The Hardy-Zuckerman 2-FeSV, a new feline retrovirus with oncogene homology to Abelson-MuLV

There is substantial evidence that oncogenes (v-onc) of acute transforming retroviruses have been acquired by transduction of cellular genes (c-onc) with retroviruses. Feline leukaemia virus (FeLV)-associated feline fibrosarcomas have proven to be extremely useful for the isolation of acute transforming retroviruses of a mammalian species. Three different v-onc genes have been identified in five acute transforming feline retroviruses. The Susan McDonough feline sarcoma virus (SMFeSV) contains the oncogene fms (ref. 4). The Snyder-Theilen (ST) and Gardner-Arnstein (GA) FeSVs contain the oncogene fes (ref. 4), which is homologous to the oncogene fps of the avian sarcoma viruses FSV, RRCII, PRCIV and 16L (refs 7, 8). The v-onc sequences of the Parodi-Irgens (PI) FeSV have recently been found to be homologous with the v-sis sequences of the simian sarcoma virus. We report here the isolation of another acute transforming feline retrovirus from a naturally occurring feline fibrosarcoma, designated the Hardy-Zuckerman 2 feline sarcoma virus (HZ2-FeSV) and demonstrate that the HZ2-FeSV and Abelson murine leukaemia virus (A-MuLV) have homologous oncogenes.     

Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses

Because of the growing incidence of AIDS (acquired immune deficiency syndrome), the need for studies on animal models is urgent. Infection of chimpanzees with the retroviral agent of human AIDS, the human immunodeficiency virus (HIV), will have only limited usefulness because chimpanzees are in short supply and do not develop the disease. Among non-human primates, both type D retroviruses and lentiviruses can be responsible for immune deficiencies. The D-type retroviruses, although important pathogens in macaque monkey colonies, are not satisfactory as a model because they differ in genetic structure and pathophysiological properties from the human AIDS viruses. The simian lentivirus, previously referred to as simian T-cell lymphotropic virus type III (STLV-III), now termed simian immunodeficiency virus (SIV) is related to HIV by the antigenicity of its proteins and in its main biological properties, such as cytopathic effect and tropism for CD4-bearing cells. Most importantly, SIV induces a disease with remarkable similarity to human AIDS in the common rhesus macaques, which therefore constitute the best animal model currently available. Natural or experimental infection of other monkeys such as African green monkeys or sooty mangabeys has not yet been associated with disease. Molecular approaches of the SIV system will be needed for biological studies and development of vaccines that could be tested in animals. We have cloned and sequenced the complete genome of SIV isolated from a naturally infected macaque that died of AIDS. This SIVMAC appears genetically close to the agent of AIDS in West Africa, HIV-2, but the divergence of the sequences of SIV and HIV-2 is greater than that previously observed between HIV-1 isolates.     

A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family

A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma. The viral genome of HZ4-FeSV contains a new oncogene designated v-kit, has the structure 5' delta gag-kit-delta pol-delta env 3' and specifies a gag-kit polyprotein of relative molecular mass 80,000. The predicted kit amino-acid sequence displays partial homology with tyrosine-specific protein kinase oncogenes. HZ4-FeSV appears to have been generated by transduction of feline c-kit sequences with feline leukaemia virus.     

Expression of the HTLV-III envelope gene by a recombinant vaccinia virus

The discovery that the aetiological agent of acquired immune deficiency syndrome (AIDS) is a retrovirus, referred to as human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) (for review see ref. 1), has raised the possibility of developing a vaccine. In this regard, the envelope (env) proteins of murine retroviruses can induce protective immunity in mice. The HTLV-III env gene specifies a primary polypeptide of approximately 860 amino acids that is glycosylated to form a precursor of relative molecular mass (Mr) 160,000 (gp160), which gives rise to mature membrane-associated proteins of Mr 120,000 (gp120) and 41,000 (gp41). The HTLV-III env gene has been expressed in Escherichia coli and by simian virus 40 (SV40) vectors but formation of the authentic proteins has not been demonstrated. Here, we describe the expression of the complete env gene by a vaccinia virus vector. Evidence is presented that synthesis, glycosylation, processing and membrane transport of the env polypeptide occurred without other HTLV-III gene functions; the env protein was recognized by sera from unrelated AIDs patients; and a single vaccination with the infectious recombinant vaccinia virus induced antibodies to gp120 in mice.     

