NEF-Induced HIV-Associated Nephropathy Through HCK/LYN Tyrosine Kinases.

HIV-1-associated nephropathy (HIVAN) is a severe complication of HIV-1 infection. To gain insight into the pathogenesis of kidney disease in the setting of HIV, a transgenic (Tg) mouse model [CD4C/HIV-negative regulator factor (Nef)] was used in which HIV-1 nef expression is under control of regulatory sequences (CD4C) of the human CD4 gene, thus allowing expression in target cells of the virus. These Tg mice develop a collapsing focal segmental glomerulosclerosis associated with microcystic dilatation, similar to human HIVAN. To identify kidney cells permissive to the CD4C promoter, CD4C reporter Tg lines were used. They showed preferential expression in glomeruli, mainly in mesangial cells. Breeding CD4C/HIV Tg mice on 10 different mouse backgrounds showed that HIVAN was modulated by host genetic factors. Studies of gene-deficient Tg mice revealed that the presence of B and T cells and that of several genes was dispensable for the development of HIVAN: those involved in apoptosis (Trp53, Tnfsf10, Tnf, Tnfrsf1b, and Bax), in immune cell recruitment (Ccl3, Ccl2, Ccr2, Ccr5, and Cx3cr1), in nitric oxide (NO) formation (Nos3 and Nos2), or in cell signaling (Fyn, Lck, and Hck/Fgr). However, deletion of Src partially and that of Hck/Lyn largely abrogated its development. These data suggest that Nef expression in mesangial cells through hematopoietic cell kinase (Hck)/Lck/Yes novel tyrosine kinase (Lyn) represents important cellular and molecular events for the development of HIVAN in these Tg mice.

HIV-1 Nef Induces Hck/Lyn-Dependent Expansion of Myeloid-Derived Suppressor Cells Associated with Elevated Interleukin-17/G-CSF Levels.

Human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection causes myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+) through largely unknown cellular and molecular pathways. The mouse cells thought to be equivalent to human CD14+ CD16+ cells are CD11b+ Gr1+ myeloid-derived suppressor cells (MDSC). We used HIV transgenic (Tg) mouse models to study MDSC, namely, CD4C/Nef Tg mice expressing nef in dendritic cells (DC), pDC, CD4+ T, and other mature and immature myeloid cells and CD11c/Nef Tg mice with a more restricted expression, mainly in DC and pDC. Both Tg strains showed expansion of granulocytic and CD11b+ Gr1low/int cells with MDSC characteristics. Fetal liver cell transplantation revealed that this expansion was stroma-independent and abrogated in mixed Tg/non-Tg 50% chimera. Tg bone marrow (BM) erythroid progenitors were decreased and myeloid precursors increased, suggesting an aberrant differentiation likely driving CD11b+ Gr1+ cell expansion, apparently cell autonomously in CD4C/Nef Tg mice and likely through a bystander effect in CD11c/Nef Tg mice. Hck was activated in Tg spleen, and Nef-mediated CD11b+ Gr1+ cell expansion was abrogated in Hck/Lyn-deficient Nef Tg mice, indicating a requirement of Hck/Lyn for this Nef function. IL-17 and granulocyte colony-stimulating factor (G-CSF) were elevated in Nef Tg mice. Increased G-CSF levels were normalized in Tg mice treated with anti-IL-17 antibodies. Therefore, Nef expression in myeloid precursors causes severe BM failure, apparently cell autonomously. More cell-restricted expression of Nef in DC and pDC appears sufficient to induce BM differentiation impairment, granulopoiesis, and expansion of MDSC at the expense of erythroid maturation, with IL-17→G-CSF as one likely bystander contributor. IMPORTANCE HIV-1 and SIV infection often lead to myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+), with the latter likely involved in neuroAIDS. We found that some transgenic (Tg) mouse models of AIDS also develop accumulation of mature and immature cells of the granulocytic lineage, decreased erythroid precursors, and expansion of MDSC (equivalent to human CD14+ CD16+ cells). We identified Nef as being responsible for these phenotypes, and its expression in mouse DC appears sufficient for their development through a bystander mechanism. Nef expression in myeloid progenitors may also favor myeloid cell expansion, likely in a cell-autonomous way. Hck/Lyn is required for the Nef-mediated accumulation of myeloid cells. Finally, we identified G-CSF under the control of IL-17 as one bystander mediator of MDSC expansion. Our findings provide a framework to determine whether the Nef>Hck/Lyn>IL-17>G-CSF pathway is involved in human AIDS and whether it represents a valid therapeutic target.

