Production of HIV particles is regulated by altering sub-cellular localization and dynamics of Rev induced by double-strand RNA binding protein

Abstract

Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1-dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication.

Figure 1. NF90ctv recognizes and exports transcripts linked to RRE.

A) Schematic representation of full-length NF90ctv and of the various truncated forms used in this study. The main domains of NF90ctv and their positions are shown. B) Quantification and Western blot results demonstrating that NF90ctv and specifically, the RCN, DRBD1/2 and RG domains bind and export mRNA-Gag linked to RRE. HeLa cells were co-transfected with the construct pCMVGag2RRE in the presence of pNF90ctv-mRFP or each of its truncated forms. pRev-GFP was used as positive control. After 24 h Western blots were performed using pr55Gag antibodies. C) Kinetic and quantification of pr55Gag expression. HeLa cells were co-transfected with pRCN-mRFP in the presence of pCMVGag2RRE. At different times, the cells were lysed and Western blots were performed using pr55Gag antibodies. D) Quantification of the results obtained by Western blots demonstrates that RCN, DRBD1/2 and RG mRNA-Gag linked to RRE export is leptomycin-dependent. HeLa cells were cultured in six-well plates; three wells for each construct of interest were used: one well for leptomycin treatment and two wells as controls at 8 and 12 h. The cells were co-transfected with pRCN-mRFP or with pDRBD1/2-mRFP, with pRG-mRFP in the presence of pCMVGag2RRE. Based on the results shown in D, 8 h later the cells of one well were harvested; in the second well, leptomycin B was added and incubated for 4 h; finally, 12 h later, the cells of the second and the third wells were harvested. Western blots were performed on the cell pellets using pr55Gag antibodies. Each assay was repeated three times. The Western blots were quantified by densitometric scanning using ImageJ and normalized using loading controls (β-tubulin). The data shown are the means and standard errors of the mean of three independent experiments.

Figure 2. The RCN, DRBD1/2 and RG domains of NF90ctv inhibit accumulation of HIV-1 p24 in the cells.

HeLa cells were co-trasnfected with each of the NF90ctv domains of interest in the presence of HIVΔEnv.GFP. After 24 h, Western blots were performed on the cell pellets using HIV-1IIIB pr55Gag antibodies. The quantification of the pr55Gag and p24 proteins was determined as described in Figure 1. The data presented are the means and standard errors of three independent experiments.

Figure 3. NF90ctv and three of its truncated forms block HIV-1 replication.

A) ELISA results show that the effect of NF90ctv and three of its deletions on HIV-1 replication is dose-dependent. HeLa cells were co-transfected with pNL4-3 and with different concentrations of pNF90ctv-mRFP, pRCN-mRFP, DRBD1/2- mRFP or pRG-mRFP. After 24 h and 48 h the supernatants from the cell cultures were collected and assayed for HIV-1 p24 accumulation by ELISA. The cells used to obtain the results observed in A, were lysed and Western blots were performed on the cell pellets using HIV-1IIIB pr55Gag antibodies. B) NF90ctv and three of its deletions inhibit accumulation of p24 in the cells.

Figure 4. The RCN, DRBD1/2 and RG domains alter the subcellular localization of the HIV-1 Rev protein.

A) Subcellular localization of Rev in cells transfected with pRev-GFP alone (left panel) or with pRev-GFP in the presence of the RRE (right panel), and subcellular localization of pDRBD1/2-mRFP alone (middle panel). The cells were fixed 24 h post-transfection and the fluorescence (green or red respectively from Rev or DRBD1/2) was registered by confocal microscopy. Rev-GFP alone is visible exclusively in nucleoli (Nu); in the presence of RRE, Rev-GFP is also visible in the cytoplasm; pDRBD1/2-mRFP is visible in the cytoplasm and nucleolus. For RCN and RG a similar subcellular localization as for DRBD1/2 was observed (results not shown). B) Subcellular localization of Rev in the presence of RCN and DRBD1/2. HeLa cells were co-transfected with pRev-GFP, with pRCN-mRFP or with pDRBD1/2 and 24 h later, the respective fluorescence was observed. Green and red signals are illustrated alone and merged in the same cell. The yellow color in the merge indicates co-localization in nucleoli and cytoplasm. A similar result was observed for the RG domain (results not shown). C) In the presence of the RRE, RCN and RG drastically alter the subcellular localization of Rev. HeLa cells were transfected as described in B, but in addition with pCMVGag2RRE (to obtain RRE), and 24 h later the cells were observed by confocal microscopy. As illustrated for RCN-mRFP (upper panel), Rev-GFP is almost excluded from the nucleoli. At low magnification, a group of cells (lower panel) illustrates the variability of the distribution of Rev in the presence of RG and RRE. A similar result was obtained for DRBD1/2 (results not shown). D) Signal intensity was quantified in different cells as illustrated in A. GFP or red fluorescence either in the nucleolus (Nu), in the nucleoplasm (Nucleopl) or in the cytoplasm (Cy) were determined for each cell. E) Signal intensity was quantified in different cells as in B, as described in D. F) Signal intensity was quantified in different cells as in C, as described in D. As control HeLa cells were co-transfected with pRev-GFP and pmRFP, but no effect on Rev localization was detected (results not shown).

Figure 5. NF90ctv, RCN, DRBD1/2 and RG alter Rev mobility.

A) Rev mobility was measured by FRAP in HeLa cells expressing Rev-GFP. The recovery of the Rev fluorescence in the nucleolar region bleached by laser was measured for 200 sec. Three images were taken before bleaching. B) FRAP analysis shows the effect of NF90ctv and its truncated forms on Rev mobility. HeLa cells were co-transfected with pRev-GFP in the presence of pNF90ctv-mRFP, of pRCN-mRFP, of DRBD1/2-mRFP or of pRG-mRFP and subjected to photobleaching 24 h post-transfection using a 488 nm laser. The average from at least 12 cells is shown. FRAP demonstrates that the FRAP rate of Rev-GFP alone is slower than in the presence of NF90ctv or each of its truncated forms, especially in the presence of RCN. C) The same as A but Rev is in the presence of RRE. D) The same strategies were used as described in B, but in addition, the cells were also transfected with pCMVGag2RRE. The FRAP analysis shows that in the presence of RRE and NF90ctv or each of the truncated forms of NF90ctv, the mobility of Rev was slower during the first 30 sec, but that afterwards Rev its mobility was faster, especially with RCN. As control HeLa cells co-transfected with pRev-GFP and pmRFP were used, but no effect on Rev mobility was detected (results not shown).