Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef.

Cytotoxic T-lymphocyte (CTL) responses to human immunodeficiency virus arise early after infection, but ultimately fail to prevent progression to AIDS. Human immunodeficiency virus may evade the CTL response by accumulating amino-acid replacements within CTL epitopes. We studied 10 CTL epitopes during the course of simian immunodeficiency virus disease progression in three related macaques. All 10 of these CTL epitopes accumulated amino-acid replacements and showed evidence of positive selection by the time the macaques died. Many of the amino-acid replacements in these epitopes reduced or eliminated major histocompatibility complex class I binding and/or CTL recognition. These findings strongly support the CTL 'escape' hypothesis.

Reinfection results in accumulation of unintegrated viral DNA in cytopathic and persistent human immunodeficiency virus type 1 infection of CEM cells.

High levels of unintegrated viral DNA accumulate during human immunodeficiency virus type 1 (HIV-1) infection of CEM T cells. Reinfection of already infected cells is required to attain these levels and reinfection also promotes the development of HIV-induced cytopathology. Rates of virus production, however, are independent of the accumulation of unintegrated viral DNA. Neutralizing antibody added soon after infection reduced viral DNA levels without appreciably affecting the production of cell-free viral p24 antigen or reverse transcriptase activity. Only 50 pM AZT were required to reduce the accumulation of unintegrated viral DNA by 50% in contrast to the 25 nM required to inhibit virus production by 50%. Cytopathology, as measured by number of syncytia in infected cell cultures, was correlated with highly elevated levels of unintegrated viral DNA. The minimal levels of unintegrated viral DNA present constitutively in the persistently infected HCEM cell line were consonant with the absence of cytopathic effects in these cells. These data demonstrate that inhibiting the reinfection of already infected cells modulates cytopathic HIV-1 infection to a form that is persistent and noncytopathic.

Human immunodeficiency virus infection of T cells and monocytes proceeds via receptor-mediated endocytosis.

The rates of internalization and uncoating of 32P-labelled human immunodeficiency virus (HIV) in the human T lymphoid cell line CEM are consonant with a receptor-mediated endocytosis mechanism of entry. This interpretation was affirmed by electron microscopic observation of virions within endosomes. Virus binding and infectivity were inhibited to the same extent by pretreatment with OKT4A antibody, therefore, the CD4 receptor-dependent pathway of internalization appears to be the infectious route of entry. The pattern of internalization by the human monoblastoid cell line U937 proved to be more complex, involving rapid and efficient CD4-independent internalization. Electron microscopy revealed the presence of large intracellular vesicles, each containing several virions. Antibody against the CD4 receptor for virus efficiently blocked infection, but did not reduce significantly HIV binding or internalization in the U937 cell line. Consequently, U937 cells have a CD4-independent pathway of virus internalization that does not coincide with the route of entry for infectious HIV.

Apoptosis as a mechanism of cell death in cultured T lymphoblasts acutely infected with HIV-1.

The mechanisms by which HIV-1 infection kills T lymphocytes are not clearly established. Apoptosis is an internally programmed cell death pathway that may regulate both T cell development and senescence, and that is characterized by cleavage of DNA at internucleosomal regions. The present experiments show that acute HIV-1 infection of MT2 lymphoblasts and activated normal peripheral blood mononuclear cells induces apoptosis. The addition of anti-gp120 neutralizing antibody, after HIV-1 infection of MT2 cells, permitted sustained high levels of viral replication, but blocked apoptosis and cell death. Apoptosis may account for the direct cytopathologic effects of HIV-1 in T cells.

Regulation of human T-lymphocyte gene expression by interleukin 2: immediate-response genes include the proto-oncogene c-myb.

Antigen-stimulated human T lymphocytes must bind the immunoregulatory hormone interleukin 2 (IL-2) if they are to transit from the G1 to the S phase of the cell cycle. Indirect methods, such as the measurement of thymidine uptake rates, were previously the only means available for exploring the mechanism of action of IL-2. Several cDNA clones have been isolated which are expressed subsequent to IL-2 binding, and the expression of two of these genes. Tact52 and Tact75, is regulated directly at the level of transcription; expression of the proto-oncogene c-myb is also regulated directly by IL-2 binding. These genes thus constitute a set which is coordinately regulated in the course of the transition from G1 to S phase of human T lymphocytes, and their expression depends on IL-2 binding.

Therapeutic approaches to HIV infection based on virus structure and the host pathogen interaction.

