INTEGRATION OF VIRUSES INTO CHROMOSOMAL DNA

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1 JOURNAL OF PATHOLOGY, VOL. 163: (1991) REVIEW ARTICLE-CHROMOSOME PATHOLOGY INTEGRATION OF VIRUSES INTO CHROMOSOMAL DNA DAVID ONIONS Director, Leukaemia Research Fund Human Virus Centre, Department of Veterinary Pathology, University of Glasgow, Glasgow G61 lqh, U.K. INTRODUCTION The elucidation of the role of viruses in human and animal diseases stands as one of the great intellectual and practical achievements of medical science. At the beginning of this century, viruses could only be characterized as filterable agents, incapable of growth on synthetic media, but with the advent of tissue culture, the spectacular nature of virus replication became evident. A single polio virus on infecting a susceptible cell will produce 1000 progeny virions within a period of hours and as these virions are released the cell is killed. Studies of lytic infections of this type became a predominant theme of virology, permitting both the analysis of viral structure and genetics, as well as leading to insights into the pathogenesis of many diseases. However, viruses display a range of more subtle interactions with their host cells, of which latency forms one of the most important, In herpesvirus infections, latency is frequent and the latently infected cell contains a circular double-stranded viral genome as an extrachromosomal episome. Transcription is limited to a few gene sets and no virus particles are produced, although productive virus replication can be induced by a number of insults to the cell. The latent state for herpesviruses does not require the integration of viral sequences into the chromosomes, but for one class of viruses-the retroviruses-the normal replication cycle and the latent state require integration of viral genetic information into chromosomal DNA. Distinct subgroups of retrovirus are recognized, of which the best characterized are the lentivirus group, which includes.4ddressee for correspondence: D. Onions, Department of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Glasgow G61 IQH, U.K. HIV-1 and -2, and the oncovirus group, which includes the human leukaemia viruses HTLV-1 and -11 and the leukaemia virus of cats (FeLV) and mice (MuLV). Although there are very important differences in the molecular organization of these viruses, their general replication patterns are similar. The diploid, single-stranded genome of the retrovirus is enclosed by an inner shell or capsid and an outer envelope derived from the cytoplasmic membrane of the host cell. Substituted into the viral envelope are viral glycoproteins which serve as receptors that bind to complementary structures on the target cell, the most studied system being the interaction of HIV gp120 with the CD4 molecule on T-cells. Once the virus has bound to the cell, the RNA genome is liberated into the cytoplasm, where a virus-specific enzyme, reverse transcriptase, transcribes the RNA genome into double-stranded DNA (Fig. 1). The DNA copy of the genome, or provirus, migrates to the nucleus as a nucleoprotein complex where the endonuclease function of the reverse transcriptase incorporates the provirus into the DNA. Integration of the provirus is influenced by the presence of an open chromatin structure and perhaps by local sequence content, but on a larger scale it is essentially a random process. Viruses like HTLV-I and -2 may only integrate one to three copies of their provirus, while for viruses like FeLV the copy number can be in excess of 30. In the lentiviruses, the importance of virus integration in the life cycle is, as yet, less clear, for in addition to integrated sequences large numbers of circular proviruses are produced. However, it is now clear that linear proviral molecules, rather than closed circles, are the precursors of the integrated form. Once integrated, the provirus may remain untranscribed, representing a latent infection within /91/ $ by John Wiley & Sons, Ltd.

2 192 D. ONIONS Fig. I-Replication the cell, or it may be transcribed by cellular RNA polymerases to produce new genomic RNA and m:rna coding for virus proteins. New virus particles are assembled at the cell surface and in the case of the oncoviruses, the release of these new particles is not usually cytopathic so that a cell may continue to divide and function normally. In the case of lentiviruses, however, the sudden burst of virus production is often lethal to the host cell. The retroviruses of man that are known to be pathogenic are all exogenous; that is, they are acquired by infection. The unique replication pattern of retroviruses has, over evolutionary history, led to the integration of retroviral genomes into the germ cells and their subsequent transmission as genetic elements. These endogenous proviruses of retroviruses are ubiquitous in the mammalian genome and constitute up to 1 per cent of the total genome of some strains of mice. In such strains, reinfection of the germ line may be occurring reasonably frequently, whereas in other cases integration may represent a rare event. For instance, cats contain several endogenous retrovirus families. of Retroviruses One is related to the exogenous leukaemogenic virus of cats, FeLV, while another is related to a retrovirus of baboons. The latter provirus is restricted to old world cats and it was presumably acquired by infection of an ancestral cat after the division of the old and new world species. Although, as discussed below, the presence of endogenous retroviral elements can have deleterious consequences, they may have had selective pressures favouring their retention. Expression of the envelope gene of a retrovirus can block the surface cellular receptor preventing superinfection of the cell. In wild mice, one resistance gene to retroviral infection has turned out to be an endogenous retroviral em gene that produces protection through this mechanism of viral interference. METHODS The study of viral genomes within cells has been dependent on advances in molecular biology that have taken place over the last decade. The principal

INTEGRATION OF VIRUSES INTO CHROMOSOMAL DNA 193 means of studying integrated viral genomes has been to make DNA libraries of infected cells, i.e., to incorporate overlapping fragments of DNA covering the whole cellular genome into a lambda phage vector.

