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.