CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration.

Abstract

The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti‐CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi‐specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post‐translational modification of CD4.

T Cell Activation and Proliferation Characteristic for HIV-Nef Transgenic Mice Is Lymphopenia Induced

“…Total thymocyte cellularity of Nef tg mice was 10-fold reduced compared with wt littermates (Fig. 1A) as shown before (17). Nef expression disturbs thymocyte development, as evidenced by changes in thymic subset distribution, resulting in low numbers of single-positive thymocytes (17, 18) (data not shown).…”

“…In these mice, HIV-1 BRU Nef gene expression is under control of the CD2 promoter and locus control region resulting in constitutive transgene expression in all T cells, starting during the earliest stages of T cell development. The most striking phenotype in these mice is a dramatic depletion of thymocytes (17). Total thymocyte cellularity of Nef tg mice was 10-fold reduced compared with wt littermates (Fig.…”

“…1B), indicative for the presence of functional Nef protein. Analysis of peripheral lymphoid organs revealed a marked decrease in T cell numbers in both PLN and spleen (17) (Fig. 1C).…”

“…To study the consequence of Nef expression in primary cells in vivo, various Nef tg murine models have been generated (6,(15)(16)(17). All, except one, which expresses Nef in CD4 T cells as well as in cells of the monocyte/macrophage lineage (6), express Nef specifically in the T cell compartment.…”

“…The CD2 Nef tg mice have been described previously (17) and were a gift from E. Dzierzak (Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands). Experiments were performed with mice 6 -12 wk of age.…”