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.     

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.     

Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160

The development of a vaccine to provide protective immunity to human immunodeficiency virus type 1 (HIV-1), the virus causing AIDS, would be the most practical method to control its spread. Subunit vaccines consisting of virus envelope glycoproteins, produced by recombinant DNA technology, are effective in preventing viral infections. We have now used this approach in the development of a candidate AIDS vaccine. Chimpanzees were immunized with recombinant forms of the HIV-1 glycoproteins gp120 and gp160 produced in Chinese hamster ovary cells, and then challenged with HIV-1. The control and the two animals immunized with the gp160 variant became infected within 7 weeks of challenge. The two animals immunized with the gp120 variant have shown no signs of infection after more than 6 months. These studies demonstrate that recombinant gp120, formulated in an adjuvant approved for human use, can elicit protective immunity against a homologous strain of HIV-1.     

Molecular cloning of lymphadenopathy-associated virus

Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated from a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS). Other LAV isolates have subsequently been recovered from patients with AIDS or pre-AIDS and all available data are consistent with the virus being the causative agent of AIDS. The virus is propagated on activated T lymphocytes and has a tropism for the T-cell subset OKT4 (ref. 6), in which it induces a cytopathic effect. The major core protein of LAV is antigenically unrelated to other known retroviral antigens. LAV-like viruses have more recently been independently isolated from patients with AIDS and pre-AIDS. These viruses, called human T-cell leukaemia/lymphoma virus type III (HTLV-III) and AIDS-associated retrovirus (ARV), seem to have many characteristics in common with LAV and probably represent independent isolates of the LAV prototype. We have sought to characterize LAV by the molecular cloning of its genome. A cloned LAV complementary DNA was used to screen a library of recombinant phages constructed from the genomic DNA of LAV-infected T lymphocytes. Two families of clones were characterized which differ in a restriction site. The viral genome is longer than any other human retroviral genome (9.1-9.2 kilobases).     

Effect of recombinant soluble CD4 in rhesus monkeys infected with simian immunodeficiency virus of macaques

The CD4 molecule is a high-affinity cell-surface receptor for the human immunodeficiency virus (HIV-1) and a soluble truncated form of CD4 produced by recombinant DNA technology is a potent inhibitor of HIV-1 replication and HIV-1-induced cell fusion in vitro. Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVMAC), a virus closely related to HIV-1, develop an AIDS-like syndrome, and so provide an important model for the evaluation of potential AIDS therapies. We have assessed the therapeutic effect of recombinant soluble CD4 in SIVMAC-infected rhesus monkeys. Virus was readily isolated from peripheral blood lymphocytes and bone marrow cells of these animals before starting treatment with soluble CD4, but became difficult to isolate soon after treatment had begun. Moreover the diminished growth of both granulocyte-macrophage and erythrocyte progenitor colonies from the bone marrow of these monkeys rose to normal levels during treatment. These findings indicate that soluble CD4 could prove valuable in the treatment of AIDS.     

Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma

Hepatitis B virus (HBV) may be one of the agents involved in the aetiology of human primary liver cancer. This hypothesis is supported by (1) the similarity between the geographical distribution of chronic carriers of the viral surface antigen (HBsAg) and that of hepatocellular carcinoma (HCC); (2) the increase in the prevalence of HBV markers in serum of patients with primary liver cancer when compared with the general population; (3) the observation that HBV infection precedes the development of the tumour. Moreover, these epidemiological indications of an association between HBV infecton and hepatocellular carcinoma are supported by the detection of HBV markers such as HBsAg or viral DNA sequences, although in a non-integrated form in tumour tissue. To study the relationship between HBV and primary liver cancer further, we looked for the presence of free or integrated viral DNA in tumour tissue of human hepatocellular carcinomas and in a HBsAg-producing human hepatoma cell line. Using the blot-transfer hybridization technique and cloned HBV DNA as a probe, we have now demonstrated that the viral DNA is integrated in the cellular genome both in tumour tissue and in a hepatoma cell line.     

Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960

Human immunodeficiency virus type 1 (HIV-1) sequences that pre-date the recognition of AIDS are critical to defining the time of origin and the timescale of virus evolution. A viral sequence from 1959 (ZR59) is the oldest known HIV-1 infection. Other historically documented sequences, important calibration points to convert evolutionary distance into time, are lacking, however; ZR59 is the only one sampled before 1976. Here we report the amplification and characterization of viral sequences from a Bouin's-fixed paraffin-embedded lymph node biopsy specimen obtained in 1960 from an adult female in Léopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo (DRC)), and we use them to conduct the first comparative evolutionary genetic study of early pre-AIDS epidemic HIV-1 group M viruses. Phylogenetic analyses position this viral sequence (DRC60) closest to the ancestral node of subtype A (excluding A2). Relaxed molecular clock analyses incorporating DRC60 and ZR59 date the most recent common ancestor of the M group to near the beginning of the twentieth century. The sizeable genetic distance between DRC60 and ZR59 directly demonstrates that diversification of HIV-1 in west-central Africa occurred long before the recognized AIDS pandemic. The recovery of viral gene sequences from decades-old paraffin-embedded tissues opens the door to a detailed palaeovirological investigation of the evolutionary history of HIV-1 that is not accessible by other methods.     

An HTLV-III peptide produced by recombinant DNA is immunoreactive with sera from patients with AIDS

Human T-cell lymphotropic retrovirus type III (HTLV-III), also called lymphadenopathy-associated virus (LAV), has been identified as the aetiological agent of acquired immune deficiency syndrome (AIDS). The sera of most patients with AIDS or AIDS-related complexes, and of asymptomatic individuals infected with HTLV-III, contain antibodies against antigens of HTLV-III. The characterization of these antibodies and their corresponding viral antigens is important not only for understanding immunity against HTLV-III and the pathology of AIDS, but also for the development of diagnostic methods and preventive vaccine for AIDS. Following the successful establishment of a long-term T-cell line permissive for HTLV-III replication, large quantities of virus have been produced, facilitating the purification of viral proteins and the development of mouse monoclonal antibodies against several viral antigens. More recently, the structure of HTLV-III proviral DNA has been elucidated. We now report the production, by genetic engineering methods, of a peptide encoded by a gene segment of HTLV-III. A 1.1-kilobase (kb) EcoRI DNA segment from an isolate of HTLV-III was inserted into a lpp and lac promoter-coupled expression vector, pIN-III-ompA. Escherichia coli transformants of this plasmid produced a peptide of relative molecular mass (Mr) 15,000 (15K) which was strongly immunoreactive with anti-HTLV-III antibodies present in sera from AIDS patients. Lysates of the clones expressing this 15K peptide inhibited the reactivity of the p31 virion protein with AIDS sera, suggesting that it is a fragment of the viral p31 protein. The peptide reacted with sera from all 20 AIDS patients but none of the 8 normal controls tested. These results suggest that the peptide may be useful for detecting anti-HTLV-III antibodies in blood samples.     

Productive dual infection of human CD4+ T lymphocytes by HIV-1 and HHV-6

Although infection by HIV-1 has been implicated as the primary cause of AIDS and related disorders, cofactorial mechanisms may be involved in the pathogenesis of the disease. For example, several viruses commonly detected in AIDS patients and capable of transactivating the long terminal repeat of HIV-1, such as herpesviruses, papovaviruses, adenoviruses and HTLV-I have been suggested as potential cofactors. Another candidate is human herpesvirus-6 (HHV-6, originally designated human B-lymphotropic virus), which has not only been identified in most patients with AIDS by virus isolation, DNA amplification techniques and serological analysis, but is also predominantly tropic and cytopathic in vitro for CD4+ T lymphocytes. Here we demonstrate that HHV-6 and HIV-1 can productively co-infect individual human CD4+ T lymphocytes, resulting in accelerated HIV-1 expression and cellular death. We also present evidence that HHV-6 transactivates the HIV-1 long terminal repeat (LTR). These observations indicate that HHV-6 might contribute directly or indirectly to the depletion of CD4+ T cells in AIDS.     

Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein

Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi-Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi-Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.     

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.     

Molecular cloning and polymorphism of the human immune deficiency virus type 2

We recently reported the isolation of a novel retrovirus, the human immune deficiency virus type 2 (HIV-2, previously named LAV-2), from patients with acquired immune deficiency syndrome (AIDS) originating from West Africa. This virus is related to HIV-1, the causative agent of the AIDS epidemic now spreading in Central and East Africa, as well as the USA and Europe (see ref. 3 for review) both by its morphology and by its tropism and in vitro cytopathic effect on CD4 (T4) positive cell lines and lymphocytes. But preliminary hybridization experiments indicated that there are substantiated differences between the sequences of the two genomes. Furthermore, the proteins of HIV-1 and HIV-2 have different sizes and their serological cross-reactivity is restricted to the major core protein, as the envelope glycoproteins of HIV-2 are not immunoprecipitated by HIV-1-positive sera. We now report the molecular cloning of the complete 9.5-kilobase (kb) genome of HIV-2, the observation of restriction site polymorphism between different isolates, and a preliminary analysis of the relationship of HIV-2 with other human and simian retroviruses.     

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.     

Superantigen implicated in dependence of HIV-1 replication in T cells on TCR V beta expression.

In the pathogenesis of AIDS it is not yet understood whether the small fraction of CD4+ T cells (approximately 1%) infected with the human immunodeficiency virus (HIV) are randomly targeted or not. Here we present evidence that human CD4 T-cell lines expressing selected T-cell antigen receptor V beta gene products can all be infected in vitro with HIV-1, but give markedly different titres of HIV-1 virion production. For example, V beta 12 T-cell lines from several unrelated donors reproducibly yielded up to 100-fold more gag gene product (p24gag antigen) than V beta 6.7a lines. This is consistent with a superantigen effect, because the V beta selectivity was observed with several divergent HIV-1 isolates, was dependent on antigen-presenting cells and on major histocompatibility complex (MHC) class II but was not MHC class II-restricted. The in vivo significance of these findings is supported by the preferential stimulation of V beta 12+ T cells by freshly obtained irradiated antigen-presenting cells from some HIV-1-seropositive but not HIV-1-negative donors. Moreover, cells from patients positive for viral antigen (gp120) were enriched in the V beta 12 subpopulation. V beta 12+ T cells were not deleted in AIDS patients, however, raising the possibility that a variety of mechanisms contribute to T-cell depletion. Our results indicate that a superantigen targets a subpopulation of CD4+ cells for viral replication.     

A structural model for the retroviral proteases

In many retroviruses the 5' end of the pol gene codes for a protease vital for the processing of the gag polyprotein into the separate core proteins. In some viruses this protease is encoded at the 3' end of the gag gene, or between the gag and pol genes in a different reading frame to either. A sequence, Asp-Thr-Gly, which is conserved in retroviral proteases is also conserved in the active sites of aspartic proteases, an observation which has led to the suggestion that the retroviral proteases could belong to this family. We have examined the sequences of the aspartic and retroviral protease families, using pattern-recognition, structure prediction and molecular modelling techniques, and conclude that the viral protease sequences probably correspond to a single domain of an aspartic protease and may function in a dimeric form. We have constructed a model of the pol-protease of human immunodeficiency virus 1 (HIV-1) to test this hypothesis.     

Genome organization and transactivation of the human immunodeficiency virus type 2

Analysis of the nucleotide sequence of the human retrovirus associated with AIDS in West Africa, HIV-2, shows that it is evolutionarily distant from the previously characterized HIV-1. We suggest that these viruses existed long before the current AIDS epidemics. Their biological properties are conserved in spite of limited sequence homology; this may help the determination of the structure-function relationships of the different viral elements.