Expression of AIDS virus envelope gene in recombinant vaccinia viruses

Acquired immune deficiency syndrome (AIDS) is an infectious disease characterized by severe impairment of the patient's cell-mediated immune system. Several lines of evidence have indicated that the aetiological agent of AIDS is a group of T-lymphotropic retroviruses, variously known as lymphadenopathy-associated virus (LAV), human T-lymphotropic virus type III (HTLV-III) and AIDS-associated retrovirus (ARV). Serological surveys have indicated that as many as one million people in the United States may have been infected by LAV/HTLV-III, and the spread of AIDS has become a global concern. The need for a better understanding of the viral immunology and for a vaccine against AIDS is self-evident. To this end, we have constructed recombinant vaccinia viruses containing the envelope (env) gene of LAV, and demonstrate here that cells infected with these viruses express immunoreactive proteins similar to those present on LAV virions. Experimental animals infected with these recombinant viruses elicited antibodies that specifically recognized LAV envelope proteins.     

Different H-2 subregions influence immunization against retrovirus and immunosuppression

Friend murine leukaemia virus complex (FV) causes an immunosuppressive retrovirus-induced disease. In certain mouse strains, FV shows striking similarities to human immunodeficiency virus (HIV) infection in man in that infected mice have severe T-cell immunosuppression but also develop virus-neutralizing antibodies incapable of eliminating infected cells. Previously we noted the influence of mouse major histocompatibility complex (H-2) genes on both FV-induced immunosuppression and on ability to protect mice against FV by immunizing with a vaccinia-Friend murine leukaemia helper virus (F-MuLV) envelope (env) recombinant virus. Here we show that different subregions of H-2 are involved in susceptibility to virus-induced immunosuppression (H-2D subregion) and protective immunization with a recombinant vaccinia virus (H-2K or I-A subregions). Thus, susceptibility to virus-induced immunosuppression does not preclude protection by vaccinia-Friend immunization. The mechanism of protection seems to involve priming of immune T cells, and not initial induction of neutralizing antibodies or cytotoxic T lymphocytes (CTL) (ref.2). Subsequent virus challenge generates a secondary response, resulting in appearance of IgG antibodies and CTL. In human HIV infection there could also be host genetic influences on elements of disease pathogenesis, such as immunosuppression, and on the success of T-cell priming by potential protective vaccines.     

Disease-specific and tissue-specific production of unintegrated feline leukaemia virus variant DNA in feline AIDS

Feline leukaemia viruses (FeLVs) have long been known to be associated with induction of proliferative and anti-proliferative diseases of domestic cats. Strains of FeLV have been recognized which specifically induce lymphosarcoma, aplastic anaemia, myelodysplastic anaemia, and, recently, feline AIDS (acquired immune deficiency syndrome), a naturally occurring immunosuppressive syndrome strikingly similar to human AIDS which is lethal in 100% of inoculated and viraemic specific-pathogen-free (SPF) cats. Here, we have analysed FeLV DNA in tissues of 22 SPF cats that had been inoculated with the feline AIDS strain (FeLV-FAIDS) and we find two classes of viral DNA--a monotypic common form which is detectable in bone marrow regardless of disease state, and variant forms, recognizable by restriction site differences, whose appearance correlates with onset of disease symptoms and persists throughout the course of the disease. FeLV-FAIDS variant DNA is detected at high concentration (10-50 copies per cell) and principally as unintegrated viral DNA (UVD) in bone marrow of cats with feline AIDS. In marked contrast high levels of UVD were not present in cats in the terminal-stages of T-cell lymphosarcoma, aplastic anaemia, or myelodysplastic anaemia induced by other FeLV strains. These results parallel recent observations in humans, where high levels of UVD were sometimes found in cells derived from AIDS patients infected with human T-lymphotropic virus type III (HTLV-III)/lymph-adenopathy-associated virus (LAV), and suggest that persistence of unintegrated variant viral DNA is a crucial indicator of retrovirus-induced cytopathic disease syndromes such as AIDS.     

Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor

Retrovirus protease is an enzyme that cleaves gag and gag-pol precursor polyproteins into the functional proteins of mature virus particles. The correct processing of precursor polyproteins is necessary for the infectivity of virus particles: in vitro mutagenesis which introduces deletions into the murine leukaemia virus genome produces a protease-defective virus of immature core form and lacking infectivity. A therapeutic drug effective against disease caused by retrovirus proliferation could likewise interfere with virus maturation. The primary structure has so far been determined for the protease of avian myeloblastosis virus, and of murine, feline and bovine leukaemia viruses. Amino acid sequencing of the retrovirus proteases, either after their purification or from prediction from the nucleotide sequence, shows that they possess the Asp-Thr-Gly sequence characteristic of the aspartyl proteinases. In this report we show that retrovirus proteases belong to the aspartyl proteinase group and demonstrate an inhibition by the aspartyl proteinase-specific inhibitor, pepstatin A, on the activity of bovine leukaemia, Moloney murine leukaemia and human T-cell leukaemia virus proteases.     

Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor

Friend spleen focus-forming virus (SFFV) is a defective murine C-type retrovirus which causes a multi-stage erythroleukaemia in mice and erythroblastosis in bone marrow cultures. The SFFV env gene encodes a membrane glycoprotein, gp55, which is located on the cell surface and in the rough endoplasmic reticulum and is essential both for the induction of leukaemia in vivo and erythroblast proliferation in vitro. The mechanism by which gp55 causes increased erythroblastosis and ultimately leukaemia is unknown, but a reasonable suggestion is that gp55 can mimic the action of erythropoietin by binding to its receptor (Epo-R), thereby triggering prolonged proliferation of erythroid cells. To test this possibility, we have co-expressed gp55 and the murine Epo-R in a fibroblast cell line. We show here that in such cells, the SFFV glycoprotein binds directly to Epo-R. Furthermore, when an interleukin-3 (IL-3)-dependent lymphoid cell line was co-infected by SFFV and a virus that carries the Epo-R gene, it could grow without IL-3. We suggest that through direct binding to Epo-R, gp55 can stimulate the receptor and by-pass the normal requirement for Epo, causing prolonged proliferation of infected erythroid cells. This could be the first step of leukaemogenesis induced by Friend virus.     

Macrophage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gp120 gene

Strains of human immunodeficiency virus type 1 (HIV-1) display a high degree of biological heterogeneity which may be linked to certain clinical manifestation of AIDS. They vary in their ability to infect different cell types, to replicate rapidly and to high titre in culture, to down-modulate the CD4 receptor, and to cause cytopathic changes in infected cells. Some of these in vitro properties correlate with pathogenicity of the virus in vivo. To map the viral determinants of the cellular host range of HIV-1, recombinant viruses were generated between biologically active molecular clones of HIV-1 isolates showing differences in infection of primary peripheral blood macrophages and established T-cell lines. We report here that a specific region of the envelope gp120 gene representing 159 amino-acid residues of glycoprotein gp120 seems to determine macrophage tropism, whereas an overlapping region representing 321 amino-acid residues determines T cell-line tropism. These studies provide a basis for relating functional domains of the HIV-1 env gene to pathogenic potential.     

Human T-lymphotropic retroviruses

The first human retroviruses have been discovered during the past six years. They cause two diseases which involve disturbances of the growth of the T4 lymphocyte, a remarkably specific target cell type. This cell, which is central to the regulation of the immune system, is induced by human T-lymphotropic virus type I (HTLV-I) to excessive proliferation (leukaemia) and by HTLV-III to premature death (acquired immune deficiency syndrome, AIDS). Both also seem to be indirectly involved in several other disorders. The genetic structures of these retroviruses and the mechanisms by which they usurp host-cell functions are novel among retroviruses.     

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.     

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.     

