Polyoma virus and simian virus 40 as cancer models: history and perspectives.

Polyoma virus and SV40 are the founding members of the Polyomaviridae. They are small viruses, with a genome consisting of around 5.3kbases of closed circular, double-stranded DNA. This simplicity, the ease with which they can be grown, and their capacity to cause cancers in newborn rodents has made them popular models for studying the molecular basis of cancer formation. As a consequence, many of the underlying principles involved in tumorigenesis have been uncovered during the study of these viruses. For instance, the discovery of p53, Rb protein function, tyrosine kinases and PI3 kinases were all made when examining polyoma virus and SV40. Here we review how these discoveries were made, and the influence they have had on our understanding of cancer development.

Complexities associated with expression of Epstein-Barr virus (EBV) lytic origins of DNA replication.

EBV has two lytic origins (oriLyt) of DNA replication lying at divergent sites on the viral genome within a duplicated sequence (DS). The latter contains potential hairpin loops, 'hinge' elements and the promoters for transcripts from viral genes BHLF1 and LF3. These genes themselves consist largely of 125 and 102 bp repetitive sequences, respectively, and encode basic proteins. We have examined these genomic regions in detail in attempts to understand why lytic replication--necessary for virus survival--is so inefficient, and to identify controlling elements. Our studies uncovered a diverse family of promoters (P) for BHLF1 and LF3, only one pair of which (P1) proved sensitive to chemical inducing agents. The others (P2-P3/4), abutting the replication 'core' origin elements in DS and extending into 5'-unique sequences, may play roles in the maintenance of viral latency. We further identified a family of overlapping small complementary-strand RNAs that transverse the replication 'core' origin elements in a manner suggesting a role for them as 'antisense' species and/or DNA replication primers. Our data are discussed in terms of alternative lytic replication models. We suggest our findings might prove useful in seeking better control over viral lytic replication and devising strategies for therapy.

Genetic diversity: frameshift mechanisms alter coding of a gene (Epstein-Barr virus LF3 gene) that contains multiple 102-base-pair direct sequence repeats.

Frameshift mutations provide recognized mechanisms for changing the coding potential of an organism. Here, multiple frameshifts are identified in repetitive sequences within an Epstein-Barr virus unspliced early gene, LF3, which is associated with the viral replicative cycle and also transcriptionally expressed in many virally associated tumors. On the DNA strand encoding LF3, there are three open reading frames, only one of which contains an initiation codon. Most (>95%) of the gene consists of numerous (>20, varying with cell source) GC-rich copies of a 102-bp direct repeat (called IR 4) flanked by small unique sequences. LF3 may express a protein if its initiation and termination codons reside in the same reading frame, but this is not always the case. Frameshifting events, occurring in short runs of pyrimidines (mainly C residues) in the repeats, give rise to mutations which may provide a mechanism for escape of an LF3 function from host surveillance. Sequence studies link these frameshifts to DNA replication errors. Notably, the number of sites in LF3 at which such mutations can occur permits a very large amount of diversity in this gene. Our data also suggest a second degeneracy mechanism within the protein itself, which influences its stability and may reflect a host defense mechanism. LF3 thus provides a potentially important model for studying the quest for supremacy between a virus and its host.

Pilot study of sodium phenylbutyrate as adjuvant in cyclophosphamide-resistant endemic Burkitt's lymphoma.

Burkitt's lymphoma (BL) accounts for the majority of childhood malignancies seen in sub-Saharan Africa. In Malawi, cyclophosphamide (CPM), the mainstay of treatment for endemic BL, is effective in around 50% of cases. Evidence exists in support of an association between activation of replication of Epstein-Barr virus (EBV) in the tumour and response to this chemotheraupeutic agent. Phenylbutyrate (PB), approved for treatment of inborn errors of the urea cycle with minimal toxicity in children, induces EBV replication and cell lysis in BL-derived cell cultures. It has also shown some success as adjuvant in treatment of chronic leukaemia and lymphoma. We tested in African BL patients with CPM-resistant tumours, and thus unlikely to survive, the hypothesis that PB can reverse this resistance. A study of five patients showed PB before CPM to induce shrinkage of CPM-resistant tumours in two of them. Findings suggested that for this effect PB pre-treatment should be given for a week before CPM treatment. A larger study is indicated.

Virus-associated RNA I-deleted adenovirus, a potential oncolytic agent targeting EBV-associated tumors.

Given the growing number of tumor types recognizably associated with EBV infection, it is critically important that therapeutic strategies are developed to treat such tumors. Replication-selective oncolytic adenoviruses represent a promising new platform for anticancer therapy. Virus-associated I (VAI) RNAs of adenoviruses are required for efficient translation of viral mRNAs. When the VAI gene is deleted, adenovirus replication is impeded in most cells (including HEK 293 cells). EBV-encoded small RNA1 is uniformly expressed in most EBV-associated human tumors and can functionally substitute for the VAI RNAs of adenovirus. It enables replication to proceed through complementation of VAI-deletion mutants. We hypothesized that VAI-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to the presence of EBV-encoded small RNA1 with no (or poor) replication in normal or EBV-negative tumor cells. In this report, we show that high levels of replication occurred in the VAI-deleted mutant in the EBV-positive tumor cells compared with low (or negligible) levels in EBV-negative and normal human primary cells. Correspondingly, high toxicity levels were observed in EBV-positive tumor cells but not in EBV-negative tumor or normal human primary cells. In vivo, VAI-deleted adenovirus showed superior antitumoral efficacy to wild-type adenovirus in EBV-positive tumor xenografts, with lower hepatotoxicity than wild-type adenovirus. Our data suggest that VAI-deleted adenovirus is a promising replication-selective oncolytic virus with targeting specificity for EBV-associated tumors.