3 INTEGRATION OF VIRUSES INTO CHROMOSOMAL DNA 193 means of studying integrated viral genomes has been to make DNA libraries of infected cells, i.e., to incorporate overlapping fragments of DNA covering the whole cellular genome into a lambda phage vector. Each phage plaque can be analysed for viral sequences by DNA hybridization and the organization of the integrated viral genome and adjacent cellular sequences can be determined by DNA sequencing. At the diagnostic level, the application of in situ hybridization for viral sequences has enabled the detection of papilloma virus DNA sequences in cervical carcinomas and their preneoplastic lesions. The recent introduction of the polymerase chain reaction (PCR) assay enables a single viral genome to be identified in a background of O6 cells compared with lo2 cells with conventional Southern hybridization. The technique is dependent on constructing primers (short stretches ofdna) to opposite strands of the viral genome a few hundred base-pairs apart. The viral genome is denatured into single-stranded DNA by heating; on cooling, the primers bind to their complementary sequences and a thermostable DNA polymerase extends the primers to produce two new copies of the viral genome. This cycle is now repeated 2040 times, each cycle being associated with a theoretical doubling of the reaction product. The specificity of this product can be confirmed by hybridization or by DNA sequencing. The beguiling simplicity of PCR and the ability to use whole cells or dewaxed paraffin sections as reaction substrates have led to a keen interest in its diagnostic potential. However, the very sensitivity of the technique makes it exquisitely sensitive to contamination, particularly from the reaction product of previous assays. Successful application of PCR requires an appreciation of the contamination problem and the introduction of rigorous systems to control it. APPLICATIONS Integration of an exogenous retroviral genome into a cellular genome could be lethal to that cell through insertional inactivation of an essential gene. However, rare events of this type are unlikely to have any observable effect unless a critical stem cell is involved. In contrast, oncogenesis by retroviruses involves an active interaction between the viral and cellular genomes. Transformation by retroviruses The structure of a typical provirus of MuLV or FeLV is shown in Fig. 2. It contains a gag gene coding for internal proteins, a pol gene coding for reverse transcriptase, and an env gene encoding the envelope spike proteins. At each end of the provirus are directly repeated sequences, the long terminal repeat or LTRs. These structures contain the transcriptional control regions of the virus, i.e., a promoter at which transcription is initiated and an enhancer that regulates the level of transcription often in a tissue-dependent fashion. From some tumours, variant viruses are isolated in which viral sequences have been replaced by cellular sequences (Fig. 2) which we now classify as oncogenes. It was the study of these defective, rapidly transforming viruses that first led to the discovery of oncogenes. However, this is not the only mode of interaction of retroviruses with cellular proto-oncogenes. At least 25 different proto-oncogenes have been shown to be activated by retroviruses. The interaction of FeLV with the cellular myc gene is shown in Fig.2. Regulation of the transcription of the myc gene is complex but exon 1 is non-coding and transcripts are often prematurely terminated within this region. In some tumours, a defective retrovirus is found integrated just upstream of exon 2, so that a hybrid message of viral and cellular sequences is produced. The important point is that the transcription of myc is under the control of the viral LTR and the normal feedback regulatory controls are disrupted. Transcripts produced from this form of promoter insertion are probably the precursors of the recombinant defective viruses. A variant form of insertional mutagenesis by retroviruses is shown in Fig. 2 in which the provirus is integrated upsteam of myc but in the opposite transcriptional orientation. In this mode, the enhancer of the LTR probably influences the transcription from the myc gene and the site of integration may also disrupt a negative regulator of myc transcription. The critical integration leading to insertional mutagenesis is a rare event and it is therefore associated with retroviruses that replicate efficiently in their hosts, infecting many tissues without causing their destruction. Transformation by HTLV-I and -11 is different. These viruses replicate less extensively in their hosts, remaining predominately as latent infections. Within the provirus of these oncoviruses are two additional genes, tax and rex (Fig. 3). The tax protein post-transcriptionally activates other cellular proteins, which in turn bind to the LTR of the provirus to up-regulate its transcription. Its effects are modulated by rex, which favours the transport to the cytoplasm and/or the stability of messenger RNA for the viral structural proteins at

