Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF

Abstract

The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2–6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8,9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.

Results and discussion

The gp41C domain interacts with the carboxy-terminal regulatory domain of p115-RhoGEF

To elucidate the function of gp41C, we performed a yeast two-hybrid screen to identify proteins that interact with it (see Supplementary material). From 5 × 10⁶ cDNA clones, four clones were identified. One encodes the carboxy-terminal 60 residues (gh60) of p115-RhoGEF and the other three all encode its carboxy-terminal 53 residues (g117; Figure 1). To confirm the interaction of p115-RhoGEF with gp41C in mammalian cells, we co-expressed p115 proteins with a gp41C protein fused to six histidine residues at the amino terminus (His₆–gp41C) and labeled the cells with [³⁵S]methionine and [³⁵S]cysteine. His₆–gp41C was precipitated with Ni–NTA beads and gp41C-associated proteins were visualized by autoradiography (Figure 1b). The full-length p115 (p115FL) was coprecipitated with gp41C (lane 4), and p115FL alone was not precipitated by the Ni–NTA beads (lane 3). The p115dC protein, which lacks the carboxy-terminal 100 residues [7], was not coprecipitated with His₆–gp41C (lane 5). These results were consistent with our findings from yeast cells, in which gp41C interacted with the carboxy-terminal 53 residues of p115 (g117, Figure 1c). Like other Dbl-family Rho GEFs, p115-RhoGEF is associated with the plasma membrane through its pleckstrin homology (PH) domain [9,10]. To show colocalization of p115-RhoGEF with HIV-1 gp41, p115FL was cotransfected with the HIV-1 provirus pNL4-3 in HeLa cells. Double staining with a rhodamine-labeled anti-p115 antiserum and a fluorescein isothiocyanate (FITC)-labeled anti-gp41C monoclonal antibody showed that gp41 and p115 were both localized in the cytoplasmic membrane region (Figure 1d). Thus, gp41 may be associated with p115 at the plasma membrane.

Figure 1.

Interaction of gp41C with the carboxyl terminus of p115-RhoGEF.

(a) Two-hybrid assay. The g117 fragment (the carboxy-terminal 53 residues of p115-RhoGEF) interacts with gp41C to activate the his3 gene and the lacZ gene. CDK6 and p18-INK4c were used as positive controls. Yeast colonies that grew on plates lacking leucine and tryptophan (−LW) were tested for the expression of lacZ (−LW/X-gal) or on histidine-deficient plates (−LWH). (b) Coprecipitation of p115 with His₆–gp41C in transfected 293T cells. The His₆–gp41C (encoded by plasmid pcHgp41C) was pelleted with Ni–NTA beads and pelleted proteins were visualized after autoradiography. Lanes 1–5 show samples from cells transfected with pcDNA3 vector alone (lane 1), pcHgp41C and pcDNA3 (lane 2), p115FL and pcDNA3 (lane 3), pcHgp41C and p115FL (lane 4), or pcHgp41C and p115dC (lane 5). Both p115FL and p115dC were efficiently expressed (data not shown). (c) Schematic representation of the structure and function of the p115 derivatives. Protein p115FL, full-length p115; p115dC, a truncated p115 fragment (residues 249–802); gh60 and g117, the gp41C-interacting fragments isolated from the two-hybrid assays (residues 853 (gh60) or 860 (g117) to 913 of p115-RhoGEF). GEF, guanine nucleotide exchanging activity on RhoA [7]; gp41C, interaction with the gp41C protein (in yeast and 293T cells); DH, Dbl homology region; PH, pleckstrin homology region. (d) Colocalization of gp41 and p115-RhoGEF. HeLa cells were cotransfected with DNAs encoding p115 and HIV-1 provirus NL4-3. Cells were stained with an anti-p115 antiserum [7] and an anti-gp41 monoclonal antibody. Secondary antibodies (rhodamine-labeled goat anti-rabbit and FITC-labeled anti-mouse) were used to detect p115 and gp41, respectively. Pre-immune or isotype controls showed no significant staining (data not shown).

