Genetic Characterization of HIV Type 1 Nef-Induced Vesicle Secretion

Abstract

The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.

Expression of a Protein Phosphatase 1 Inhibitor, cdNIPP1, Increases CDK9 Threonine 186 Phosphorylation and Inhibits HIV-1 Transcription

“…The 2-kb fragment containing the linker was subcloned back into the Nco-BamH1 sites of pNL4-3 vector. The insert into the nef gene leads to the deletion of the first 36 amino acids of nef, which are critical for its function (32). The pNL4-3 398 vector was used as positive control in the infection experiment.…”

HIV Type 1 Nef Is Released from Infected Cells in CD45+Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

“…49,50 Some studies have separated microvesicles from HIV-1 virions using (Optiprep) iodixonal gradients clearly showing the capacity to discriminate between microvesicles, virions, and cell debris. 51,52 CD45 (human leukocyte antigen) is highly expressed on the surface of vesicles derived from hematopoietic cells but is not incorporated into HIV-1 virions. 53,54 This differential incorporation of CD45 has been used to remove contaminating vesicles from virion preparations and we have used this method conversely to capture microvesicles.…”

Exosomes in Viral Disease

“…This would further explain how Nef + exosomes enhance HIV infectivity. More recently, Luo et al [95] dispute the incorporation of Nef into exosomes at all, despite several reports finding motifs within Nef that are necessary for exosomal incorporation [96,97]. It is a subject that requires further investigation.…”