Nonspecific integration of the HTLV provirus genome into adult T-cell leukaemia cells

Human T-cell leukaemia virus (HTLV), previously also reported as ATLV, is a recently identified retrovirus which is closely associated with adult T-cell leukaemia (ATL) endemic in southwestern Japan and the Caribbean. Determination of the total nucleotide sequence of the HTLV genome has revealed no typical onc gene acquired from the cellular sequence. Screening of the HTLV provirus genome in tumour cells has shown that in all cases of ATL examined, the primary tumour cells contained the provirus genome and were monoclonal with respect to the integration site of the provirus. These findings suggest that ATL leukaemogenesis may be due to insertional mutagenesis in which the provirus genome is integrated into a specific locus on the chromosomal DNA and then activates an adjacent cellular onc gene, a mechanism already demonstrated in avian lymphoma and erythroblastosis induced by avian leukosis viruses. A common site of HTLV provirus integration in leukaemic cells among some ATL patients was reported by Hahn et al. but subsequently retracted. However, this retraction does not imply the random integration of the proviruses. Independently, we have been testing this insertional mutagenesis model in ATL and report here that the provirus did not have a common locus of integration in 35 ATL patients and did not integrate on the same chromosome in 2 ATL patients.     

Common site of integration of HTLV in cells of three patients with mature T-cell leukaemia-lymphoma

Human T-cell leukaemia-lymphoma virus (HTLV) was first isolated in the United States from a patient with an aggressive form of cutaneous T-cell lymphoma (CTCL) and was later found associated with clusters of adult T-cell leukaemia-lymphomas (ATL) in various parts of the world, including Japan and the Caribbean. Leukaemic cells of the HTLV-positive patients seem to be clonal expansions of single infected cells since the provirus(es) are found at the same sites in a given patient. In avian leukosis virus-induced B-cell lymphomas, the provirus very frequently integrates at several discrete sites in a common domain near the cellular gene, c-myc, that it activates and it has been speculated that the same would hold true for other chronic leukaemia viruses. We report here that cultured cells from two US patients with CTCL and fresh leukaemia cells of a Japanese patient with ATL contained an HTLV provirus integrated at the same site. In addition, a cord blood T-lymphocyte cell line established by co-cultivation with one of the two HTLV-positive CTCL cell lines also contained HTLV provirus contiguous with the same flanking cellular sequences. Ten other HTLV-positive cell samples did not show integration of HTLV at this site, suggesting that there is more than one discrete site of HTLV integration in tumour cells.     

Dilute (d) coat colour mutation of DBA/2J mice is associated with the site of integration of an ecotropic MuLV genome

The single endogenous DBA/2J ectropic provirus segregated concordantly with the dilute (d) coat colour mutation on chromosome 9 in 53/53 DBA/2J-derived recombinant inbred mouse strains and all seven inbred and mutant strains tested that carry the d allele. Analysis of DNA from a spontaneous DBA/2J d revertant (d+2J) showed that these mice lack ecotropic-specific MuLV DNA sequences and suggested that the dilute mutation resulted from integration of an ecotropic provirus into the mouse genome.     

Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity

The pattern of DNA methylation changes during development of eukaryotes, and hypomethylation frequently correlates with gene expression (for reviews see refs 1-4). A causal relationship between hypermethylation and gene inactivity has been established for retroviral genomes which are methylated de novo when inserted into the germ line of mice (ref. 5; for review, see ref. 6). The mutual interaction of the provirus with the host genome can influence virus expression and can result in inactivation of the host gene by insertional mutagenesis. We report here that the insertion of a provirus can change the methylation pattern of the host DNA. Sequences flanking the provirus become methylated de novo within 1 kilobase (kb) of the integration site. In Mov-13 mice, which carry a lethal mutation of the alpha 1(I) collagen gene, de novo methylation of host DNA is associated with a change in chromatin conformation. This suggests that virus-induced DNA methylation can alter DNA-protein interactions and thereby interfere with correct gene activation during embryonic development.     

Interferon inhibits transformation by murine sarcoma viruses before integration of provirus

Neoplastic transformation by C-type retroviruses requires synthesis of a DNA copy (the provirus) of the RNA genome and its integration into the host cell DNA. We have previously shown that interferon (IFN) can stably prevent transformation of murine fibroblasts by the Kirsten strain of murine sarcoma virus (KiMSV), a murine leukaemia virus (MLV). A series of cell clones (IFN clones), isolated in the presence of IFN (10(4) U ml-1) from cultures of NIH-3T3 cells which had been treated with IFN, and then infected with KiMSV (KiMLV) in conditions where every cell was infected, were shown to be phenotypically untransformed. These untransformed cells did not produce virus or contain rescuable KiMSV. However, cells isolated using an identical procedure, but in the absence of IFN, were uniformly transformed and all produced KiMSV (KiMLV) or contained rescuable KiMSV. It was concluded that IFN either prevents synthesis or integration of the provirus, or else that in the presence of IFN the provirus is integrated such that it is not expressed. We now show that five representative clones contain no detectable KiMSV proviral DNA, and also that the initial stages of infection by KiMSV (KiMLV) are inhibited by IFN treatment. IFN seems to act before integration, preventing either the synthesis or the integration of proviral DNA.     

