Zaher Hanna

Nef Harbors a Major Determinant of Pathogenicity for an AIDS-like Disease Induced by HIV-1 in Transgenic Mice

Transgenic (Tg) mice expressing the complete coding sequences of HIV-1 in CD4+ T cells and in cells of the macrophage/dendritic lineages develop severe AIDS-like pathologies: failure to thrive/weight loss, diarrhea, wasting, premature death, thymus atrophy, loss of CD4+ T cells, interstitial pneumonitis, and tubulo-interstitial nephritis. The generation of Tg mice expressing selected HIV-1 gene(s) revealed that nef harbors a major disease determinant. The latency and progression (fast/slow) of the disease were strongly correlated with the levels of Tg expression. Nef-expressing Tg thymocytes were activated and alpha-CD3 hyperresponsive with respect to tyrosine phosphorylation of several substrates, including LAT and MAPK. The similarity of this mouse model to human AIDS, particularly pediatric AIDS, suggests that Nef may play a critical role in human AIDS, independently of its role in virus replication.

The Pathogenicity of Human Immunodeficiency Virus (HIV) Type 1 Nef in CD4C/HIV Transgenic Mice Is Abolished by Mutation of Its SH3-Binding Domain, and Disease Development Is Delayed in the Absence of Hck

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P72XXP75XXP78) was mutated to A72XXA75XXQ78. This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck−/− (knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.

HIV-1 Genes vpr and nef Synergistically Damage Podocytes, Leading to Glomerulosclerosis

This study aimed to identify the causative gene for HIV-1 associated nephropathy, a paradigmatic podocytopathy. A previous study demonstrated that transgenic expression of nonstructural HIV-1 genes selectively in podocytes in mice with FVB/N genetic background resulted in podocyte injury and glomerulosclerosis. In this study, transgenic mice that expressed individual HIV-1 genes in podocytes were generated. Five of six transgenic mice that expressed vpr developed podocyte damage and glomerulosclerosis. Analysis of an established vpr transgenic line revealed that transgenic mice on FVB/N but not on C57BL/6 genetic background developed podocyte injury by 8 wk of age, with later glomerulosclerosis. Four of 11 transgenic mice that expressed nef also developed podocyte injury. One transgenic line was established from the nef founder mouse with the mildest phenotype. Transgenic mice in this line developed mesangial expansion at 3 wk of age and mild focal podocyte damage at 10 wk of age. Mating with FVB/N mice did not augment nephropathy. None of the transgenic mice that expressed vif, tat, rev, or vpu in podocytes, even with the FVB/N genetic background, developed podocyte injury. For testing effects of simultaneous expression of vpr and nef, these two lines were mated. All nef:vpr double-transgenic mice showed severe podocyte injury and glomerulosclerosis by 4 wk of age. In contrast, all vpr or nef single-transgenic mice in the same litter uniformly showed no or much milder podocyte injury. These findings indicate that vpr and nef each can induce podocyte injury with a prominent synergistic interaction.

Ahi-1, a Novel Gene Encoding a Modular Protein with WD40-Repeat and SH3 Domains, Is Targeted by the Ahi-1 and Mis-2 Provirus Integrations

ABSTRACT The Ahi-1 locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-myb proto-oncogene. Since no significant alteration of c-myb expression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the Ahi-1 locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (Ahi-1) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3′ end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the Ahi-1 gene. The Ahi-1 cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The Ahi-1 gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The Ahi-1 gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in Ahi-1, truncated Ahi-1/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore, Ahi-1 is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus, Ahi-1 may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-abl and c-myc) or with a tumor suppressor gene (Nf1), since Ahi-1 insertion sites were identified in tumors harboring v-abl defective retroviruses or a c-myc transgene or in tumors exhibiting deletion of Nf1.

The AIDS Disease of CD4C/HIV Transgenic Mice Shows Impaired Germinal Centers and Autoantibodies and Develops in the Absence of IFN-γ and IL-6

The mechanisms responsible for degeneration of germinal centers (GC) and follicular dendritic cell (FDC) networks during progression to AIDS remain elusive. Here, we show that CD4(+) T cells from CD4C/HIV-1 Tg mice, which develop a severe AIDS-like disease, express low levels of CD40 ligand. Accordingly, GC formation, FDC networks, and immunoglobulin isotype switching are impaired in these animals. However, Tg B cells respond to in vitro CD40 stimulation. Total serum IgG levels are reduced in Tg mice, whereas total IgM levels are increased with a significant amount showing DNA specificity. IFN-gamma- and IL-6-deficient CD4C/HIV Tg mice also develop the AIDS-like disease and produce auto-Ab. Thus, CD4C/HIV Tg mice have immune dysfunction accompanied by autoimmune responses.

Expression of Simian Immunodeficiency Virus nef in Immune Cells of Transgenic Mice Leads to a Severe AIDS-Like Disease

ABSTRACT In order to study the functions of simian immunodeficiency virus (SIV) Nef in vivo in a small-animal model, we constructed transgenic (Tg) mice expressing the SIVmac239 nef gene in the natural target cells of the virus under the control of the human CD4 gene promoter (CD4C). These CD4C/SHIV-nefSIV Tg mice develop a severe AIDS-like disease, with manifestations including premature death, failure to thrive or weight loss, wasting, thymic atrophy, an especially low number of peripheral CD8+ T cells as well as a low number of peripheral CD4+ T cells, diarrhea, splenomegaly, and kidney (interstitial nephritis, segmental glomerulosclerosis), lung (lymphocytic interstitial pneumonitis), and heart disease. In addition, these Tg mice fail to mount a class-switched antibody response after immunization with ovalbumin, they produce anti-DNA autoantibodies, and some of them develop Pneumocystis carinii lung infections. All these results suggest a generalized Nef-induced immunodeficiency. The low numbers of peripheral CD8+ and CD4+ T cells are likely to reflect a thymic defect and may be similar to the DiGeorge-like “thymic defect” immunophenotype described for a subgroup of human immunodeficiency virus type 1-infected children. Therefore, it appears that SIV Nef alone expressed in mice, in appropriate cell types and at sufficient levels, can elicit many of the phenotypes of simian and human AIDS. These Tg mice should be instrumental in studying the pathogenesis of SIV Nef-induced phenotypes.

