Biomedical subjects
Publications and source records attributed to M S Reitz.
Three related, but very different, retroviruses have recently been identified in man. Human T-cell leukaemia virus (HTLV) type I apparently causes adult T-cell leukaemia. HTLV type II has been isolated only rarely and has not been identified with a disease. HTLV type III is very likely the causative agent in acquired immunodeficiency syndrome.
Tetanus-toxoid specific helper-inducer T-cell clones, which had been infected and transformed by human T-cell leukemia-lymphoma virus (HTLV-I), were obtained from an antigen-specific human T cell line by using a limiting dilution technique in the presence of the virus. These HTLV-I-infected T-cell clones proliferated specifically in response to soluble tetanus toxoid but, unlike normal T cells, they could do so in the absence of accessory cells. The HTLV-I-infected T-cell clones did not present the antigen to autologous antigen-specific T cells that were not infected with HTLV-I. The capacity of helper-inducer T cells to retain antigen-specific reactivity after infection by HTLV-I, while losing the normal T-cell requirement for accessory cells, has clinical and theoretical implications.
The nucleotide sequence of a transforming human c-sis complementary DNA shows an open reading frame 723 base pairs in length located downstream from an in-phase terminator thymine-guanine-adenine codon. Sequences within this region were identical to those previously determined for the exons of the normal human c-sis gene. Thus, the predicted transforming product, a protein of 27,281 daltons, may be the actual precursor for normal human platelet-derived growth factor chain A.
Human T-cell leukemia-lymphoma virus (HTLV) is a human C-type retrovirus that can transform T lymphocytes in vitro and is associated with certain T-cell neoplasms. Recent data suggest that, in the United States, patients with acquired immunodeficiency syndrome (AIDS), homosexual men with lymphadenopathy, and hemophiliacs have had significant exposure rates to HTLV, whereas matched and unmatched control American subjects have rarely been exposed to this agent. In the present experiments, T cells specifically reactive against HTLV were propagated from a patient whose HTLV-bearing lymphoma was in remission. The T cells were cloned in the presence of the virus and an HTLV-specific cytotoxic T-cell clone was isolated. This clone was infected and transformed by the virus, with one copy of an HTLV-I provirus being integrated into the genome. This T-cell clone did not exhibit the normal dependence on T-cell growth factor (interleukin-2) and proliferated spontaneously in vitro. Exposure of the clone to HTLV-bearing, autologous tumor cells specifically inhibited its proliferation and resulted in its death. These results may have implications for HTLV-associated inhibition of T-cell responses.
Gibbon ape leukemia virus, SEATO strain (GaLV-SEATO), a virus that induces myeloid leukemia in gibbon apes, and GaLV, San Francisco strain (GaLV-SF), a virus associated etiologically with lymphocytic leukemia in gibbon apes, have been molecularly cloned. The complete nucleotide sequence of the large terminal repeats (LTRs) of both viruses are reported and compared to the previously published nucleotide sequence of the LTR of another member of the same virus group, the simian sarcoma virus (SSV). Substantial homology is evident among all three LTR sequences. The most striking feature of the GaLV-SEATO LTR is the presence of a 45-bp tandem direct repeat in the U3 region, an area likely to contain transcriptional enhancers. Both GaLV-SEATO and GaLV-SF contain a deletion in U3 when compared to SSV. Each of the three LTRs differ from the other two by short deletions in R-U5 and short additions in U3, as well as by numerous point mutations. The possibility that the structural changes observed in the LTR contribute to the differences in the pathogenic effects of these viruses is discussed.
Leukemic peripheral blood lymphocytes from individuals infected with the human T-cell leukemia/lymphoma virus (HTLV) were found to express little or no viral RNA before being put into tissue culture. Within 24-48 hr, viral RNA expression increased at least four- to eightfold. Established HTLV-infected cell lines constitutively express viral RNA. Southern blots of DNA from HTLV-infected cells digested with the methylation-sensitive restriction enzyme HpaII showed that the proviral DNA was methylated in all of the uncultured peripheral blood cells tested. In contrast, no proviral methylation was detected in any of the cell lines examined, suggesting a functional correlation between methylation and viral RNA expression. However, DNA from HTLV-infected lymphocytes cultured for 48 hr (by which time increases in viral RNA expression are evident) did not differ detectably with respect to proviral DNA methylation from uncultured cells, suggesting that the increase in viral RNA expression after short-term culture is mediated by mechanisms independent of changes in DNA methylation.