4 194 D. ONIONS Cell DNA - LTR gag POI env LTR Organisation ;$ of FeLV, MuLV proviruses r 2 LTR contains Codes for Codes for Codes for transcriptional control internal proteins reverse transcriptase envelope proteins elements 2 z c-myc organisation Promoters P1 and P2 LTR t Insertion of Retroviral LTR promotes transcription of c-myc leading to hybrid viral-myc mrna LTR LTR - Provirus integrated upstream and in opposite transcriptional orientation to c-myc Exon Enhancer in viral LTR influences transcription from c-rnyc promoters Promoter insertion activation of c-myc Insertional mutagenesis activation of c-myc LTR gag A POI v-myc LTR : Exons 2 and 3 of myc incorporated into retrovirus genome. Viruses of this type are rapidly oncogenic and defectlve for replication Transduction of myc Fig. 2-Interaction of retroviral with oncogenes the expense of those for rex and tax. The transactivation properties of tax are not limited to the LTR and expression of tux is associated with the induction of the IL-2 and IL-2 receptor genes. This phenomenon may account for the in vitro immortalizing effects of HTLV-1 on infection of T-cells. However, the absence of tax transcription in leukaemias produced by HTLV-1 indicates that other processes are involved in the development of the malignant phenotype in vivo. Endogenous retroviruses and transposable genetic elements Laboratory mice are unusual in possessing endogenous retroviruses that may infect their own cells (ecotropic viruses). In certain strains like the AKR, these viruses are expressed efficiently and they may undergo recombination with other retroviral genomes. The frequent reinfection of lymphoid cells by these viruses leads to a high incidence of lymphoma in such strains. In addition, reinfection of the germ line can occur and mutations associated with this process have been documented. In addition to these fully infectious retroviral genomes the murine genome contains intracisternal type-a particles (IAPs). IAPS lack an envelope and are therefore not infectious. However, they can act as transposable elements within a cell by reverse transcription and reintegration of new proviruses. Mice contain about 1000 copies of IAP proviral sequences per haploid genome and they have been associated with the activation mos and m y oncogenes in myelomas as well as the IL-3 gene in a myeloid leukaemia line. Clearly their unregulated expression would constitute a major threat to genomic stability. However, many IAPs are integrated in inactive regions of chromatin and are methylated, a feature common in inactive genes. In addition, there appear to be post-transciptional mechanisms suppressing their expression. Mobile genetic elements like IAPs have been classified as type t retroposons to distinguish them from distantly related class I1 retroposons which again code for reverse transcriptase but lack LTRs. The LINE- 1 elements are a family of abundant class I1 retroposons in mammalian DNA and constitute up to 3 per cent of the DNA in the human genome.

5 INTEGRATION OF VIRUSES INTO CHROMOSOMAL DNA 195 gag pol env PX I I H. In Vitro Transformation - Cellular protein interaction IL-' IL-R GM-CSF f0s T-cell AutoCrine stimulation Fig. 3-HTLV structure and transcription However. ike IAPs most L JE- 1 sequences are not expressed and they have not been associated with insertional mutagenesis in man. The human genome also contains several families of retroviral genomes, most of which are defective and none are known to be expressed as infectious particles. Class I human endogenous retroviruses are related to type C retroviruses like MuLV and FeLV, and class I1 are related to the murine mammary tumour virus (MMTV). Retroviral transcripts from class I proviruses have been detected in placentae, normal spleens, and colon carcinomas, although the expression of viral related proteins remains to be shown. Similarly, class I1 and class I transcripts have been detected in xeast carcinomas. While these genomes have not been shown to express infectious particles, their role as insertional mutagens remains to be resolved. Integration of DNA virus genomes While one group of RNA viruses, the retroviruses, have been shown to be oncogenic, a number of DNA virus groups have been shown to have this property. During productive replication, DNA viruses do not integrate into the chromosomal DNA and they posses genes that maintain them as unintegrated episomal elements. Since the replication of most DNA viruses is a lytic event,

196 D. ONIONS Late gene function Viral DNA replication -2 L d v v i/ <-) Early gene funciion associated with proliferation of basal cells Episomal HPV DNA in nucleus Fig.

