The Effects of HIV-1 Nef on CD4 Surface Expression and Viral Infectivity in Lymphoid Cells Are Independent of Rafts

Abstract

The HIV-1 Nef protein is a critical virulence factor that exerts multiple effects during viral replication. Nef modulates surface expression of various cellular proteins including CD4 and MHC-I, enhances viral infectivity, and affects signal transduction pathways. Nef has been shown to partially associate with rafts, where it can prime T cells for activation. The contribution of rafts during Nef-induced CD4 down-regulation and enhancement of viral replication remains poorly understood. We show here that Nef does not modify the palmitoylation state of CD4 or its partition within rafts. Moreover, CD4 mutants lacking palmitoylation or unable to associate with rafts are efficiently down-regulated by Nef. In HIV-infected cells, viral assembly and budding occurs from rafts, and Nef has been suggested to increase this process. However, using T cells acutely infected with wild-type or nef-deleted HIV, we did not observe any impact of Nef on raft segregation of viral structural proteins. We have also designed a palmitoylated mutant of Nef (NefG3C), which significantly accumulates in rafts. Interestingly, the efficiency of NefG3C to down-regulate CD4 and MHC-I, and to promote viral replication was not increased when compared with the wild-type protein. Altogether, these results strongly suggest that rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication.

Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle

“…This role has been, however, questioned, because CD4 molecules unable to associate with rafts still allow virus entry (Percherancier et al, 2003;Popik and Alce, 2004). Moreover, Sol-Foulon and coworkers proposed that the effects of Nef on CD4 downregulation and the increase in viral infectivity were independent of lipid rafts (Sol-Foulon et al, 2004). Discrepancies in methodological approaches from these authors with the reports described before could account for the observed differences on this effect of Nef.…”

Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef

“…In all experiments, a Gly-to-Ala point mutation was introduced by PCR at residue position 2 of the Nef consensus sequence; this mutation has been shown to obviate Nef-mediated major histocompatibility complex (MHC) class I down-regulation. 28 Except for residue G2, the predicted 207-residue translation product of the consensus Nef sequence is 100% identical to that of an array of synthetic overlapping peptides (OLPs) covering the 2001 B consensus Nef sequence, allowing a direct comparison of immune responses to mRNA and synthetic peptide.…”

Human Immunodeficiency Virus Type 1 Nef Incorporation into Virions Does Not Increase Infectivity

“…In addition, various proteins fused to Vpr were shown to be efficiently incorporated into HIV-1 particles while retaining their original properties (17,32,57,93,94). Since the myristoylation of Nef is important for most of the functions of Nef investigated so far (8,82,91), we decided to fuse Vpr to the C-terminal end of Nef as depicted in Fig. 1.…”