The HIV-1 infection of central nervous system, with attendant neuropathy and dementia, poses a unique challenge for antiviral therapy. For practical considerations, it is important to define carefully the precise therapeutic objectives. (1) Is it necessary to inhibit spreading HIV-1 infection in the central nervous system? (2) What is the role of inflammatory responses in central nervous system disease during HIV-1 infection? (3) Is there a correlation between pathology and dementia? (4) Are virions or virus gene products toxic in the central nervous system? (5) Is there a role for immune suppression and opportunistic pathogens in AIDS dementia? The development of therapeutic agents for HIV-1 infection is guided by our knowledge of virus structure, the function of viral proteins, the interactions with host components, and detailed features of the virus life cycle. In each case, unique features of the virus can be identified and established as targets for unique antiviral compounds. Drugs acting as inhibitors of virus enzymatic functions are plagued by the rapid development in vivo of drug-resistant virus variants, although combination or alternating chemotherapeutic regimens may obviate some of these concerns. Novel approaches to inhibiting virus are flourishing. In vitro studies show the value of agents as diverse as molecular decoys for tat activity to efforts to mutagenize integrated proviruses by modified oligonucleotides that form triple helices with chromosomal genes. As each particular clinical situation is better defined, the design and application of these agents can be refined to inhibit HIV-1 replication and reduce the associated morbidity.

Vitamin D3 compounds regulate human immunodeficiency virus type 1 replication in U937 monoblastoid cells and in monocyte-derived macrophages.

We studied the effects of vitamin D3 compounds on the replication of human immunodeficiency virus type 1 (HIV-1) in the monoblastoid cell line U937 and in primary monocyte-derived macrophage cultures to understand how modulators of monocyte/macrophage effector function might affect the pathogenesis of HIV-1 infection. U937 cell cultures exposed to 1, alpha 25-dihydroxyvitamin D3 prior to HIV-1 infection showed enhanced virus replication that was apparently due to increased cellular resistance to viral cytopathic effects; a marked inhibition of virus replication was noted in cells exposed to 1 alpha,25-dihydroxyvitamin D3 subsequent to infection. Exposure of blood-derived monocyte/macrophages to vitamin D3 compounds prior to infection also affected virus growth; in most cases, substantial inhibition of HIV-1 replication was noted in vitamin D3-treated macrophage cultures. Our results demonstrate that vitamin D3 compounds with recognized abilities to induce cellular differentiation can modulate HIV-1 infection of human macrophages.

Innate T-cell immunity in HIV infection: the role of Vgamma9Vdelta2 T lymphocytes.

There is growing interest in the use of innate immune reactions in the therapy and prophylaxis of various diseases. Natural T (NT) lymphocytes that recognize infected cells or microbial compounds without the classical genetic restriction by polymorphic MHC molecules are crucial components of innate immunity. NT cells bearing the Vgamma9Vdelta2 T-cell receptor (TCR) are broadly reactive against intracellular pathogens, can lyse human immunodeficiency virus (HIV) infected cells, and release cytokines capable of regulating HIV replication. The potent antiviral activities of Vgamma9Vdelta2 T cells may help to contain viral spread during acute HIV infection and/or to prevent the establishment of viral persistence. Substantial changes in the composition and function of circulating gammadelta T-cell pools occur in HIV-infected patients. These changes a) may contribute to the etiopathogenesis of opportunistic infections and neoplasms, and b) are partly reversed by highly active anti-retroviral therapy (HAART). In addition to direct antiviral activities, activated gammadelta T cells influence dendritic cell maturation and the adaptive alphabeta T-cell response. Vgamma9Vdelta2 T cells can be stimulated in vivo and in vitro by various nonpeptidic antigens (NpAgs) and recent animal experimental data suggest that activated Vgamma9Vdelta2 T cells may help to control SIV replication. Currently, NpAgs are being assessed as potential therapeutic agents in AIDS, tuberculosis and certain cancers susceptible to Vgamma9Vdelta2 T-cell effector mechanisms.

Rapid and slow progressors differ by a single MHC class I haplotype in a family of MHC-defined rhesus macaques infected with SIV.

Highly polymorphic HLA class I molecules may influence rates of disease progression of HIV-infected individuals. Recent evidence suggests that individuals who mount vigorous CTL responses to multiple HIV-1 epitopes have reduced viral loads, and survive longer than individuals that make a less robust or less diverse CTL response. It has been difficult, however, to define associations between particular HLA class I alleles and rates of disease progression. This may be due, in part, to the uncontrolled variables associated with naturally acquired HIV infections. Studies using MHC-defined, non-human primates infected with well characterized viral stocks should help to clarify this relationship. To explore the possibility that MHC class I polymorphism can influence disease progression, we infected four Mamu-DRB-identical individuals from a family of MHC-defined rhesus macaques intravenously with 40 TCID50SIVmac239. Two of these macaques developed severe wasting and were euthanized within 80 days of infection, while the other two survived for more than 400 days without showing any symptoms of disease. Since all four of these macaques were Mamu-DRB-identical, we were able to exclude the MHC class II DRB loci as determinant of disease progression. Interestingly, both of the slow progressors made CTL responses to the same three SIV CTL epitopes, which were restricted by two molecules (Mamu-B*03 and B*04) encoded by their common maternal haplotype. The two rapid progressors did not share this haplotype with the slow progressors, and we were unable to detect CTL responses in these two siblings. These observations implicate products of the Mamu-B*03 and B*04 alleles in resistance to disease progression in this family of SIV-infected macaques, and provide additional evidence that certain MHC class I-restricted CTL responses may play a significant role in delaying the onset of AIDS.

