV infection did not react to K, even though the serum contained anti-M titers above 1:1000. Lymphoid cells from one such patient carried a normal gene for K and made K protein of correct size. Therefore, in this patient the absence of antibody to K had not resulted from a viral mutation that destroyed the K protein. These serologic studies show that some patients with chronic active EBV infection have an abnormal immune response to a specific viral gene product.

Differences in the extent of activation of Epstein-Barr virus replicative gene expression among four nonproducer cell lines stably transformed by OriP/BZLF1 plasmids

Lymphoid cell lines were established which stably carry the Epstein-Barr viral (EBV) BZLF1 gene on an extrachromosomal plasmid. These lines, which spontaneously synthesize the BZLF1 gene product, ZEBRA, were examined for expression of EBV genes which were activated by ZEBRA. Cell lines which acquired oriP plasmids without BZLF1 served as controls. The extent of activation differed among derivatives of four cell lines. X50-7 cells, which harbor a standard latent EBV, could be induced by ZEBRA to produce transforming virus; a cellular subclone of this line was induced to express EBV late antigens but did not release transforming virus. In two other cell lines, Raji and ER, ZEBRA activated only a group of early antigens. Using immunofluorescence and immunoblotting with monoclonal antibodies and Northern analysis five EBV early genes were shown to be induced in cells stably transformed by oriP/BZLF1 plasmids. ZEBRA itself was activated; thus BZLF1 is autostimulatory. Four other activated genes were components of the diffuse (EA-D) and restricted (EA-R) early antigens (BMRF1, BMLF1, BHRF1, and BORF2). Stable cell lines with extrachromosomal BZLF1 expression vectors will ultimately be useful in a variety of experiments designed to study regulation of this gene, to analyze the effects of mutations on ZEBRA protein function, and to define the full spectrum of viral and cellular genes which are activated by and interact with the ZEBRA protein.

Role of The Zebra Protein in the Switch Between Epstein-Barr Virus Latency and Replication

Experiments will be summarized which address two general questions about the BZLF1 gene. The first is, “what factors regulate expression of the gene?” The second is “which other EB viral genes are activated as the result of ZEBRA expression?”

Disruption of Latency by a Fragment of Rearranged EBV DNA: Evidence for Host Cell Regulation

In B lymphocytes immortalized by EBV the genome is latent. Only certain viral genes are expressed; most are inhibited. The tightness of this regulation varies among cells from different hosts. Neonatal human lymphocytes usually maintain latency in a tight state; lymphocytes from older humans are more permissive. Those from certain New World primates especially cotton-top marmosets are most permissive. Thus latency seems to be maintained through regulatory event(s) imposed upon the viral genome by the host cell (1).