Retroviral transduction of T-cell antigen receptor beta-chain and myc genes

Support for multistage models of oncogenesis has been provided by several highly leukaemogenic retrovirus isolates that have transduced more than one host cell gene. Where functional studies have been performed, these retroviral oncogenes show synergy for in vitro transformation and leukaemogenesis. In naturally occurring feline leukaemias associated with feline leukaemia virus (FeLV), retroviral transduction of myc is a frequent oncogenic mechanism. But evidence suggesting that the FeLV v-myc genes might be insufficient for leukaemogenesis was provided by the latency (12 weeks) and clonality of FeLV/v-myc-induced tumours and the absence of demonstrable in vitro transformation by these viruses. In the search for secondary leukaemogenic events in FeLV/v-myc tumours, we have identified a case of FeLV transduction of a T-cell antigen receptor beta-chain gene. The proviruses carrying this gene (which we have named v-tcr) were a separate population from those carrying v-myc. In its normal role, the T-cell receptor beta-chain forms part of a multimeric complex involved in antigen recognition and T-cell activation. We suggest that v-tcr is a novel viral oncogene which assisted v-myc in the genesis of a naturally occurring case of thymic lymphosarcoma.     

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.     

mRNA transcripts related to full-length endogenous retroviral DNA in human cells

Mammalian cells contain multiple copies of endogenous type C retroviral DNA sequences. Among these sequences are complete, potentially infectious proviruses, proviral DNA that is expressed only in the form of viral antigens, retroviral segments that may contribute portions of envelope (env) genes during the generation of recombinant polytropic viruses, and many subgenomic viral DNA segments that may not be expressed at all. We have previously reported the identification and molecular cloning of type C retroviral sequences from human DNA and have shown that the partial nucleotide and deduced amino acid sequences of one of the clones obtained (lambda 51) are homologous to Moloney MuLV (MoMuLV) in the gag and pol regions. The lambda 51 clone as well as several others isolated from a human DNA library contained approximately 4.3 kilobases (kb) of retroviral sequences, were deleted in the env region, and were flanked by tandem repeats unlike the long terminal repeats (LTRs) typically found in proviral DNAs (P.E.S., in preparation). We describe here the characterization of a full-length human retroviral clone (lambda 4-1) containing LTR elements as well as a putative env region. DNA-RNA hybridization experiments reveal that human cells contain species of poly(A)+ RNA that anneal to segments of the full-length retroviral DNA clone.     

Isolation of a replication-defective murine leukaemia virus from cultured AKR leukaemia cells.

Mice of the AKR strain are characterised by a high incidence of spontaneous thymic lymphomas. AKR chromosomes contain the genomes of ecotropic murine leukaemia virus (MuLV) at two loci, termed Akv-1 and Akv-2 (refs 2-6). Shortly after birth, the normal tissues of AKR mice begin to produce high levels of this XC-positive MuLV (ref. 7) (that is, one that forms XC plaques). A second class of MuLV, termed mink cell focus-inducing virus (MCF), is produced specifically by preleukaemic and leukaemic AKR thymocytes. Nowinski et al. have established a series of tissue culture lines from AKR leukaemias and reported that the resulting cell lines produce virus particles, but that these particles, surprisingly, do not give rise to XC plaques. We have analysed the virus particles produced by one of these cell lines, termed AKRSL2. We show here that, unlike most or all of the nonmalignant tissues in the AKR mouse, these cultured lymphoma cells produce very little non-defective ecotropic MuLV; however, they do produce replication-defective ecotropic MuLV.     

Spi-1 is a putative oncogene in virally induced murine erythroleukaemias

Retroviral insertional mutagenesis has been proposed as an efficient mechanism to turn on or to increase the expression of oncogenes in several avian or mammal models. Integration site studies of avian leukosis virus, murine leukaemia and murine mammary tumour viruses led to the coleutification of highly conserved genes whose expression is induced or increased during leukaemogenesis, probably through enhancer elements present in the retroviral long terminal repeats. This is reminiscent of the activation of cellular proto-oncogenes or putative oncogenes in numerous human tumours and leukaemias as a result of chromosomal translocations or DNA rearrangements. Here we report the characterization of a new putative oncogene isolated from a murine erythroleukaemia induced by the acute leukaemogenic retrovirus spleen focus forming virus (SFFV). An important and unusual feature of this genomic locus Spi-1 (for SFFV proviral integration) is that rearrangements due to SFFV integration were found in 95% of the erythroid tumours studied. A 4.0-kilobase messenger RNA was detected in rearranged tumours. No Spi-1 rearrangement was detected in other virally induced myeloid, lymphoid or erythroid tumours tested.     

