Paul Jolicoeur

We previously reported that CD4C/human immunodeficiency virus (HIV)^Neftransgenic (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^Nefand 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^NefTg mice. Mice from all five strains developed persistent oral carriage ofCandida 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.

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.

Some murine leukemia viruses (MuLVs), among them Cas-Br-E andts-1 MuLVs, are neurovirulent, inducing spongiform myeloencephalopathy and hind limb paralysis in susceptible mice. It has been shown that theenvgene of these viruses harbors the determinant of neurovirulence. It appears that neuronal loss occurs by an indirect mechanism, since the target motor neurons have not been found to be infected. However, the pathogenesis of the disease remains unclear. Several lymphokines, cytokines, and other cellular effectors have been found to be aberrantly expressed in the brains of infected mice, but whether these are required for the development of the neurodegenerative lesions is not known. In an effort to identify the specific effectors which are indeed required for the initiation and/or development of spongiform myeloencephalopathy, we inoculated gene-deficient (knockout [KO]) mice withts-1 MuLV. We show here that interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS), ICE, Fas, Fas ligand (FasL), and TNF-R1 KO mice still develop signs of disease. However, transgenic mice overexpressing Bcl-2 in neurons (NSE/Bcl-2) were largely protected from hind limb paralysis and had less-severe spongiform lesions. These results indicate that motor neuron death occurs in this disease at least in part by a Bcl-2-inhibitable pathway not requiring the ICE, iNOS, Fas/FasL, TNF-R1, and IL-6 gene products.

Vacuolar myelopathy (VM) is a frequent central nervous system complication of human immunodeficiency virus type 1 (HIV-1) infection. We report here that transgenic (Tg) mice expressing even low levels of Nef in oligodendrocytes under the regulation of the myelin basic protein (MBP) promoter (MBP/HIV ^Nef ) developed VM similar to the human disease in its appearance and topography. The spinal cords of these Tg mice showed lower levels of the myelin proteins MAG and CNPase and of the 21-kDa isoform of MBP prior to the development of vacuoles. In addition, Tg oligodendrocytes in primary in vitro cultures appeared morphologically more mature but, paradoxically, exhibited a less mature phenotype based on O4, O1, CNPase, and MBP staining. In particular, mature CNPase ⁺ MBP ⁺ Tg oligodendrocytes were less numerous than non-Tg oligodendrocytes. Therefore, Nef appears to affect the proper differentiation of oligodendrocytes. These data suggest that even low levels of Nef expression in human oligodendrocytes may be responsible for the development of VM in HIV-1-infected individuals.

CD4C/human immunodeficiency virus (HIV) transgenic mice develop an AIDS-like disease. We used this model to study the effects of HIV-1 on dendritic cells (DC). We found a progressive decrease in total DC numbers in the lymph nodes, with a significant accumulation of CD11b^Hi DC. In the thymus, the recovery of transgenic CD8α⁺ DC had a tendency to be lower. Spleen DC were augmented in the marginal zone. Transgenic DC showed a decreased capacity to present antigen in vitro, consistent with their reduced major histocompatibility complex class II expression and impaired maturation profile. The accumulation of immature DC may contribute to disease and may reflect an adaptive advantage for the virus by favoring its replication and preventing the generation of fully functional antiviral responses.

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 .

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 SIV_mac239nefgene in the natural target cells of the virus under the control of the human CD4 gene promoter (CD4C). These CD4C/SHIV-nef^SIVTg 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 developPneumocystis cariniilung 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.

To identify the regulatory elements controlling expression of the human CD4 (hCD4) gene in different cell types of the immune system, deletion and chimeric (human/murine) reporter genes were constructed and tested in transgenic (Tg) mice. Regulatory elements required for the proper hCD4 expression in the immature double-positive thymic T cells were identified in the enhancer and in the 3′ end of intron 1. Expression of hCD4 in macrophages is controlled by at least 2 sets of regulatory elements: one present in front of exon 1 and the second at the 5′ end of intron 1. The hCD4 elements required for expression on both myeloid and lymphoid CD8α⁺ dendritic cells (DCs) from lymph node and thymus were found to be different from those required for macrophage expression. The results indicate that expression of hCD4 in T cells, macrophages, and DCs is controlled by distinct regulatory elements.

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 (P ₇₂ XXP ₇₅ XXP ₇₈ ) was mutated to A ₇₂ XXA ₇₅ XXQ ₇₈ . 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.