Extrachromosomal HIV-1 DNA in persistently infected U937 cells.

Persistent human immunodeficiency virus type 1 (HIV-1) infection of U937 monocytic cells resulted in the accumulation of novel forms of extrachromosomal viral DNA. These DNA species are larger than the genome size of HIV-1 and persist indefinitely. The extrachromosomal viral DNA species (E-DNA) were shown to be structurally stable by subcloning of infected cell lines and restriction fragment analysis. Similar E-DNA structures were observed in independent infections. Persistently infected monocytic cells had low levels of viral antigens, reflecting the low levels of viral RNA that were detected. These results support a role for E-DNA in persistent HIV-1 infection of monocytic cells.

Mucosal transmission of virulent and avirulent lentiviruses in macaques.

Sexual transmission of human immunodeficiency virus provides an efficient mode for virus spread and poses unique challenges to vaccine developers. Host and viral factors that affect transmission have been studied by epidemiological approaches in the human population, and some of these factors have been modeled with experimental infection of nonhuman primates. Basic principles have emerged regarding transmission and viral virulence. These ideas may be beneficial for designing a safe and effective vaccine.

Quinolinic acid and lymphocyte subsets in the intrathecal compartment as biomarkers of SIV infection and simian AIDS.

Cerebrospinal fluid (CSF) samples were collected from monkeys infected with SIVmac251 (SIV) or HIV-1/SIVmac chimeric viruses (SHIV(HXBc2) and SHIV(89.6P)) to investigate quinolinic acid (QUIN) levels in the intrathecal compartment. CSF levels of QUIN were elevated in the SIV-infected monkeys, especially in animals with end-stage disease, and in those infected with pathogenic SHIV(89.6P), but not after infection with the nonpathogenic construct SHIV(HXBc2). QUIN elevations occurred in association with reduced CD4+ and increased CD8+ lymphocytes, cellular alterations that were more pronounced in CSF than in the blood. These findings support the view that the intrathecal compartment provides a unique window on viral infection, and are in keeping with the a priori prediction that QUIN increases primarily in response to more pathogenic viral strains.

The lymphocytosis-promoting agent pertussis toxin affects virus burden and lymphocyte distribution in the SIV-infected rhesus macaque.

Pertussis toxin from the gram-negative bacterium Bordetella pertussis is an ADP-ribosylase that modifies Gi proteins in mammalian lymphocytes and inhibits their capacity to traffic from blood into lymphoid tissues. We used this compound to induce lymphocytosis in rhesus macaques and to study its effects on SIV infection. Pertussis toxin injected at 25 micrograms/kg induced a transient lymphocytosis that peaked 3-8 days after administration and caused a rapid, transient decrease in the frequency of infectious cells in blood as judged by in vitro virus isolation assays. Lymphocyte subsets were altered during the lymphocytosis interval and sustained changes in CD8+ T cell levels were noted as long as 53 days after pertussis toxin injection. In situ hybridization studies showed that pertussis toxin altered the distribution of viral RNA in lymph nodes during the interval of lymphocytosis, and caused long-term changes with decreased virus replication in some tissue specimens.

Macaque models for early immunosuppression and T cell loss during acute immunodeficiency virus infections.

The interval of acute infection with immunodeficiency viruses is critically important for determining the long-term rate of disease progression. The steps of initial infection, systemic dissemination, and explosive replication of pathogenic SIV or SHIV in macaques are being mapped to show the mechanisms responsible for remodeling host immunity, for establishing the persistent infection, and for promoting disease progression. Here, we describe recent studies on two ways in which CD4+ T cell populations are depleted during acute infection. Initially, we discuss recent work on the mechanisms for CD4+ T cell-mediated, MHC-unrestricted cytolysis. This mechanism shows how even soluble viral antigens such as the envelope glycoprotein, can prime CD4+ lymphocytes to be both effector and target cells in an unrestricted cytolysis mechanism. The consequence of unrestricted cytolysis is a more rapid destruction of the CD4+ T cell population. Secondly, we discuss the broader issue of T cell hyperactivation during acute infection. Inappropriate activation of this lymphocyte population renders cells susceptible to activation induced cell death and also increases the rate of virus replication. Macaque immunization studies have shown a clear role for extracellular Tat in hyperactivation. These two mechanisms, unrestricted cytolysis and T cell hyperactivation, are components of the acute infection that remodel host immunity and dictate the rate of progression to AIDS.