Defective IL-17- and IL-22-dependent mucosal host response to Candida albicans determines susceptibility to oral candidiasis in mice expressing the HIV-1 transgene.

BACKGROUND: The tissue-signaling cytokines IL-17 and IL-22 are critical to host defense against oral Candida albicans infection, by their induction of oral antimicrobial peptide expression and recruitment of neutrophils. Mucosal Th17 cells which produce these cytokines are preferentially depleted in HIV-infected patients. Here, we tested the hypothesis that defective IL-17- and IL-22-dependent host responses to C. albicans determine the phenotype of susceptibility to oropharyngeal candidiasis (OPC) in transgenic (Tg) mice expressing HIV-1. RESULTS: Naïve CD4+ T-cells and the differentiated Th1, Th2, Th17, Th1Th17 and Treg lineages were all profoundly depleted in cervical lymph nodes (CLNs) of these Tg mice. However, naive CD4+ cells from Tg mice maintained the capacity to differentiate into these lineages in response to polarizing cytokines in vitro. Expression of Il17, Il22, S100a8 and Ccl20 was enhanced in oral mucosal tissue of non-Tg, but not of Tg mice, after oral infection with C. albicans. Treatment of infected Tg mice with the combination of IL-17 and IL-22, but not IL-17 or Il-22 alone, significantly reduced oral burdens of C. albicans and abundance of Candida hyphae in the epithelium of tongues of infected Tg mice, and restored the ability of the Tg mice to up-regulate expression of S100a8 and Ccl20 in response to C. albicans infection. CONCLUSIONS: These findings demonstrate that defective IL-17- and IL-22-dependent induction of innate mucosal immunity to C. albicans is central to the phenotype of susceptibility to OPC in these HIV transgenic mice.

HIV Nef expression favors the relative preservation of CD4+ T regulatory cells that retain some important suppressive functions.

HIV-1 infection causes depletion and/or dysfunction of distinct CD4(+) T cell subsets and may affect these differently. Using the CD4C/HIV-1(Nef) transgenic (Tg) mice as a model, we report that HIV-1 Nef causes depletion of total CD4(+) T cells, but preserves and relatively enriches CD4(+) regulatory T cells (Treg). We found that Nef-mediated CD4(+) Treg enrichment is the direct result of Nef expression in CD4(+) T cells, occurs independently of Nef-induced lymphopenia, and most likely results from multiple mechanisms: lower apoptosis, enhanced cell division, and increased generation from precursors. Interestingly, Tg Treg relative enrichment could be reversed by enhancing Lck activity. Most importantly, we show that, in contrast to Tg helper CD4(+) T cells that have lost their function, Nef-expressing CD4(+) Treg retain their regulatory function in vitro and also in vivo, under some settings. In particular, we found that Treg prevent expansion of Tg B and non-Treg T cells in vivo. Our study reveals that Nef affects distinct CD4(+) T cell subsets differently and uncovers the high proliferative potential of B and non-Treg T cells in this mouse model.

Normal development and function but impaired memory phenotype of CD8⁺ T cells in transgenic mice expressing HIV-1 Nef in its natural target cells.

We studied the impact of HIV Nef on CD8(+) T cells in a mouse model of AIDS, the CD4C/HIV(Nef) transgenic (Tg) mice. We found that negative and positive thymic selections of CD8(+) T cells proceeded normally in Nef Tg mice bred respectively with HY or OT-1 TCR Tg mice. Tg peripheral CD8(+) T cells showed an activated phenotype and enhanced cell division in vivo and proliferated efficiently when stimulated in vitro with antigenic peptide. When challenged with LCMV(Armstrong), Nef Tg mice developed a strong acute CD8(+) T cell response and cleared the virus as efficiently as wild-type mice. However, maintenance of LCMV-specific CD8(+) memory T cells was impaired in Nef Tg mice, a defect partially rescued by adoptive transfer of non-Tg naïve CD4(+) T cells. Thus, despite severe abnormalities of their precursors, the double-positive CD4(+)CD8(+) thymocytes, Tg CD8(+) T cells have conserved important functions.

