Anne G. Laurent-Crawford

The cytopathic effect of HIV is associated with apoptosis.

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.

Autoantibodies typical of non-organ-specific autoimmune diseases in Hiv-seropositive patients

ObjectiveTo analyse serological aspects of systemic autoimmunity in HIV-1-seropositive patients and in individuals at risk for AIDS. Design and methodsThe reactivity of antibodies in the serum of 100 HIV-1-seropositive patients was investigated by enzyme-linked immunosorbent assay (ELISA) using a series of antigens known to be recognized by antibodies from patients with multisystemic autoimmune diseases, such as systemic lupus erythematosus, mixed-connective tissue disease and Sjogrens syndrome. ResultsHigh levels of immunoglobulin G (IgC) antibodies reacting with double-stranded DNA (dsDNA), synthetic peptides of ubiquitinated histone H2A, Sm-D antigen, U1-A RNP antigen and 60 kD SSA/Ro antigen were found in 44–95% of HIV-infected patients. Among histone antibodies, the most frequent reactions were towards the carboxy-terminal region of histone H1 and to histone H2B and its amino-terminal domain 1–25. Eight HIV-1-seropositive patients at different stages of disease according to the Centers for Disease Control classification were also studied. In most cases, no obvious fluctuations were observed over several years. Antibodies were found early, and their specificity and apparent level of activity remained relatively constant. There was no evidence of such an autoimmune response in the serum of high-risk homosexual seronegative men. ConclusionsAlthough the aetiology of AIDS is known, in general the aetiology of multisystemic autoimmune diseases remains to be determined, and the sequence of events taking place remains obscure in both cases. It is possible that the large spectrum of antibodies found in HIV-infected patients reflects a specific stimulation of B-cells by nuclear antigens released by apoptosis during an early stage of disease.

Membrane-expressed HIV envelope glycoprotein heterodimer is a powerful inducer of cell death in uninfected CD4+ target cells.

HIV infection of CD4+ T cells in culture results in the production of virus and induction of cell killing by apoptosis. Such a cytopathic effect is observed during infection with syncytium-inducing or non-syncytium-inducing HIV isolates. Apoptosis is triggered by the interaction of the cell membrane-expressed HIV envelope glycoprotein heterodimer gp120-gp41 complex (external and transmembrane glycoprotein complex) with the CD4 receptor. Here we demonstrate an experimental model for the induction of apoptosis independent of HIV infection, using transiently transfected HeLa cells with the HIV1 env gene as effector cells and the CD4+ MOLT4-T4 T cells as target cells. Results obtained confirm that the induction of apoptosis requires the membrane expression of the two HIV env gene products, gp120 and gp41. Single amino acid point mutations of the envelope products that affect binding to the CD4 receptor or the fusion process abrogate the capacity of the gp120-gp41 complex to induce apoptosis. Interestingly, a point mutation in the V3 loop which inhibits fusion without affecting CD4 binding also results in the abrogation of apoptosis. These observations indicate that the induction of apoptosis is an intrinsic property of the cell membrane-expressed gp120-gp41 complex, and thus should be considered as one of the functions of HIV env gene products.

The cytopathic effect of human immunodeficiency virus is independent of high levels of unintegrated viral DNA accumulated in response to superinfection of cells

Large quantities of genome-sized viral DNA are detected in the nucleoplasm of CD4+ T cells infected with human immunodeficiency virus type 1 (HIV-1). This unintegrated HIV DNA is in the form of both circular and linear species. Accumulation of such DNA occurs gradually during a 5 day HIV infection and is correlated with the proportion of cells involved in the production of HIV proteins. To pinpoint the stage in a synchronized HIV infection during which accumulation of HIV DNA occurs, high titres of HIV were employed to infect CEM cells to infect the majority of cells by the input virus. By this latter infection, more than 95% of cells became producers of HIV proteins at 48 h post-infection (p.i.) concomitantly with the development of the c.p.e. of HIV, manifested by formation of syncytia and induction of cell death by apoptosis. Addition of azidothymidine (AZT) or neutralizing anti-gp120 monoclonal antibodies at 8 h p.i. did not alter the course of virus infection nor the amount of virus produced at 48 h p.i. but the accumulation of unintegrated HIV DNA was drastically reduced. These results indicate that viral DNA accumulates as a result of superinfection of cells late in the virus cycle. The development of the c.p.e. of HIV was inhibited in the presence of neutralizing antibodies, whereas in the presence of AZT the accumulation of unintegrated HIV DNA was completely blocked without apparent effect on the c.p.e. These observations indicate that the c.p.e. of the HIV infection, which is manifested by syncytium formation and apoptosis, does not require superinfection of cells or accumulation of unintegrated viral DNA.