The endocytic pathway for human immunodeficiency virus infection.

We have presented data supporting the view that HIV infection of T cells and monocytes proceeds via receptor-mediated endocytosis. Biochemical analysis of the pathway for internalization of 32P-labeled HIV revealed the presence of intact, intercellular virions and provided evidence for subsequent uncoating of these particles and release of viral RNA to the cytoplasm. Electron microscopy demonstrated virions within vesicles and documented fusion between viral and endosomal membranes. Others have provided evidence supporting the notion that endocytosis is not required for HIV infection. In consequence of this apparent paradox, it has been suggested that both mechanisms, i.e. direct fusion to the plasma membrane and endocytosis, are functional pathways for HIV entry. I do not favor this position. Examination of the biology of CD4, its role in lymphocyte activation, and its function as the receptor for HIV imply a more active role for this molecule in virus infection than simply as a transient anchor for fusion events. The principal experiment of concern is the report by Maddon, et al., that human CD4- expressing murine cell lines remain resistant to infection by VSV pseudotypes bearing the HIV envelope glycoprotein. This paper and a subsequent review of HIV entry by Marsh and Dalgleish comment on the possibility that endocytosis of human CD4 may not occur in murine cells hence, the block to infectious entry of these pseudotype virions. Resolution of the mechanism for HIV infection is more than rationalization or semantics. The pathway of endocytosis presents an unique feature of cell biology. Should HIV infection be proven to occur via this mechanism, then appropriate assays for novel classes of antiviral agents could be developed. Other features of the endosomal vesicle, such as the unique composition of its membrane or the possible presence of specific proteolytic activities might play important roles in HIV infection and thus, present new targets for the development of antiviral agents. Indeed, the synthetic liposome AL721 is an effective inhibitor of HIV infection in vitro. This antiviral agent probably acts by decreasing the cholesterol content of the virion envelope. It was shown previously that the envelope is specifically enriched in cholesterol, a property not associated with the plasma membrane but clearly involved in Semliki Forest virus fusion within the endosome.(ABSTRACT TRUNCATED AT 400 WORDS)

Human Vgamma2Vdelta2 T cells contain cytoplasmic RANTES.

The adult human Vgamma2Vdelta2 T cell repertoire is a product of chronic selection in the periphery. Endogenous antigens drive the expansion of cells expressing the Vgamma2Vdelta2 TCR. Thus, we would expect the majority of circulating Vgamma2Vdelta2 T cells to be antigen experienced and to have memory phenotype, in contrast to the alpha/beta TCR+ subsets that include a substantial fraction of naive cells. We sought to characterize functional aspects of Vgamma2Vdelta2 T cells that might show whether circulating cells are memory or naive. For these studies, we focus on the expression of the CC chemokine regulated upon activation normal T cell expressed and secreted (RANTES). In naive alphabeta T cells, an initial stimulus triggers the onset of RANTES transcription followed later by protein expression. In memory CD8+ alphabeta T cells, RANTES mRNA is already present in unstimulated cells and protein expression is triggered immediately by TCR signaling; some cells may also contain RANTES protein in cytoplasmic stores. We show here that the vast majority of circulating human T cells contain RANTES protein in cytoplasmic stores and the chemokine is secreted rapidly after TCR signaling. Primary Vgamma2Vdelta2 T cell lines obtained after in vitro stimulation with phosphoantigens behaved similarly to circulating Vgamma2Vdelta2 T cells and contained both RANTES mRNA and protein, but only very low levels of mRNA or protein for macrophage inflammatory protein (MIP)-1alpha or MIP-1beta. The presence of stored RANTES shows that circulating Vgamma2Vdelta2 T cells are mostly memory phenotype and capable of rapid chemokine responses to phosphoantigen stimulation. Considering that one of 40 circulating CD3+ lymphocytes is Vgamma2Vdelta2+, they comprise the largest circulating memory population against a single antigen, and phosphoantigen stimulation will trigger a rapid activation with immediate release of RANTES.

HIV persistence in monocytes leads to pathogenesis and AIDS.

An hypothesis of the pathogenic mechanism leading to acquired immunodeficiency syndrome (AIDS) that places special emphasis on the potential for infected monocytes to act as the reservoir of a persistent human immunodeficiency virus (HIV) infection has been developed. Monocytes may mediate directly the infection and ultimate destruction of helper T cells; this establishes a direct relationship between antigen presentation and HIV dissemination, thus accounting for the cytopathogenic effects and immune system debilitation associated commonly with AIDS. The possibility that this mode of virus dissemination can account for the depletion of helper-T-cell subsets based on their antigen specificity is considered and may explain why the cellular immune response to the virus is ineffective. This concept and may also elucidate the role of intercurrent infections in the development of disease and it suggests mechanistic explanations for the conversion from prodromal to fulminant AIDS.