No detectable XMRV in subjects with chronic fatigue syndrome from Quebec.

We investigated the presence of XMRV in a cohort of Quebec patients with chronic fatigue syndrome (CFS). DNA was purified from activated peripheral blood mononuclear cells (PBMCs) and PCR was used to detect XMRV gag and env in 72 patients. Anti-XMRV antibodies were searched in sera of 62 patients by Western blot analysis. Attempts to detect XMRV antigens was made, using immunofluorescence with Gag anti-p30 antiserum on activated PBMC from 50 patients. Plasma viremia was measured by RT-PCR on 9 subjects. Finally, detection of infectious virus in 113 CFS subjects was made by co-culture of PHA+IL-2 activated PBMC with human LNCaP carcinoma cells, and by infecting the same susceptible cells with plasma, using a reverse transcriptase (RT) assay as a readout in both experiments. No detection of XMRV footprints nor infectious virus was detected with any of the approaches, in any of the tested individuals.

HIV-1 Nef disrupts maturation of CD4+ T cells through CD4/Lck modulation.

The HIV-1 Nef protein is a major determinant of HIV-1 pathogenicity. It has been found to induce thymocyte depletion, but the mechanisms involved are not completely understood. Also, nothing is known about its effects on thymocyte selection. We used the CD4C/HIV(Nef) transgenic (Tg) mice, which develop a profound CD4(+) T cell lymphopenia, to study their thymic development. We report that HIV-1 Nef causes depletion of double-positive thymocytes and impairs selection and lineage commitment of CD4(+) single-positive thymocytes. This latter defect could be relieved by increasing the affinity of the TCR-MHC interaction or by allowing CD4(+) T cell maturation to proceed in absence of the CD4 tail, in double-Tg (Nef × CD4(tailless)) mice or in the presence of constitutively active Tg Lck(Y505F). These rescue strategies also resulted in reversal of peripheral CD4(+) T cell lymphopenia. Our data indicate that impairment of Lck-mediated CD4 coreceptor signaling by Nef is an important in vivo mechanism of HIV-1 pathogenesis.

Adult AIDS-like disease in a novel inducible human immunodeficiency virus type 1 Nef transgenic mouse model: CD4+ T-cell activation is Nef dependent and can occur in the absence of lymphophenia.

CD4C/HIV(nef) transgenic (Tg) mice express Nef in CD4+ T cells and in the cells of the macrophage/monocyte/dendritic lineage, and they develop an AIDS-like disease similar to human AIDS. In these mice, Nef is constitutively expressed throughout life. To rule out the contribution of any developmental defects caused by early expression of Nef, we generated inducible human immunodeficiency virus type 1 (HIV-1) Nef Tg mice by using the tetracycline-inducible system. Faithful expression of the Nef transgene was induced in (CD4C/rtTA x TRE/HIV(Nef)) or (CD4C/rtTA2S-M2 x TRE/HIV(Nef)) double-Tg mice upon doxycycline (DOX) treatment in drinking water. Long-term treatment of these mice with DOX also led to loss, apoptosis, and activation of CD4+ T cells, this latter phenotype being observed even with low levels of Nef. These phenotypes could be transferred by bone marrow (BM) transplantation, indicating a hematopoietic cell autonomous effect. In addition, in mixed Tg:non-Tg BM chimeras, only Tg and not non-Tg CD4+ T cells exhibited an effector/memory phenotype in the absence of lymphopenia. Finally, the DOX-induced double-Tg mice developed nonlymphoid organ diseases similar to those of CD4C/HIV(Nef) Tg mice and of humans infected with HIV-1. These results show for the first time that adult mice are susceptible to the detrimental action of Nef and that Nef-mediated T-cell activation can be independent of lymphopenia. These Tg mice represent a unique model which is likely to be instrumental for understanding the cellular and molecular pathways of Nef action as well as the main characteristics of immune reconstitution following DOX withdrawal.

Selective expression of human immunodeficiency virus Nef in specific immune cell populations of transgenic mice is associated with distinct AIDS-like phenotypes.

We previously reported that CD4C/human immunodeficiency virus (HIV)(Nef) transgenic (Tg) mice, expressing Nef in CD4(+) T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4(+) T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4(+) T cells, DCs, and macrophages (CD4E/HIV(Nef)); in CD4(+) T cells and DCs (mCD4/HIV(Nef) and CD4F/HIV(Nef)); in macrophages and DCs (CD68/HIV(Nef)); or mainly in DCs (CD11c/HIV(Nef)). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV(Nef) Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4(+) T cells showed CD4(+) T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4(+) T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.