We have previously shown that two human T-cell lines (HSB and 8402) derived from patients with childhood T-cell ALL (T-ALL) do not synthesize detectable mRNA for HLA-DR alpha. The DR alpha genes in both cell lines are hypermethylated relative to the same genes in T-cell lines infected with human T-cell leukemia virus (HTLV) and derived from patients with adult T-cell leukemia/lymphoma (ATL). These latter cell lines do express HLA-DR alpha-mRNA, as well as HLA-DR surface antigens. We report here that the genes for HLA class I antigens are also highly methylated in the T-ALL T-cell lines relative to the same genes in the ATL T-cell lines, normal peripheral blood T cells, and autologous normal B-cell lines. In spite of substantial differences in the extent of methylation of class I-related genes, no obvious differences exist among these cell types in their levels of expression of HLA-A and -B antigens. The data clearly indicate, however, that the class I and class II components of the major histocompatibility complex are unusually hypermethylated in several T-ALL-derived cell lines, while ATL T-cell lines do not substantially differ in this respect from normal peripheral blood T-cells.
B-cell lines established from two individuals with T-cell acute lymphocytic leukemia (T-ALL) express HLA-DR antigens, whereas the isogenic T-cells do not. The lack of expression correlates with a lack of detectable HLA-DR mRNA. All of the DR alpha DNA sequences detected by a cloned DR alpha cDNA probe are contained in a BglII fragment which varies slightly in size (4.0 to 4.8 kilobases) from one individual to another. In DNA from the T-cells not expressing DR alpha mRNA, all of the potential HpaII sites within the BglII fragment appeared to be methylated. In contrast, at least some of these sites were not methylated in DNA from the B-cells expressing high levels of DR alpha mRNA. Treatment of these T-cells with 5-azacytidine resulted in the induction of DR surface antigen expression, the appearance of DR alpha mRNA, and the partial demethylation of the DR alpha DNA sequences. T-cell lines established from human T-cell leukemia-lymphoma virus associated T-cell neoplasias, in contrast to the T-cell acute lymphocytic leukemia cell lines, expressed both DR antigens and DR alpha mRNA; the HpaII sites within the BglII fragment of DR alpha DNA of these human T-cell leukemia-lymphoma virus-positive T-cell lines were in all cases at least partially unmethylated. Uncultured peripheral blood T-cells from human T-cell leukemia-lymphoma virus-infected individuals expressed DR antigens at a low level, and the DR alpha locus was partially unmethylated. After 48 h in culture, DR antigen expression was substantially increased, but no significant changes were observed in methylation of the DR alpha locus or in the amount of DR mRNA which was present. This suggests that expression of DR antigens also can be modulated post-transcriptionally.
Human T-cell leukemia-lymphoma virus (HTLV) has now been isolated from many different patients with cutaneous T-cell lymphoma and leukemia, as judged by detection of media reverse transcriptase and virus particles and of antigenic determinants related to those of viral structural proteins p24 and p19. Molecular hybridization experiments with HTLV cDNA to viral mRNA or proviral DNA to ascertain the relatedness of four of these new isolates to the first HTLV isolate have been used. By these assays, three appear virtually indistinguishable from the original isolate, HTLV-I(CR), the second U.S. isolate (HTLV-I[MB]), and the Japanese ATLV isolates. Proviral sequences indistinguishable from those of HTLV-I(CR) were also detected in uncultured leukemic blood leukocytes from a patient of Japanese origin with adult T-cell leukemia. These viral isolates thus form a closely related virus group, HTLV-I. In contrast, however, RNA and DNA from one cell line derived from a patient with a T-cell variant of hairy cell leukemia, which expresses media reverse transcriptase and antigenic determinants related to but distinguishable from HTLV p24, did not hybridize substantially with HTLV cDNA. This latter virus appears to represent a second type of HTLV (HTLV-II), related to but substantially different from HTLV-I.