6 196 D. ONIONS Late gene function Viral DNA replication -2 L d v v i/ <-) Early gene funciion associated with proliferation of basal cells Episomal HPV DNA in nucleus Fig. &State I Integrated papilloma virus genome - - E6 Open reading frames Coil DNA Pap,lloma NU* genome EZ E5 L L2 El E} Transcription of E6 probably required for transformation of cell E2 disrupted by integration of papilloma virus genomes in warts and carcinomas oncogenic transformation by DNA viruses occurs in the absence of productive replication and is associated with integration of a part of, or the whole of, a virus genome. The major DNA virus groups associated with oncogenesis in their natural hosts are the herpesviruses, papovaviruses (which include the papilloma viruses), and the hepadnaviruses. The hepadnaviruses, to which hepatitis-b virus belongs, are intriguing in that they show a genetic arrangement similar to that of retroviruses and although they are DNA viruses, their replication involves an RNA intermediate and reverse transcription of this RNA into DNA. Evidence for integration of hepatitis-b genomes has been found in most, but not all, hepatocellular carcinomas and efforts have been directed at resolving whether this event could be associated with activation of proto-oncogenes or whether genes within the viral genome like the X gene could have transactivating properties like HTLV- 1 tux. The papilloma viruses are deceptively simple in structure, containing a small circular doublestranded DNA genome. Transcription of this genome is divided into early (E) genes preceding DNA replication and late genes coding for the viral structural proteins. In papillomas, the replication cycle of the papillomavirus genome is in lock step with epithelial differentiation, the genome being maintained as a multicopy plasmid (Fig. 4). In cervical carcinomas, integration of the circular genome is usually associated with opening of and disruption of the ElLE2 region of the genome, whereas the E6 and E7 regions remain intact and may be expressed. The E6 region of the bovine papilloma virus BPV-1 has been shown to be able to transform mouse cells, and this suggests that for transformation to occur, the integration event must permit expression of this region. CONCLUSION Most viruses have probably evolved from eukaryotic genetic elements. The first stage in this process could have been the acquisition of independent replication followed later by the formation of infectious particles. The reintegration of these elements into somatic cells can subvert the cell, leading to its neoplastic transformation, while the infection of germ cells has brought the cycle of viral evolution full circle and resulted in the formation of genetically transposable elements in the mammalian genome. REFERENCES Greene WC, Bohnlein E, Ballard DW. HIV, HTLV-I and normal T-cell growth: transcriptional strategies and surprises. Immunol Today 1989; Yoshida M, Inoue J, Fujisawa J, Seiki M. Trans-regulation of HTLV- 1 gene expression. In: Franza BR, Cullen BR, Wong-Staal F, eds. The Control of Human Retrovirus Gene Expression. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1988; Onions DE, Jarrett 0. Viral oncogenesis: lesions from naturally occurring animal viruses. In: Onions DE, and Jarrett 0, eds. Cancer Surveys, Vol. 6, No. 1. Oxford: Oxford University Press, 1987; Kuff EL. Factors.affecting retrotransposition of intracisternal A- particle proviral elements. In: Lambert ME, McDonald JF, Weinstein

7 INTEGRATION OF VIRUSES INTO CHROMOSOMAL DNA 197 IB, eds. Eukaryotic Transposable Elements as Mutagenic Agents: 30th Banbury Report. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1988; Singer MF, Skowronski J, Fanning TG, Mongkolsuk S. The functional potential of the human LINE-I family of interspersed repeats. In: Lambert ME, McDonald JF, Weinstein IB, eds. EukaryoticTransposable Elements as Mutagenic Agents: Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1988; Robinson WS, Marion PL. Biological features of hepadna viruses. In: Zuckerman AJ, ed. Viral Hepatitis and Liver Disease. New York: Alan R. Liss, 1988; Schwarr E, Schneider-Gadicke A. Expression of human papillomavirus type I8 DNA in cervical carcinoma cell lines. In: Botchan M, Grodzicker T, Sharp PA, eds. DNA Tumor Viruses: Cancer Cells 4. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1986; Sudgen B. Herpesviruses: latent and oncogenic infections by human herpesviruses. In: Botchan M, Grodzicker T, Sharp PA, eds. DNA Tumor Viruses: Cancer Cells 4. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1986; Saiki RK, Gelfand DH, Stoffel S, ef ul. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 237: PCR Protocols. A Guide to Methods and Applications. Innis MA, Gelfand DM, Sninsky JJ, White TJ, eds. New York: Academic Press, 1990.

Viruses Tomasz Kordula, Ph.D.

Viruses Tomasz Kordula, Ph.D. Resources: Alberts et al., Molecular Biology of the Cell, pp. 295, 1330, 1431 1433; Lehninger CD Movie A0002201. Learning Objectives: 1. Understand parasitic life cycle of