Macrophage-mediated responses to Candida albicans in mice expressing the human immunodeficiency virus type 1 transgene.

The critical impairments of innate and adaptive immunity that cause susceptibility to mucosal candidiasis in human immunodeficiency virus (HIV) infection have not been fully determined. We therefore conducted an analysis of macrophage-mediated responses to Candida albicans in transgenic (Tg) mice expressing Nef, Env, and Rev of HIV type 1 (HIV-1) in CD4(+) T cells, dendritic cells, and macrophages and developing an AIDS-like disease (CD4C/HIV(MutA) Tg mice). Macrophages were successfully recruited to the oral and gastric mucosae of these Tg mice in response to chronic carriage of C. albicans and displayed polarization toward an alternatively activated phenotype. Functionally, peritoneal macrophages from uninfected Tg mice exhibited increased phagocytosis of C. albicans and enhanced production of interleukin 6 and monocyte chemoattractant protein 1, demonstrating that the HIV-1 transgene independently activates selected macrophage functions. Production of H(2)O(2) by macrophages from Tg mice primed with gamma interferon and treated with phorbol 12-myristate 13-acetate or C. albicans was moderately reduced, but expression of the HIV-1 transgene did not alter production of nitric oxide or reduce killing of C. albicans. A knockout of the inducible nitric oxide synthase (NOS2) gene in these Tg mice did not augment oral or gastrointestinal burdens during chronic carriage of C. albicans or cause systemic dissemination, likely due to a redundancy provided by partially preserved production of H(2)O(2) and oxygen-independent candidacidal mechanisms. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine the susceptibility to mucosal candidiasis.

Oral mucosal cell response to Candida albicans in transgenic mice expressing HIV-1.

Controlled studies on the immunopathogenesis of mucosal candidiasis in HIV infection have been hampered by the lack of a relevant animal model. We have previously reported that oral Candida infection in CD4C/HIV transgenic mice expressing gene products of HIV-1 in immune cells and developing an AIDS-like disease closely mimics oropharyngeal candidiasis in human HIV infection. The role of defective dendritic cells and CD4+ T cells in impaired induction of protective immunity and in the phenotype of chronic oral carriage of C. albicans can now be investigated under controlled conditions in these transgenic mice.

Primary human immunodeficiency virus type 1 nef alleles show major differences in pathogenicity in transgenic mice.

We previously reported that the human immunodeficiency virus type 1 NL4-3 Nef is necessary and sufficient to induce a severe AIDS-like disease in transgenic (Tg) mice when the protein is expressed under the regulatory sequences of the human CD4 gene. We have now assayed additional Nef alleles (SF2, JR-CSF, YU10x, and NL4-3 [T71R] Nef alleles), including some from long-term nonprogressors (AD-93, 032an, and 039nm alleles) in the same Tg system and compared their pathogenicities. All these Nef alleles downregulated cell surface CD4 in human cells in vitro and also, with the exception of Nef(YU10x), in Tg CD4(+) T cells. Depletion of double-positive and single-positive thymocytes occurred with all alleles but was less pronounced in Nef(YU10x) Tg mice. A loss of peripheral CD4(+) T cells was observed with all alleles but was minimal in Nef(YU10x) Tg mice. In Nef(032an) and Nef(SF2) Tg mice, T-cell loss was severe despite lower levels of Tg expression, suggesting a higher virulence of these alleles. All Nef alleles except the Nef(YU10x) and Nef(NL4-3(T71R)) alleles induced an enhanced activated memory (CD25(+) CD69(+) CD44(high) CD45RB(low) CD62L(low)) and apoptotic phenotype. Also, all could interact with and/or activate PAK2 except the Nef(JR-CSF) allele. Organ (lung and kidney) diseases were present in Nef(NL4-3(T71R)), Nef(032an), Nef(039nm), and Nef(SF2) Tg mice, despite very low levels of Tg expression for the last strain. However, no organ disease or minimal organ disease developed in Nef(YU10x) and Nef(AD-93) Tg mice and Nef(JR-CSF) Tg mice, respectively, despite high levels of Tg expression. Our data show that important differences in the pathogenicities of various Nef alleles can be scored in Tg mice. Interestingly, our results also revealed that some phenotypes can segregate independently, such as CD4(+) T-cell depletion and activation, as well as severe depletion of thymic CD4(+) T cells and peripheral CD4(+) T cells. Therefore, expression of Nef alleles in Tg mice under the CD4C regulatory elements represents a novel assay for measuring their pathogenicity. Because of the very high similarity of this murine AIDS-like disease to human AIDS, this assay may have a predictive value regarding the behavior of Nef in infected humans.