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Human T (thymus-derived)-cell leukemia/lymphoma virus (HTLV) is a new retrovirus first isolated from T-cell lines from a patient with cutaneous T-cell lymphoma from the southeastern United States. Closely related viruses have since been isolated from several patients with adult T-cell leukemia and lymphoma (and some normal persons) from different areas of the world. HTLV is not a genetically transmitted endogenous virus of humans, but it rather is acquired by postzygotic infection. Natural antibodies to several purified viral proteins have been observed in infected individuals. HTLV is transmissible in vitro to human cord blood T cells, and infection results in an increased growth rate, a reduced requirement for (and often independence from) T-cell growth factor, and an abrogation of the crisis period that usually occurs a month after the establishment of normal T-cell cultures. These data suggest that HTLV is the etiologic agent in some human cases of leukemia and lymphoma.
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A retrovirus designated RPMI 8226V, isolated in 1973 from the human myeloma cell line RPMI 8226 has been characterized by competition radioimmunoassay (RIA) for the major viral structural protein and by nucleic acid hybridization analysis using cDNA of the virus. The virus is highly related to the squirrel monkey type D retrovirus, SMRV. In the homologous RIA using rabbit anti-RPMI 8226V and 125I-labelled p37 of RPMI 8226V, RPMI 8226V and SMRV exhibited competition of 81% and 73% respectively. Similarly, in the homologous system for SMRV p36, these viruses competed 98 and 100%. Reagents made from the type D retrovirus. Mason Pfizer Monkey Virus (MPMV), known to be related but distinct from SMRV, were used in assays designed to detect interspecies determinants of type D retroviruses. In assays using goat anti-MPMVp26 vs SMRV 125I-p36, RPMI 8226V, SMRV and MPMV competed to the same extent (93%). Hybridization analysis of RPMI 8226V cDNA showed significant homology to cellular RNA and DNA of mink, bat, and human cell infected with RPMI 8226V and to DNA or SMRV infected cells but not to uninfected cells or cells infected with other viruses. These results taken together clearly indicate that RPMI 8226V and SMRV are very closely related to each other. The finding of a type D retrovirus in this human myeloma cell line that had been used in EBV studies (the usual source of EBV being the marmoset cell line B95-8) prompted a survey of RPMI 8226V in some human and marmoset cell lines. The assays included the RIA for p36, nucleic acid hybridization using cDNA of RPMI 8226V, reverse transcriptase analysis and electron microscopy (EM). The results clearly show that in addition to RPMI 8226, human Burkitt lymphoma cells BJAB/B-95-8/K which were supertransformed by EBV from B-95-8/K marmoset cells as well as marmoset cell lines [(B-95-8/K and B-95-8/N) obtained from Stockholm and Uppsala, Sweden] were positive for the RPMI 8226V. Similar lines obtained elsewhere were negative. The results obtained clearly indicate that RPMI 8226V is a serious laboratory contamination in some widely used human cell lines. The possible impact of this viral contamination for some virological and cell biological studies is discussed.
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A human type C retrovirus, designated HTLV, previously was isolated from or identified in some patients with leukemias and lymphomas of mature T lymphocytes. HTLV is genetically and serologically distinct from any known animal retroviruses. The absence of HTLV proviral sequences in DNA from normal humans showed that HTLV is not a ubiquitous endogenous (germ-line transmitted) virus of humans. Antibodies to HTLV core proteins have been identified in some people with T-cell neoplasias and are particularly prevalent in Japanese with adult T-cell leukemia, suggesting that HTLV is acquired horizontally. However, it was possible that HTLV is transmitted through the germ line of some (possibly rare) families and is then expressed in the HTLV- positive malignancies. An opportunity to study this question was provided by the development of several T-cell lines and a B-cell provided by the development of several T-cell lines and a B-cell line from one HTLV-positive patient with a cutaneous T-cell lymphoma. Here we report that HTLV proteins or nucleic acids (or both) are found in three independently derived T-cell lines, all shown by HLA typing to have originated from the patient. In contrast, the B-cell line, the identity of which was also ascertained by HLA typing, contained no detectable HTLV protein, RNA, or proviral DNA. Because the sensitivity of the latter assay is more than sufficient to detect one proviral equivalent per haploid genome, the results indicate that HTLV was not transmitted to this patient through the germ line but rather was acquired by infection.
Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or point mutations or both exist throughout the entire unique portion of the genome among these viruses.