The HIV-1 Nef protein binds argonaute-2 and functions as a viral suppressor of RNA interference

The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR)

Transcriptomic Analysis of mRNAs in Human Monocytic Cells Expressing the HIV-1 Nef Protein and Their Exosomes

The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression to AIDS. Besides its well-studied effects on intracellular signaling, Nef also functions through its secretion in exosomes, which are nanovesicles containing proteins, microRNAs, and mRNAs and are important for intercellular communication. Nef expression enhances exosome secretion and these exosomes can enter uninfected CD4 T cells leading to apoptotic death. We have recently reported the first miRNome analysis of exosomes secreted from Nef-expressing U937monocytic cells. Here we show genome-wide transcriptome analysis of Nef-expressing U937 cells and their exosomes. We identified four key mRNAs preferentially retained in Nef-expressing cells; these code for MECP2, HMOX1, AARSD1, and ATF2 and are important for chromatin modification and gene expression. Interestingly, their target miRNAs are exported out in exosomes. We also identified three key mRNAs selectively secreted in exosomes from Nef-expressing U937 cells and their corresponding miRNAs being preferentially retained in cells. These are AATK, SLC27A1, and CDKAL and are important in apoptosis and fatty acid transport. Thus, our study identifies selectively expressed mRNAs in Nef-expressing U937 cells and their exosomes and supports a new mode on intercellular regulation by the HIV-1 Nef protein

Characterization of Nef-expressing cell lines.

<p>U937 cells stably expressing either a Nef-EYFP fusion protein or EYFP were established as described in Materials and Methods. (A) Western blot for expression of EYFP (lanes Y6-A to -D) and Nef-EYFP (lanes NY-19 and -20) in selected U937 stable clones. (B) Flow cytometric analysis of U937/Nef-EYFP and U937/EYFP cells for surface expression of the indicated proteins. (C) The U937/Nef-EYFP cells were (i) cultured with 5 µM NRhPE, or (ii) stained with anti-Ago2 as described in Materials and Methods. U1 cells were activated with PMA and labeled with anti-Nef and either (iii) anti-Ago2 or (iv) anti-P bodies as described in Materials and Methods. The white arrowheads in the merged image represent colocalization points. The images are representative of three independent experiments. The boxed regions are expanded. (D) Correlation analysis of the colocalization of Nef with Ago2 in (i) U937/Nef-EYFP cells and (ii) activated U1 cells showing Pearson’s coefficient (PC) and Manders coefficients (M1, M2). The coefficients represent an average of three independent images, each consisting of at least 10 cells, calculated using the JACoP plugin within the Image J software. M1 represents the fraction of Ago2-red overlapping with Nef-green and M2 represents the fraction of Nef-green overlapping with Ago2- red. Bars represent ± SD.</p

Nef interacts with Ago2.

<p>(A) Lysates of U937/Nef-EYFP or U937/EYFP cells were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. The lane marked NIC represents IP of U937/Nef-EYFP lysates with an irrelevant antibody. (B) U1 and J1.1 cells were treated with DMSO (–) or PMA (+) and cell lysates were either immunoblotted for p24 and Nef (i, ii), or IP/IB with anti-Nef and anti-Ago2 (iii, iv), as described in Materials and Methods. U937 and Jurkat cells served as background controls and antibody heavy chain as loading control. (C) Lysates were prepared from the U937 stable cell lines using Buffer D as described in Materials and Methods. From this, 150 µg lysate was passed through a pre-equilibrated Sephacryl S200HR column with a 3 ml bed volume. The column was eluted and fractions of 3 drops (∼120 µl) were collected, which were then precipitated with acetone, separated by SDS-PAGE and western blotting was done with Ago2 and GFP antibodies. (D) Lysates from activated U1 cells were prepared and fractionated as in (C) followed by SDS-PAGE and western blotting with Ago2 and Nef antibodies. The profiles shown are representative of three separate experiments.</p

Nef does not affect siRNA loading into RISC, but inhibits its slicing activity.

<p>(A) Lysates from the U937 stable cell lines were prepared in Buffer D as described in Materials and Methods. From this, 25 µg of lysate was incubated with ³²P-labeled duplex siRNA (without or with 50-fold molar excess of unlabeled siRNA) in binding buffer for 30 min at 4°C. The complexes were separated on a 6% non-denaturing polyacrylamide gel. (B) The gel shift assay was set up as in (A) except that lysates immunodepleted for either Ago2 or Dicer were also included. The positions of the mobility shifted complexes and free probe are indicated. (C) Slicer activity was assayed using immunoprecipitated Ago2 from U937/Nef-EYFP or U937/EYFP cell lysates and a ³²P-labeled <i>in vitro</i> transcribed let7a RNA, as described in Materials and Methods. The positions of full-length RNA and the two sliced products are shown. The 59 nt marker oligonucleotide was run on the same gel, but a lower exposure is shown. (D) Densitometric analysis of autoradiograms from three independent experiments was carried out using Image J (version 1.4.1). The slicing activity in Nef-EYFP lysates is represented as a percentage of that in EYFP lysates.</p

Mutations in Nef GW motifs abrogate its interaction with Ago2 and reduce virus yield and infectivity.

<p>(A) Alignment of Nef sequences. All 2660 Nef amino acid sequences available in the Los Alamos HIV database were analyzed. Regions encompassing the two conserved GW motifs (bold and underline) at positions 12–13 and 140–141 are shown for consensus and ancestral (.anc) sequences for various HIV-1 M group clades (A–H), and consensus sequences for various recombinant circulating forms. (B) HEK293T cells were transiently transfected to express wild type or GW mutant Nef proteins. Cell lysates were precipitated with anti-Nef and blotted with anti-Ago2. Nef and Actin were expression and loading controls, respectively. (C) HEK293T cells were transfected with either pNL4-3 or pNL4-3ΔNef; cells were also co-transfected with pNL4-3ΔNef and expression vectors for either the wild type or GW mutant Nef proteins. After 36 hr the culture supernatants were quantified for p24. Purified viruses equivalent to 100ng p24 were also used to infect 0.5 million 1G5 Jurkat indicator cells, and 48 hr later the cells were harvested, lysed and luciferase assay was performed as described in Methods. Error bars represent mean ± SD from three independent experiments, and p-values calculated using the Student’s <i>t</i>-test were as follows: * 0.002; # 0.023.</p

Nef blocks the sorting of GW182 into exosomes.

<p>Exosomes were purified from the spent culture media of the two cell lines as detailed in Materials and Methods. (A) Western blotting was done using 40 µg of cellular and exosomal lysates using antibodies that detect marker proteins for exosomes (Alix, Tsg101, CD81), mitochondria (VDAC), endoplasmic reticulum (Calnexin) and apoptotic bodies (Cytochrome C). GAPDH and Actin were used as loading controls and anti-GFP antibodies were used to detect Nef-EYFP and EYFP. (B) Cellular and exosomal lysates were subjected to SDS-PAGE and western blotting to detect GW182, Ago2 and Nef; GAPDH was used as a loading control.</p