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.

Structurally distinct ligand-binding or ligand-independent Notch1 mutants are leukemogenic but affect thymocyte development, apoptosis, and metastasis differently.

We previously found that provirus insertion in T cell tumors of mouse mammary tumor virus/c-myc transgenic (Tg) mice induced two forms of Notch1 mutations. Type I mutations generated two truncated molecules, one intracellular (IC) (Notch1(IC)) and one extracellular (Notch1(EC)), while in type II mutations Notch1 was deleted of its C terminus (Notch1(DeltaCT)). We expressed these mutants in Tg mice using the CD4 promoter. Both Notch1(IC) and Notch1(DeltaCT), but not Notch1(EC), Tg mice developed double-positive (DP) thymomas. These disseminated more frequently in Notch1(DeltaCT) Tg mice. Double (Notch1(IC) x myc) or (Notch1(DeltaCT) x myc) Tg mice developed thymoma with a much shorter latency than single Tg mice, providing genetic evidence of a collaboration between these two oncogenes. FACS analysis of preleukemic thymocytes did not reveal major T cell differentiation anomalies, except for a higher number of DP cells and an accumulation of TCR(high)CD2(high)CD25(high) DP cells in Notch1(IC), and less so in Notch1(DeltaCT) Tg mice. This was associated with enhanced in vivo thymocyte proliferation. However, Notch1(IC), but not Notch1(DeltaCT), DP thymocytes were protected against apoptosis induced in vivo by dexamethasone and anti-CD3 and in vitro by anti-CD3/CD28 Abs. This indicates that the C terminus of Notch1 and/or the conserved regulation by its ligands have a significant impact on the induced T cell phenotype. Therefore, Notch1(IC) and Notch1(DeltaCT) behave as oncogenes for T cells. Because these two Notch1 mutations are very similar to those described in some forms of human T cell leukemia, these Tg mice may represent relevant models of these human leukemias.

Altered CD4+ T cell phenotype and function determine the susceptibility to mucosal candidiasis in transgenic mice expressing HIV-1.

The impairments of protective mucosal immunity which cause susceptibility to oropharyngeal candidiasis (OPC) in HIV infection remain undefined. This study used a model of OPC in CD4C/HIV MutA transgenic (Tg) mice expressing Rev, Env, and Nef of HIV-1 to investigate the role of transgene expressing dendritic cells (DCs) and CD4+ T cells in maintenance of chronic oral carriage of Candida albicans. DCs were depleted in the Tg mice and had an immature phenotype, with low expression of MHC class II and IL-12. CD4+ T cells were quantitatively reduced in the oral mucosa, cervical lymph nodes (CLNs) and peripheral blood of the Tg mice, and displayed a polarization toward a nonprotective Th2 response. Proliferation of CLN CD4+ T cells from infected Tg mice in response to C. albicans Ag in vitro was abrogated and the cells failed to acquire an effector phenotype. Coculture of C. albicans-pulsed DCs with CD4+ T cells in vitro showed that Tg expression in either or both of these cell populations sharply reduced the proliferation of CD4+ T cells and their production of IL-2. Finally, transfer of naive non-Tg CD4+ T cells into these Tg mice restored proliferation to C. albicans Ag and sharply reduced oral burdens of C. albicans. Overall, these results indicate that defective CD4+ T cells primarily determine the susceptibility to chronic carriage of C. albicans 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.

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/HIV(Mut) 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/HIV(MutA) 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/HIV(MutG) 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/HIV(MutG) 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.

Activation of p21-activated kinase 2 and its association with Nef are conserved in murine cells but are not sufficient to induce an AIDS-like disease in CD4C/HIV transgenic mice.