T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses

There is much interest in developing vaccines against acquired immune deficiency syndrome (AIDS), which is caused by a retrovirus termed human immunodeficiency virus (HIV). Isolates of this virus include human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Several approaches towards the development of an AIDS vaccine result in the production of antibodies in subprimates. These methods involve the use of: antigens isolated from the AIDS virus; viral antigens expressed by transfected cells or by recombinant vaccinia viruses; and particular synthetic peptides of viral antigens. Because T-cell-mediated immunity (in addition to antibodies) is involved in resistance to diseases and death caused by various enveloped viruses, we sought to determine whether potential AIDS vaccines can induce T-cell responses against the AIDS virus. Here we report that immunization of non-human primates, Macaca fascicularis (macaques), with recombinant vaccinia viruses that express LAV envelope glycoproteins gp41 and gp110 results not only in the production of antibodies against the LAV envelope antigens but also in the generation of T-cells that proliferate and produce the lymphokine interleukin-2 (IL-2), in response to stimulation with purified LAV. We believe this is the first report demonstrating T-cell-mediated immunity to the virus that causes AIDS.     

Deletion of the human T-cell receptor delta-gene by a site-specific recombination

The newly described T-cell receptor (TCR) delta locus is located inside the TCR alpha locus, between variable region (V)alpha and joining region (J)alpha. Although the delta and alpha TCR genes are physically linked on the same chromosome, they are sequentially expressed during T-cell development. This implies the existence of a highly efficient regulatory mechanism by which these two genes are independently rearranged. We have recently described a genetic element 'T early alpha' (TEA) in humans transcribed in foetal thymocytes, spliced alternatively to constant region (C)alpha, and located between the TCR-delta locus (5') and the group of J alpha segments (3'). Importantly, TEA flanks a common site of rearrangement in the thymus, and distinguishes cells using TCR-gamma/delta (TEA in germline configuration) from cells using TCR-alpha/beta (TEA deleted on both chromosomes). In order to understand this TEA-associated recombination we analysed genomic clones representing these thymic rearrangements. We show that the TEA-associated recombination deletes the delta locus before productive (V delta D delta J delta) rearrangement. The diversity (D)delta and J delta regions, which provide the major source of delta gene diversity, are eliminated as a consequence of delta gene deletion and cannot then be used in conjunction with an alpha-TCR. We propose that the TEA-associated deletion of TCR-delta precedes the formation of an alpha-TCR and could down-regulate TCR-delta formation in maturing thymus.     

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.     

BIV92044功能片段的克隆和序列分析

本文克隆了BIV92044毒株的RT段（位于pol基因2254nt～2497nt，编码病毒反转录酶）和env段（位于env基因6060nt～6683nt，编码外膜精蛋白gp100）序列，并对这两段克隆进行了序列分析，结果表明，BIV92044的上述两段与BIVR29的差异很小，序列同源性分别为99％和96％．     

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.     

HIV infection is blocked in vitro by recombinant soluble CD4

The T-cell surface glycoprotein, CD4 (T4), acts as the cellular receptor for human immunodeficiency virus, type 1 (HIV-1), the first member of the family of viruses that cause acquired immunodeficiency syndrome. HIV recognition of CD4 is probably mediated through the virus envelope glycoprotein (gp120) as shown by co-immunoprecipitation of CD4 and gp120 (ref.5) and by experiments using recombinant gp120 as a binding probe. Here we demonstrate that recombinant soluble CD4(rsT4) purified from the conditioned medium of a stably transfected Chinese hamster ovary cell line is a potent inhibitor of both virus replication and virus-induced cell fusion (syncytium formation). These results suggest that rsT4 is sufficient to bind HIV, and that it represents a potential anti-viral therapy for HIV infection.     

