Beverly E. Griffin

Burkitt's lymphoma

Burkitts lymphoma is a highly aggressive B-cell non-Hodgkin lymphoma and is the fastest growing human tumour. The disease is associated with Epstein-Barr virus and was one of the first tumours shown to have a chromosomal translocation that activates an oncogene (c-MYC). Burkitts lymphoma is the most common childhood cancer in areas where malaria is holoendemic. The incidence is very high in immunosuppressed patients in non-endemic areas, especially when associated with HIV infection. Outcome with intensive chemotherapy has improved and is now excellent in children, but the prognosis is poor in elderly adults. The success of intensive treatment relies on good supportive care. The therapy offered in oncology units in low-income countries is not as aggressive as in centres in high-income countries and outcomes are less successful. Adjuvant monoclonal antibody therapy with rituximab shows promise for improved outcomes and reduced toxic effects in the future.

EBV gene expression in an NPC-related tumour.

A nasopharyngeal carcinoma tumour (designated C15) propagated in nude mice has been used to generate a large cDNA library that we have analysed for Epstein‐Barr virus (EBV) gene expression. No gross alterations exist in viral DNA from C15 relative to other human isolates and the large deletion present in the B95‐8 ‘prototype’ viral strain established in marmoset cells is not found; C15 contains no linear virion DNA. In the cDNA library, of the six EBV nuclear antigens (EBNAs) expressed in latently infected B‐lymphocytes, only clones for EBNA‐1 are found. These data are confirmed by immunoblotting. Sequence analysis shows the EBNA‐1 mRNA splicing pattern in the carcinoma to differ from that observed in B‐lymphocytes. Further, contrary to observations with B‐cell lines, most viral transcription in the tumour is localized onto the ‘rightmost’ region of the conventional EBV physical map. Transcripts identified corresponding to known genes include those for the latent membrane protein (LMP), the alkaline DNA exonuclease and probably the terminal protein; major transcripts are also derived from the BamHI D fragment and the region deleted in B95‐8 EBV DNA. Novel transcripts have also been identified that proceed in an anti‐sense direction to genes encoding functions associated with replication, such as the viral DNA polymerase. They contain a large, hitherto unidentified, open reading frame in the viral genome that is complementary to the putative function known as BALF3 and a smaller open reading frame complementary to BALF5 (the DNA polymerase gene). From the present studies we can conclude that: (i) EBV transcription patterns in the epithelial cells vary markedly from those identified previously in B‐cells, reflecting differential use of promoters or splicing patterns. (ii) Transcription is tightly regulated and restricted in the C15 tumour with many latent genes, notably EBNAs 2‐6, being ‘switched off.’ (iii) A family of cytoplasmic RNAs are transcribed in an antisense direction to a number of existing open reading frames in the EBV genome. (iv) There are a number of mutations in C15 transcripts relative to the B95‐8 genome, some of which could result in amino acid alterations in proteins.

Coding potential and regulatory signals of the polyoma virus genome

The complete DNA sequence of the A2 strain of polyoma virus has been determined. It consists of 5,292 base pairs. The sequence is analysed in terms of its coding potential and sites of possible functional significance or structural interest. The polyoma virus genome is compared with those of related tumour viruses, simian virus 40 and BK virus.

Structure and Coding Content of CST (BART) Family RNAs of Epstein-Barr Virus

ABSTRACT CST (BART BARF0) family viral RNAs are expressed in several types of Epstein-Barr virus (EBV) infection, including EBV-associated cancers. Many different spliced forms of these RNAs have been described; here we have clarified the structures of some of the more abundant splicing patterns. We report the first cDNAs representing a full-length CST mRNA from a clone library and further characterize the transcription start. The relative abundance of splicing patterns and genomic analysis of the open reading frames (ORFs) suggest that, in addition to the much studied BARF0 ORF, there may be important products made from some of the upstream ORFs in the CST RNAs. Potential biological functions are identified for two of these. The product of the RPMS1 ORF is shown to be a nuclear protein that can bind to the CBF1 component of Notch signal transduction. RPMS1 can inhibit the transcription activation induced through CBF1 by NotchIC or EBNA-2. The protein product of another CST ORF, A73, is shown to be a cytoplasmic protein which can interact with the cell RACK1 protein. Since RACK1 modulates signaling from protein kinase C and Src tyrosine kinases, the results suggest a possible role for CST products in growth control, perhaps consistent with the abundant transcription of CST RNAs in cancers such as nasopharyngeal carcinoma.

Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments

A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned. Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned. Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA. Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells. In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322. The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases.

Protein kinase activity associated with polyoma virus middle T antigen in vitro

Abstract A protein kinase activity can be detected in immunoprecipitates of extracts from polyoma virus (Py)-infected cells using antiserum raised against Py-transformed cells (anti-T serum). The activity is not detected in uninfected cells or when using control serum. Using rat anti-T serum both Py middle T and the heavy chain of rat IgG are phosphorylated, whereas using hamster anti-T serum only Py middle T is phosphorylated. Experiments using a number of different mutants of Py indicate that the kinase activity detected is under viral control and is associated with Py middle T. Consistent with this the kinase, like middle T, can be detected in purified preparations of plasma membranes. The kinase can also be detected in a large number of Py-transformed cells, but not in untransformed cells or in cells transformed by other viruses. Some of the Pytransformed cells which contain kinase activity lack full sized Py large T but all contain middle T. Kinase activity is not detected in a cell line (18.37) which contains integrated viral DNA of a nontransforming hr-t deletion mutant and which contains Py large T but not middle T or small t. These results show that Py middle T or a protein which specifically binds to it has protein kinase activity in vitro. Although these results raise the possibility that protein kinases play an essential role in Py-induced transformation, however, thus far we have no data which show unequivocally that the results are physiologically significant.