A well conserved feature of human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus (SIV) Nef is the interaction with and activation of the human p21-activated kinase 2 (PAK2). The conservation of this interaction in other species and its significance for Nef pathogenesis in vivo are poorly documented. In the present study, we measured these parameters in Nef-expressing thymocytes, macrophages, and dendritic cells of a transgenic (Tg) mouse model of AIDS (CD4C/HIV). We found that Nef binds to and activates PAK2, but not PAK1 and -3, in these three cell subsets. Nef associates with only a small fraction of PAK2. The Nef-PAK2 complex also comprises beta-PIX-COOL. The impact of the Nef-PAK2 association on disease development was also analyzed in Tg mice expressing 10 different Nef mutant alleles. CD4C/HIV Tg mice expressing Nef alleles defective in Nef-PAK2 association (P69A, P72A/P75A, R105A/R106A, Delta56-66, or G2A (myristoylation site)) failed to develop disease of the non-lymphoid organs (kidneys and lungs). Among these, only Tg mice expressing Nef(P69A) and Nef(G2A) showed some depletion of CD4(+) T cells, although a down-regulation of the CD4 surface protein was documented in all these Tg lines, except those expressing Nef(Delta56-66). Among other Tg mice expressing Nef mutants having conserved the Nef-PAK2 association (RD35AA, D174K, P147A/P150A, Delta8-17, and Delta25-65), only Tg mice expressing Nef(Delta8-17) develop kidney and lung diseases, but all showed partial CD4(+) T cell depletion despite some being defective for CD4 down-regulation (RD35AA and D174K). Therefore, Nef can activate murine PAK2 and associate with a small fraction of it, as in human cells. Such activation and binding of PAK2 is clearly not sufficient but may be required to induce a multiorgan AIDS-like disease in Tg mice.

HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice.

HIV-1 Nef has the ability to downmodulate CD4 cell surface expression. Several studies have shown that CD4 downregulation is required for efficient virus replication and high infectivity. However, the pathophysiological relevance of this phenomenon in vivo, independently of its role in sustaining high virus loads, remains unclear. We studied the impact of the CD4 downregulation function of Nef on its pathogenesis in vivo, in the absence of viral replication, in the CD4C/HIV transgenic (Tg) mouse model. Two independent Nef mutants (RD35/36AA and D174K), known to abrogate CD4 downregulation, were tested in Tg mice. Flow cytometry analysis showed that downregulation of murine CD4 was severely decreased or abrogated on Tg T cells expressing respectively Nef(RD35/36AA) and Nef(D174K). Similarly, the severe depletion of double-positive CD4+CD8+ and of single-positive CD4+CD8- thymocytes, usually observed with Nef(Wt), was not detected in Nef(RD35/36AA) and Nef(D174K) Tg mice. However, both mutant Tg mice showed a partial depletion of peripheral CD4+ T cells. This was accompanied, as previously reported for Net(Wt) Tg mice, by the presence of an activated/memory-like phenotype (CD69+, CD25+, CD44+, CD45RB(Low), CD62(Low)) of CD4+ T cells expressing Nef(RD35/36AA) and to a lesser extent Nef(D174K). In addition, both mutants retained the ability to block CD4+ T cell proliferation in vitro after anti-CD3 stimulation, but not to enhance apoptosis/death of CD4+ T cells. Therefore, it appears that Nef-mediated CD4 downregulation is associated with thymic defects, but segregates independently of the activated/memory-like phenotype, of the partial depletion and of the impaired in vitro proliferation of peripheral CD4+ T cells. Histopathological assessment revealed the total absence of or decrease severity and frequency of organ AIDS-like diseases (lung, heart and kidney pathologies) in respectively Nef(RD35/36AA) and Nef(D174K) Tg mice, relative to those developing in Nef(Wt) Tg mice. Our data suggest that the RD35/36AA and D174K mutations affect other Nef functions, namely those involved in the development of lung and kidney diseases, in addition to their known role in CD4 downregulation. Similarly, in HIV-1-infected individuals, loss of CD4 downregulation by Nef alleles may reflect their lower intrinsic pathogenicity, independently of their effects on virus replication.

The Nef-mediated AIDS-like disease of CD4C/human immunodeficiency virus transgenic mice is associated with increased Fas/FasL expression on T cells and T-cell death but is not prevented in Fas-, FasL-, tumor necrosis factor receptor 1-, or interleukin-1beta-converting enzyme-deficient or Bcl2-expressing transgenic mice.