Relation of HTLV-4 to simian and human immunodeficiency-associated viruses

Human immunodeficiency virus type 1 (HIV-1) is the aetiologic agent of AIDS (acquired immune deficiency syndrome) in most countries and probably originated in Central Africa like the AIDS epidemic itself. Evidence for a second major group of human immunodeficiency-associated retroviruses came from a report that West African human populations like wild-caught African green monkeys had serum antibodies that reacted more strongly with a simian immunodeficiency virus (STLV-3Mac) (ref.6) than with HIV-1. Novel T-lymphotropic retroviruses were reported to have been isolated from healthy Senegalese West Africans (HTLV-4) (ref. 4) and from African green monkeys (STLV-3AGM) (ref. 7), and a different retrovirus (HIV-2) was identified in other West African AIDS patients. Genomic analysis of HIV-2 clearly distinguished it from STLV-3 (ref. 9), but restriction enzyme site-mapping of three different HTLV-4 isolates and six different STLV-3AGM isolates showed them to be essentially indistinguishable. In this report we clone, restriction map, and partially sequence three isolates of HTLV-4 (PK82, PK289, PK190) (ref. 4). We find that these viruses differ in nucleotide sequence from each other and from three isolates of STLV-3AGM (K78, K6W, K1) (ref. 7) by 1% or less. We also report the isolation of a T-lymphotropic retrovirus from the peripheral blood of a healthy Senegalese woman which hybridizes preferentially to HIV-2 specific DNA probes. We conclude that HTLV-4 (ref. 4) and STLV-3AGM (ref. 7) are not independent virus isolates and that HIV-2 is present in Senegal as it is in other West African countries.     

Surface phenotypes in T-cell leukaemia are determined by oncogenic retroviruses

Spontaneous thymic leukaemia in experimental mice is the result of a complex series of genetically controlled events. An important step in this process involves the production by thymocytes of recombinant polytropic retroviruses (MCF viruses). These leukaemogenic agents arise by recombination of genes from the env regions of endogenous precursor viruses. Sequences in these regions encode the envelope glycoprotein gp70 (ref. 6). Thus far, each cloned isolate of recombinant virus from AKR and HRS/J mice has been found to possess unique oligonucleotide sequences in its env region, as well as clone-specific peptides in its gp70 (refs 7,8). Therefore, the polytropic viruses of these leukaemia-susceptible mice are extremely diverse. These findings suggest that random recombination of env genes gives rise to leukaemogenic polytropic viruses. McGrath and Weissman have proposed that thymocytes with cell surface receptors for the gp70 of a particular leukaemogenic virus are the target cells for malignant transformation by that specific virus. In view of the diversity of polytropic viral gp70, their hypothesis would predict extensive phenotypic diversity among spontaneous thymic leukaemias. In contrast, leukaemias induced by a particular leukaemogenic recombinant virus would always have the same phenotype. Here we verify these predictions experimentally.     

Recovery of immunodeficient mice from a vaccinia virus/IL-2 recombinant infection

Vaccinia virus recombinants that express cloned genes encoding antigens of unrelated infectious agents, such as hepatitis B virus and human immunodeficiency virus (HIV), provide a new approach to the development of live vaccines. Although there is evidence that genetically engineered vaccinia viruses have reduced pathogenicity a major obstacle to their use as vaccines is that severe complications can occur after vaccination, especially in immunodeficient individuals. We describe here a recombinant vaccinia virus expressing murine interleukin-2 (IL-2) and show that athymic nude mice infected with the recombinant virus resolve the virus infection rapidly whereas mice infected with control virus develop a progressive vaccinal disease. By incorporating the gene for IL-2 in live virus vaccines it may be possible to prevent the severe complications that arise in recipients with an impaired immune system.     

A survey of human leukaemias for sequences of a human retrovirus

Human T-cell leukaemia-lymphoma virus (HTLV) is an exogenous human retrovirus distinct from all known animal retroviruses. HTLV is closely linked to a subtype of adult T-cell malignancies and except for isolated cases, has not been found associated with any other form of leukaemia, lymphoma or other cancers (see refs 1, 2 for review). HTLV can be transmitted to cord blood T lymphocytes in vitro and the infected cells exhibit characteristics of transformed neoplastic T cells. We have recently cloned DNA sequences derived from approximately 1 kilobase (kb) of the 5' and 3' termini of the HTLV genome, as well as a 4-5-kb defective HTLV provirus flanked by cellular sequences. The availability of these probes has enabled us to carry out a limited survey of different fresh or cultured cells from patients of different lymphoid and myeloid malignancies for HTLV-related DNA sequences. The results presented here show that cells from all Japanese patients with adult T-cell leukaemia and several patients with various mature T-cell malignancies from elsewhere contained one or more copies of a highly conserved HTLV genome. The infected cells are of clonal origin. Fresh cells from 1 of the 10 myeloid leukaemic patients contained exogenous DNA sequences distantly related to HTLV.