Promiscuous expression of Epstein-Barr virus genes in Burkitt's lymphoma from the central African country Malawi

Primary BL in Malawian children has a very high frequency association, approaching 100%, with the human herpesvirus EBV. A detailed study carried out on viral gene expression in these tumours, using both fresh material and methanol‐fixed FNAs, showed, contrary to prediction, that most belong to a variant “class II” latency category, with lytic cycle–related genes also expressed. That is, in addition to EBNA1 expression, membrane proteins (LMP1/2A), immediate early (BZLF1) and early (IR2 and IR4) genes, a putative viral oncogene (BARF1), CST (BART) antisense transcripts and the viral bcl‐2 homologue are expressed in a high proportion of the BLs. Most, but not all, express the small viral (EBER) RNAs. Two other significant observations were made: (i) in addition to expression of cellular cytokine (IL‐10) transcripts in all tumours investigated, the normally silent viral IL‐10 homologue was expressed in some tumours; (ii) whereas EBNA1 expression from its restricted Qp promoter was generally observed, the nonrestricted Cp/Wp promoter was also active in some tumours. Viral gene expression in the Malawian [endemic (e)] BLs appears to be more promiscuous than predicted from other studies, but expression accords with the cytopathologic picture of eBLs as a rapidly proliferating cell population accompanied by considerable necrosis, and a clinically diverse disease. A small‐scale study of relapse Malawian BLs revealed a different picture of viral association, more akin to systemic BL than eBL, where EBV appears to be absent or present only at very low levels. The significance of these findings is considered.

Sequence from early region of polyoma virus DNA containing viral replication origin and encoding small, middle and (part of) large T antigens

The sequence of about one third of the polyoma virus genome is presented. This sequence covers the origin of replication of two large plaque strains (A2 and A3) of polyoma virus. The two strains differ by 11 bp in the origin region. A model for replication is suggested. The sequence probably also covers the entire coding region of two of the polyoma virus early proteins--small and middle T antigens--as well as part of the coding region for large T antigen. Over a small region of the DNA, all three coding frames contain termination codons, which argues a need for spliced early messenger RNAs. In another region of the DNA, two coding frames can be used. Correlation with protein data suggests that one frame codes for part of middle T antigen and the other for part of large T antigen.

EBNA-1, the major nuclear antigen of Epstein-Barr virus, resembles 'RGG' RNA binding proteins.

Nuclear antigen 1 (EBNA‐1) is one of the key functions of the oncogenic DNA virus, Epstein‐Barr virus (EBV), and is the only viral protein consistently expressed in EBV‐associated malignancies. EBNA‐1 binds in a site‐specific manner to the viral DNA and is essential for viral replication, as well as for maintaining the genome as an extrachromosomal episome within infected cells. EBNA‐1 is not recognized by the cellular immune system. Here we demonstrate that, in addition to its known DNA binding properties, EBNA‐1 can also act as a strong RNA binding protein, interacting with diverse substrates in vitro, including the EBV‐encoded RNA polymerase III transcript EBER1 and the HIV‐encoded transactivation response (TAR) element. We also show that EBNA‐1 can bind exon sequences derived from its own RNA expressed from the Fp promoter, as found in Burkitts lymphoma‐related cells and in nasopharyngeal carcinomas. EBNA‐1 has been identified as a component in an RNA complex; moreover, an anti‐EBNA‐1 antibody 1H4‐1, that does not inhibit DNA binding, blocks binding to RNA. Arginine/glycine‐containing (so‐called ‘RGG’) motifs have been found in an increasing number of proteins that interact with RNA. The EBV antigen contains three potential ‘RGG’ motifs located around an internal glycine/alanine‐rich repetitive sequence in the protein, and outside the region of EBNA‐1 mapped previously as essential for viral DNA replication and other functionally defined properties. These motifs could be involved in the observed binding between EBNA‐1 and RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Organization of the Genomes of Polyoma Virus and SV40

Publisher Summary The major stimulus for the studies on two papovaviruses—polyoma virus and simian virus 40 (SV40)—has been their oncogenic potential. Both viruses cause tumors in a variety of rodents in vivo. In tissue-culture systems, polyoma virus transforms number of different types of rodent cells, whereas SV40 can transform, in addition to rodent cells, monkey and human cells. These viruses do not appear to be important tumor-causing agents in the wild. However, large numbers of transformed cells are produced in vitro in a simple and reproducible manner after viral infection. Thus, these viruses appear to offer a good model system for elucidating in vitro some of the events that lead to in vivo tumor formation. The genetic information of both polyoma virus and SV40 resides in their DNAs, which have molecular weights of about 3.4 x l06 daltons. The DNAs exist in “native” form as covalently closed circular superhelical molecules with a coding capacity for about 200,000 daltons of protein. Three distinct genes have so far been identified. One can be correlated with an early function concerned with viral DNA synthesis and the other two with proteins found in the viral capsid.