CD4(+)- and CD8(+)-T-cell death is a frequent immunological dysfunction associated with the development of human AIDS. We studied a murine model of AIDS, the CD4C/HIV transgenic (Tg) mouse model, to assess the importance of the apoptotic pathway in human immunodeficiency virus type 1 (HIV-1) pathogenesis. In these Tg mice, Nef is the major determinant of the disease and is expressed in immature and mature CD4(+) T cells and in cells of the macrophage/myeloid lineage. We report here a novel AIDS-like phenotype: enhanced death, most likely by apoptosis (as assessed by 7-aminoactinomycin D and annexin V/propidium iodide staining), of Tg thymic and peripheral CD4(+) and CD8(+) T cells. The Tg CD4(+) and CD8(+) T cells were also more susceptible to cell death after activation in vitro in mixed lymph node (LN) cultures. However, activation-induced cell death was not higher in Tg than in non-Tg-purified CD4(+) T cells. In addition, expression of Fas and FasL, assessed by flow cytometry, was increased in CD4(+) and CD8(+) T cells from Tg mice compared to that of non-Tg littermates. Despite the enhanced expression of Fas and FasL on Tg CD4(+) and CD8(+) T cells, Fas (lpr/lpr) and FasL (gld/gld) mutant CD4C/HIV Tg mice developed an AIDS-like disease indistinguishable from lpr/+ and gld/+ CD4C/HIV Tg mice, including loss of CD4(+) T cells. Similarly, CD4C/HIV Tg mice homozygous for mutations of two other genes implicated in cell death (interleukin-1beta-converting enzyme [ICE], tumor necrosis factor receptor 1 [TNFR-1]) developed similar AIDS-like disease as their respective heterozygous controls. Moreover, the double-Tg mice from a cross between the Bcl2/Wehi25 and CD4C/HIV Tg mice showed no major protection against disease. These results represent genetic evidence for the dispensable role of Fas, FasL, ICE, and TNFR-1 on the development of both T-cell loss and organ disease of these Tg mice. They also provide compelling evidence on the lack of protection by Bcl2 against Tg CD4(+)-T-cell death. In view of the high resemblance between numerous phenotypes observed in the CD4C/HIV Tg mice and in human AIDS, our findings are likely to be relevant for the human disease.

In vivo mutational analysis of the N-terminal region of HIV-1 Nef reveals critical motifs for the development of an AIDS-like disease in CD4C/HIV transgenic mice.

HIV-1 Nef is a critical determinant of pathogenicity in humans and transgenic (Tg) mice. To gain a better understanding of the molecular mechanisms by which Nef induces an AIDS-like disease in Tg mice, a mutational analysis of the N-terminal domain, involved in anchoring Nef to the plasma membrane, was carried out. The pathogenic effects of these Nef mutant alleles were evaluated in Tg mice by FACS analysis and by histopathological assessment. Mutation of the myristoylation site (G2A) completely abrogated the development of the AIDS-like organ disease in Tg mice, although partial downregulation of the CD4 cell surface protein and depletion of peripheral CD4+ T-cells, but not of CD4(+)CD8+ thymocytes, still occurred. Despite that, the peripheral CD4+ T cells expressing Nef(G2A) show normal spontaneous proliferation in vivo or after stimulation in vitro, including in an allogenic mixed leukocyte reaction (MLR). Three other internal deletion mutants of Nef, spanning amino acids 8-17 (Nef(Delta8-17)), 25-35 (Nef(Delta25-35)), and 57-66 (Nef(Delta57-66)), were also studied. Nef(Delta8-17) retained full pathogenic potential, although Nef(Delta25-35) and Nef(Delta57-66) Tg mice were free of organ disease. However, Nef(Delta25-35) Tg mice exhibited disorganization of thymic architecture and a partial depletion of peripheral CD4+ T cells. These data indicate that myristoylation and other regions at the N-terminus of Nef (aa 25-35 and 57-66) are involved in mediating severe T-cell phenotypes and organ disease, although residues 8-17 are dispensable for these Nef functions. In addition, these results indicate that at least some of the CD4+ T-cell phenotypes can develop independently of the other AIDS-like organ phenotypes. This apparent segregation of different Nef-mediated phenotypes suggests distinct mechanisms of Nef action in different populations of target cells, and may be relevant to human AIDS.