The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus

Acquired immune deficiency syndrome (AIDS) is characterized by opportunistic infections and by 'opportunistic neoplasms' (for example, Kaposi's sarcoma). Persistent generalized lymphadenopathy (PGL) is epidemiologically associated with AIDS, especially in male homosexuals. A subset of T lymphocytes positive for the CD4 antigen (also termed T4 antigen), is depleted in AIDS and PGL patients. A retrovirus found in T-cell cultures from these patients is strongly implicated in the aetiology of AIDS because of the high frequency of isolation and the prevalence of specific antibodies in the patients. Here we have detected cell-surface receptors for the AIDS retrovirus (human T-cell leukaemia virus-III (HTLV-III) and lymphadenopathy-associated virus-1 (LAV-1) isolates) by testing the susceptibility of cells to infection with pseudotypes of vesicular stomatitis virus bearing retroviral envelope antigens, and by the formation of multinucleated syncytia on mixing virus-producing cells with receptor-bearing cells. Receptors were present only on cells expressing CD4 antigen; among 155 monoclonal antibodies tested, each of the 14 anti-CD4 antibodies inhibited formation of syncytia and blocked pseudotypes. Productive infection of CD4+ cells with HTLV-III or LAV-1 markedly reduced cell-surface expression of CD4. In contrast, receptors for HTLV-I and HTLV-II were not restricted to CD4+ cells, were not blocked by anti-CD4 antibodies; cells productively infected with HTLV-I and HTLV-II expressed surface CD4. Hence, we conclude that the CD4 antigen is an essential and specific component of the receptor for the causative agent of AIDS.     

High frequency of unequal recombination in pseudoautosomal region shown by proviral insertion in transgenic mouse

The mammalian X and Y chromosomes, in contrast to the autosomes, pair during male meiosis only near the telomeres. Alleles localized in this region can undergo reciprocal exchange during meiosis. Because such sequences do not show strict sex-linked inheritance, they have been termed pseudoautosomal. In man, several DNA sequences have been described which show pseudoautosomal transmission and which are localized in the pairing region at the ends of the short arms of both the X and Y chromosomes (refs 6-9, and D. Page, unpublished results). We now show that the transgenic mouse strain, Mov-15, contains a single Moloney murine leukaemia virus (M-MuLV) genome in its germline, and genetic evidence indicates that the provirus is integrated into the pseudoautosomal region of the sex chromosome. Proviral copies are lost or gained in 7% of male meioses in this strain, and mouse sequences flanking the provirus are tandemly repeated and highly variable. We conclude that unequal recombination events occur with high frequency in the pairing region, possibly because of the presence of repeated sequences.     

Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus

The 9,213-nucleotide structure of the AIDS/lymphadenopathy virus has been determined from molecular clones representing the integrated provirus and viral RNA. The sequence reveals that the virus is highly polymorphic and lacks significant nucleotide homology with type C retroviruses characterized previously. Together with an analysis of the two major viral subgenomic RNAs, these studies establish the coding frames for the gag, pol and env genes and predict the expression of a novel gene at the 3' end of the genome unrelated to the X genes of HTLV-1 and -II.     

Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus

Proviruses cloned from rat cells infected with mouse mammary tumour virus, a B-type retrovirus regulated by glucocorticoid hormones, show the structural features of transposable elements: short inverted repeats conclude long direct repeats at the ends of viral DNA, and short sequences of cellular DNA are duplicated during integration and flank each provirus. The integrative mechanism joins a precise site in viral DNA to non-homologous sites in host DNA.     

Lack of evidence for involvement of known human retroviruses in multiple sclerosis

The recent report by Koprowski et al. that human T-cell lymphotropic retroviruses (HTLVs) may be involved in the development of multiple sclerosis (MS) has aroused much interest. The report was based largely on immunological evidence, using enzyme-linked immunosorbent assays (ELISAs) with viral antigens or disrupted virions. We have accordingly sought confirmation by screening sera and cerebrospinal fluid (CSF) samples from MS patients against cell lines infected respectively with adult T-cell leukaemia (ATL) virus (ATLV/HTLV-I) of Japanese cells (MT-1 and MT-2 lines), our own isolate from British black patients with ATL, the MoT cell line which produce HTLV-II, and our own T-cell line containing a local isolate of acquired immune deficiency syndrome (AIDS) virus (C-LAV/HTLV-III). We have failed to find antibodies against these retroviruses in the sera or CSF. Furthermore, neither virus could be isolated from the peripheral white blood cells of two MS patients.     

Molecular characterization and expression of the gene encoding human erythroid-potentiating activity

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lymphotropic virus type II (HTLV-II). This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA). Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.     

Detection of virus-specific sequences in Drosophila melanogaster mutants induced by injection of RSV DNA into early embryos

The injection of purified Rous sarcoma virus (RSV) (Prague strain) into Drosophila melanogaster (Oregon R line) eggs changes the fly phenotype in certain cases, and RSV-specific sequences can be identified in the Drosophila genome (ref. 1 and preceding paper). Here we have used Southern blotting to analyse in greater detail the proviral DNA present in several mutant lines of D. melanogaster produced by microinjection of intact RSV or plasmid DNA containing the viral insert. In certain populations of flies, RSV provirus was found to be incorporated into cellular DNA, and in one mutant family the unintegrated form of plasmid DNA was identified. Generally, the presence of injected genetic material in fly cells correlated with morphological changes in Drosophila.     

JD病毒基因组片段的克隆和序列分析

扩增并克隆了ＪＤ病毒的ｇａｇ 基因片段（位于３００ｎｔ～５６４ｎｔ,编码基质蛋白ＭＡ）和ｐｏｌ基因片段（位于３４６７ｎｔ～３６９１ｎｔ,编码反转录酶ＲＴ）,测定其序列并同ＢＩＶ、ＢＦＶ 和ＢＬＶ 的相应基因进行比较．所建立的方法能够特异性扩增ＪＤＶ序列,适用于ＪＤＶ的检测