牛免疫缺陷病毒(BIV)在中美两国流行情况的比较(英文)

牛免疫缺陷病毒是反转录病毒科慢病毒属的重要成员,已在一些国家相继发现ＢＩＶ的感染和蔓延。本文以重组ＢＩＶ衣壳蛋白和包膜蛋白为抗原,通过免疫印迹技术发现,不仅我国进口奶牛及其后代存在一定比例的ＢＩＶ感染（血清阳性率为２．３２％）,而且ＢＩＶ已开始在我国北方牛群中传播（阳性率约３．８％）。上述结果已被聚合酶链式反应及其产物的狭缝杂交所证实,并与美国中部及南部地区ＢＩＶ流行情况进行了比较。     

Genetic recombination between RNA components of a multipartite plant virus

Genetic recombination of DNA is one of the fundamental mechanisms underlying the evolution of DNA-based organisms and results in their diversity and adaptability. The importance of the role of recombination is far less evident for the RNA-based genomes that occur in most plant viruses and in many animal viruses. RNA recombination has been shown to promote the evolutionary variation of picornaviruses, it is involved in the creation of defective interfering (DI) RNAs of positive- and negative-strand viruses and is implicated in the synthesis of the messenger RNAs of influenza virus and coronavirus. However, RNA recombination has not been found to date in viruses that infect plants. In fact, the lack of DI RNAs and the inability to demonstrate recombination in mixedly infected plants has been regarded as evidence that plants do not support recombination of viral RNAs. Here we provide the first molecular evidence for recombination of plant viral RNA. For brome mosaic virus (BMV), a plus-stranded, tripartite-genome virus of monocots, we show that a deletion in the 3' end region of a single BMV RNA genomic component can be repaired during the development of infection by recombination with the homologous region of either of the two remaining wild-type BMV RNA components. This result clearly shows that plant viruses have available powerful recombinatory mechanisms that previously were thought to exist only in animal hosts, thus they are able to adapt and diversify in a manner comparable to animal viruses. Moreover, our observation suggests an increased versatility of viruses for use as vectors in introducing new genes into plants.     

Expression of endogenous murine leukaemia viruses in AKR/J streaker mice

MICE of the AKR/J strain show high rates of spontaneous thymic lymphoma and are chronically infected with murine leukaemia viruses. Total thymectomy in young mice of this strain results in a marked decrease in the incidence of leukaemia. The finding of athymic mutant mice on the AKR/J background (referred to as streaker mice) is of importance in the study of genetic factors which influence virus expression and leukaemogenesis. Here we report that mice of both normal and mutant genotypes vary in the expression of a particular class of murine leukaemia virus. This lower virus expression in conjunction with the absence of a thymus leads to a lower tumour incidence in streaker mice.     

Variable and conserved neutralization antigens of human immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1, HTLV-III/LAV), the retrovirus responsible for acquired immune deficiency syndrome (AIDS), shows a high degree of genetic polymorphism, particularly in the env gene. We have examined sera from rabbits and guinea pigs immunized with gp130, a recombinant env glycoprotein, and sera from HIV-1-infected subjects, to test their capacity to neutralize a panel of genetically divergent HIV-1 isolates. The sera raised against recombinant antigen specifically neutralized the virus strain from which the env gene was cloned (HTLV-IIIB), but not an independent isolate (HTLV-IIIRF). One rabbit serum tested on seven isolates cross-neutralized two at lower titres. In contrast, human sera from Britain and Uganda, chosen for ability to neutralize HTLV-IIIRF, cross-neutralized six other HIV-1 isolates. When serum and isolate were derived from the same subject, the serum was in some cases effective at slightly lower concentrations (higher titres). Human complement did not affect neutralization titres. These findings indicate that genetically diverse HIV-1 isolates carry both variable and widely conserved antigenic epitopes for neutralizing antibodies. The identification of shared epitopes may help the development of protective vaccines.     

Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus

The 'Spanish' influenza pandemic of 1918-19 was the most devastating outbreak of infectious disease in recorded history. At least 20 million people died from their illness, which was characterized by an unusually severe and rapid clinical course. The complete sequencing of several genes of the 1918 influenza virus has made it possible to study the functions of the proteins encoded by these genes in viruses generated by reverse genetics, a technique that permits the generation of infectious viruses entirely from cloned complementary DNA. Thus, to identify properties of the 1918 pandemic influenza A strain that might be related to its extraordinary virulence, viruses were produced containing the viral haemagglutinin (HA) and neuraminidase (NA) genes of the 1918 strain. The HA of this strain supports the pathogenicity of a mouse-adapted virus in this animal. Here we demonstrate that the HA of the 1918 virus confers enhanced pathogenicity in mice to recent human viruses that are otherwise non-pathogenic in this host. Moreover, these highly virulent recombinant viruses expressing the 1918 viral HA could infect the entire lung and induce high levels of macrophage-derived chemokines and cytokines, which resulted in infiltration of inflammatory cells and severe haemorrhage, hallmarks of the illness produced during the original pandemic.     

The trans-activator gene of HTLV-III is essential for virus replication

Studies of the genomic structure of human T-lymphotropic virus type III (HTLV-III) and related viruses, implicated as the causal agent of acquired immune deficiency syndrome (AIDS), have identified a sixth open reading frame in addition to the five previously known within the genome (gag, pol, sor, env and 3'orf). This gene, called tat-III, lies between the sor and env genes and is able to mediate activation, in a trans configuration, of the genes linked to HTLV-III long terminal repeat (LTR) sequences. We now present evidence that the product of tat-III is an absolute requirement for virus expression. We show that derivatives of a biologically competent molecular clone of HTLV-III, in which the tat-III gene is deleted or the normal splicing abrogated, failed to produce or expressed unusually low levels of virus, respectively, when transfected into T-cell cultures. The capacity of these tat-III-defective genomes was transiently restored by co-transfection of a plasmid clone containing a functional tat-III gene or by introducing the TAT-III protein itself. As HTLV-III and related viruses are the presumed causal agents of AIDS and associated conditions, the observation that tat-III is critical for HTLV-III replication has important clinical implications, and suggests that specific inhibition of the activity of tat-III could be a novel and effective therapeutic approach to the treatment of AIDS.     

Profound block in thymocyte development in mice lacking p56lck.

The protein Lck (p56lck) has a relative molecular mass of 56,000 and belongs to the Src family of tyrosine kinases. It is expressed exclusively in lymphoid cells, predominantly in thymocytes and peripheral T cells. Lck associates specifically with the cytoplasmic domains of both CD4 and CD8 T-cell surface glycoproteins and interacts with the beta-chain of the interleukin-2 receptor, which implicates Lck activity in signal transduction during thymocyte ontogeny and activation of mature T cells. Here we generate an lck null mutation by homologous recombination in embryonic stem cells to evaluate the role of p56lck in T-cell development and activation. Lck-deficient mice show a pronounced thymic atrophy, with a dramatic reduction in the double-positive (CD4+CD8+) thymocyte population. Mature, single-positive thymocytes are not detectable in these mice and there are only very few peripheral T cells. These results illustrate the crucial role of this T-cell-specific tyrosine kinase in the thymocyte development.     

Antigenic peptides recognized by T lymphocytes from AIDS viral envelope-immune humans

T-lymphocyte immunity is likely to be an important component of the immune defence against the AIDS virus, because helper T cells are necessary for the antibody response as well as the cytotoxic response. We have previously predicted two antigenic sites of the viral envelope protein gp120 likely to be recognized by T lymphocytes, based on their ability to fold as amphipathic helices, and have demonstrated that these are recognized by T cells of mice immunized with gp120 (ref. 1). A peptide corresponding to one of these sites can also be induce immunity in mice to the whole gp120 protein. Because many clinically healthy seropositive blood donors have already lost their T-cell proliferative response to specific antigen, we tested the response to these synthetic peptides of lymphocytes from 14 healthy human volunteers who had been immunized with a recombinant vaccinia virus containing the AIDS viral envelope gene and boosted with a recombinant fragment. Eight of the 14 responded to one peptide, and four to the other peptide, not included in the boost. These antigenic sites recognized by human T cells may be useful components of a vaccine against AIDS. We also found a correlation between boosting with antigen-antibody complexes (compared to free antigen) and higher stimulation indices, suggesting a more effective method of immunization.