Hypothesis: a novel route for immortalization of epithelial cells by Epstein-Barr virus.

Transfection of primate tissue explants with a specific sub-fragment (p31) of EBV DNA results in epithelial (but no other) cells proliferating indefinitely (becoming 'immortalized') without evidence of a 'growth crisis'. Molecular evidence supports integration of viral information into the host chromosome, and an early genotypic alteration involving specific amplification of a sub-component (IR1) of p31 DNA, followed by apparent loss of viral DNA from chromosomes, consistent with a 'hit and run' mechanism. However, analysis at the individual cell level during long-term culture, by FISH techniques, reveals chromosomal alterations, and viral sequences surviving within double minute (DM) bodies. Changing growth patterns occurring at different stages during propagation (>a year in culture) may be explained by sporadic reintegration of surviving viral DNA into the host chromosome. Notably, throughout culture, telomere lengths in chromosomal DNAs do not alter but rather retain the length observed in the primary cell populations. Introduction of a growth stimulating function of EBV, BARF1, into the immortalized, non-clonable epithelial cells under conditions which permit overexpression, allows clonal populations to be derived. Based on the data, mechanisms of immortalization, in the absence of a proven viral oncogene in p31 DNA, and possible genes involved, are considered.

Burkitt's lymphoma: maximising the use of fine needle aspirates by long-term preservation for diagnosis and research.

Fine needle aspirates from Burkitt's lymphoma and other tumours transferred directly into ThinPrep® PreservCyt® (Cytyc UK Ltd, Crawley, UK) buffered alcohol fixative retain their cellular and viral antigens and nucleic acids for many months at ambient temperatures. Despite the presence of blood and debris, cells dried onto slides from droplets and post-fixed in formalin, or sections of paraffin-embedded cell blocks from formalin post-fixed pellets, prove adequate for morphology, immunocytochemistry, in-situ hybridization and molecular biological analyses. Where there is lack of expertise in making thin smears or hospitals lack pathology laboratories and services, PreservCyt® provides an excellent medium for transport elsewhere for diagnosis and research.

[Nasopharyngeal carcinoma in sub-Saharan Africa: a tribute to Mr. Peter Clifford].

Poorly differentiated nasopharyngeal carcinoma (NPC) is a major malignancy in certain areas of Asia. It exists also in Africa, notably among largely Arab populations in North Africa, and in "hotspots" in East Africa among "native" Africans. This article deals with the latter, as studied and defined in depth during the 1960s and 70s by the Irish-born surgeon, Peter Clifford. Through his published works, he has influenced and helped define the field of head and neck cancer as it exists in Kenya. He also did pioneering work on the African childhood malignancy, Burkitt's lymphoma (BL). Both BL and NPC have been ultimately shown to be associated with the human herpes Epstein-Barr virus (EBV). This article is written as a tribute to Peter Clifford, focusing on his work on NPC, where he first defined the disease "hotspots" in the Kenyan Highlands, studied how best to treat the malignancy in the absence of radiotherapy, looked at possible NPC predisposing factors in the Kenyan setting, and ultimately addressed how the cancer cells interact with EBV. Peter Clifford's pioneering work was cut short by accident. Although outside East Africa he remains largely an 'unsung hero' in the field, his influence has been great. It begs to be re-addressed and reconsidered.

Effect of dose and long-term storage on the immunogenicity of murine polyomavirus VP1 virus-like particles.

We have analysed the stability and immunogenicity of murine polyomavirus virus-like particles (VLPs) following intranasal administration without adjuvant. No morphological or immunological changes were observed in a preparation of these VLPs stored for 9 weeks at room temperature. Strong humoral and cellular (Th1) responses were obtained after a single 5.55 microg dose immunisation, which are efficiently boosted after a second dose. However, at dose concentrations above 0.22 microg/microl, these VLPs appear to aggregate and, when used for immunisations, they fail to induce a strong cellular response, even though the humoral response is unaffected. These results may reflect the differential processing of VLP aggregates by the immune system or, alternatively, VLP neutralisation by antibodies induced after a primary immunisation.

Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins.

Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2- and VP3- viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr- viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used. In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.

Immunization of T-cell deficient mice against polyomavirus infection using viral pseudocapsids or temperature sensitive mutants.

A murine experimental model system aimed at developing potential vaccines to papovavirus infection in immunosuppressed individuals was explored. A VP1-pseudocapsid based on the major capsid protein of the murine polyomavirus A2 strain and a mutant, M17-pseudocapsid as well as four temperature sensitive (ts)-mutants were used as immunogens. T-cells deficient CD4-/-8-/- mice were immunized four times with each immunogen and then together with non-immunized control mice challenged with polyomavirus. In contrast to all control mice, only half of the immunized mice exhibited presence of polyoma DNA when assayed by PCR. The results indicate that pseudocapsids and ts-mutant immunization may potentially protect mice with an impaired T-cell function from polyomavirus infection.

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.