Cardiac disease in transgenic mice expressing human immunodeficiency virus-1 nef in cells of the immune system.

We previously reported that a severe acquired immune deficiency syndrome-like disease develops in transgenic (Tg) mice expressing the human immunodeficiency virus-1 in its natural target cells: immature and mature CD4(+) T cells and cells of the macrophage/dendritic lineage. Here, we show that these mice also develop cardiac disease, characterized most prominently by a focal myocytolysis, occasionally by myocarditis and by deposition of endogenous immunoglobulin on cardiomyocytes. Microfil perfusion demonstrated widespread coronary arteriospasm and echocardiographic analysis revealed depressed cardiac function in Tg mice. A higher (but still modest) level of cardiomyocyte apoptosis was detected in Tg as compared to non-Tg hearts. Tg expression was detected in some of the infiltrating mononuclear cells, but not in cardiomyocytes or in cells of the heart vessels, suggesting a human immunodeficiency virus-1-induced disease process mediated by cells of the immune system. The similarity of the heart disease observed in these Tg mice to that observed in acquired immune deficiency syndrome patients suggests a common pathogenesis.

Mucosal Candidiasis in Transgenic Mice Expressing Human Immunodeficiency Virus Type 1

The availability of CD4C/HIV(MutA) transgenic (Tg) mice expressing human immunodeficiency virus type 1 in immune cells and developing an AIDS-like disease has provided the opportunity to devise a model of mucosal candidiasis that closely mimics the clinical and pathologic features of candidal infection in human AIDS. After intraoral infection with Candida albicans, oral burdens were strikingly elevated in the Tg mice, compared with non-Tg littermates (P<.05), during primary infection, a 6-10-week carrier state, and a marked terminal outgrowth preceding death. The chronic carrier state was absent in the non-Tg mice because of clearing of C. albicans. Candida hyphae penetrated the epithelium of the oral cavity, esophagus, and cardial-atrium fold of the stomach, accompanied by a mononuclear cell infiltrate. Immunohistochemical analysis suggested that decreased frequencies of major histocompatibility complex class II-expressing cells, combined with reduced CD4+ cells, may underlie the susceptibility to mucosal candidiasis in these Tg mice.

CD4+ T Cells from CD4C/HIVNef Transgenic Mice Show Enhanced Activation In Vivo with Impaired Proliferation In Vitro but Are Dispensable for the Development of a Severe AIDS-Like Organ Disease

ABSTRACT The cellular and molecular mechanisms of dysfunction and depletion of CD4+ T lymphocytes over the course of human immunodeficiency virus type 1 (HIV-1) infection are still incompletely understood, but chronic immune activation is thought to play an important role in disease progression. We studied CD4+ T-cell biology in CD4C/HIV transgenic (Tg) mice, in which Nef expression is sufficient to induce a severe AIDS-like disease including a preferential decrease of CD4+ T cells. We show here that Nef-expressing Tg CD4+ T cells exhibit an activated/memory-like phenotype which appears to be independent of antigenic stimulation, as documented in experiments involving breeding with AD10 TcR Tg mice. In addition, in vivo bromodeoxyuridine incorporation showed that a larger proportion of Tg than non-Tg CD4+ T cells entered the S phase. However, in vitro, Tg CD4+ T cells were found to have a very limited capacity to divide in response to stimulation with anti-CD3 and anti-CD28 or in allogeneic mixed leukocyte reactions. Interestingly, despite these observations, the deletion of Tg CD4+ T cells had little impact on the development of other AIDS-like organ phenotypes. Thus, the Nef-induced chronic activation of CD4+ T cells may exhaust the T-cell pool and may contribute to the thymic atrophy and the low number of CD4+ T cells observed in these Tg mice.

CD8+ T cells but not polymorphonuclear leukocytes are required to limit chronic oral carriage of Candida albicans in transgenic mice expressing human immunodeficiency virus type 1

ABSTRACT Candida albicans causes oropharyngeal candidiasis (OPC) but rarely disseminates to deep organs in human immunodeficiency virus (HIV) infection. Here, we used a model of OPC in CD4C/HIVMut transgenic (Tg) mice to investigate the role of polymorphonuclear leukocytes (PMNs) and CD8+ T cells in limiting candidiasis to the mucosa. Numbers of circulating PMNs and their oxidative burst were both augmented in CD4C/HIVMutA Tg mice expressing rev, env, and nef of HIV type 1 (HIV-1), while phagocytosis and killing of C. albicans were largely unimpaired compared to those in non-Tg mice. Depletion of PMNs in these Tg mice did not alter oral or gastrointestinal burdens of C. albicans or cause systemic dissemination. However, oral burdens of C. albicans were increased in CD4C/HIVMutG Tg mice expressing only the nef gene of HIV-1 and bred on a CD8 gene-deficient background (CD8−/−), compared to control or heterozygous CD8+/− CD4C/HIVMutG Tg mice. Thus, CD8+ T cells contribute to the host defense against oral candidiasis in vivo, specifically in the context of nef expression in a subset of immune cells.