Expression of gp41C disrupts actin stress fiber organization

To test whether gp41C affected p115 activity, we studied the effect of gp41C on actin stress fiber formation in Swiss 3T3 cells [11]. Cells overexpressing p115FL or p115dC showed enhanced actin stress fiber formation and increased phalloidin staining (Figure 2a,g). Non-injected cells had low levels of actin stress fibers. Expression of gp41C led to a disruption of the actin stress fibers (Figure 2c). Co-expression of p115FL or p115dC with gp41C led to reorganized actin stress fibers (Figure 2e,g). Therefore, gp41C inhibited actin stress fiber formation. Co-expression of active p115-RhoGEF counteracted the inhibitory activity of gp41C.

Figure 2.

Inhibition of p115-RhoGEF-mediated RhoA activation by gp41C.

(a–h) Disruption of actin stress fiber organization by gp41C. Swiss 3T3 cells were micro-injected with plasmid DNA and serum-starved for 12–16 h before fixation and staining of actin stress fibers. (a,b) Cells injected with Myc-tagged p115FL alone; (c,d) cells injected with a plasmid encoding enhanced green fluorescent protein under the control of the CMV promoter (pCMV–eGFP) and pcHgp41C; (e,f) cells injected with p115FL and pcHgp41C; (g,h) cells injected with Myc-tagged p115dC and pcHgp41C. The injected cells (arrows) were identified either (d) by GFP signals or (b,f,h) by immunostaining of the Myc epitope-tagged p115 proteins. The rhodamine–phalloidinstained stress fibers were visualized in (a,c,e,g). Representatives of about 20 microinjected cells for each sample are shown. Two independent experiments were performed. (i) Inhibition of p115-RhoGEF-mediated activation of SRF by gp41C. A plasmid with a luciferase gene controlled by a mutant c-Fos serum response element (SREm2-Luc) that no longer responds to ternary complex factor (TCF) and measures only SRF activity (see Supplementary material) was co-expressed with plasmids encoding fragments of gp41 and/or p115, and luciferase activity was measured. Co-expression of gp41C inhibited p115FL-mediated activation by 70% (p115+gp41C), but did not inhibit p115dC-mediated activation (p115dC+gp41C) or FGD1-activated SRF (FGD1+gp41C). The experiments were performed in triplicate and repeated three times. Error bars indicate standard deviations.

Ectopic expression of gp41C inhibits p115-mediated SRF activation

We further showed that gp41C inhibited p115-mediated activation of SRF, which is activated by Rho GTPases [12]. An SRF-responsive reporter gene was activated by p115 in serum-starved NIH 3T3 cells (Figure 2i). Co-expression of gp41C inhibited this p115-mediated activation. This inhibition depended on the interaction between gp41C and p115 because p115dC, which did not interact with gp41C, also activated the SRF reporter but gp41C co-expression failed to inhibit its activity (Figure 2i). Furthermore, gp41C showed no inhibition of SRF activation mediated by other Rho GEFs such as FGD1 [13] (Figure 2i) and Dbl (data not shown). Therefore, gp41C interacted with p115-RhoGEF to inhibit its activity. Further investigation of the interaction between gp41C and p115 should help to elucidate the mechanism of GEF-mediated Rho activation.

Inhibition of p115-mediated activation of RhoA by gp41C has significant implications for HIV-1 pathogenesis. First, inhibition of RhoA activity has been reported to enhance the spreading and migration of monocytes and macrophages [14]. Thus, gp41C may interact with p115-RhoGEF to modulate target-cell migration from the initial site of infection. Second, RhoA is also involved in regulating cell survival and cell-cycle progression [15,16]. Inhibition of p115-RhoGEF (or RhoA) activity by gp41C may lead to depletion of human T cells and progenitor cells [17,18].

A putative leucine-zipper motif in gp41C is involved in the interaction with p115-GEF

Several structural motifs implicated in protein–protein interaction and signal transduction are present in gp41C (Figure 3a). Two lentivirus lytic peptides (LLP1 and LLP2) interact with calmodulin [19] and inhibit Ca²⁺-dependent T-cell activation [20]. A leucine-zipper motif (790–811) has recently been shown to bind lipid membranes [21]. To map the region of gp41C required for the interaction with p115, gp41C was dissected by deletion analysis (Figure 3a). Deletion of 12 or more residues at the carboxyl terminus (dC1–dC6) abolished interaction with p115 (g117). Deletion of the amino-terminal 52 residues of gp41C (dN1), however, had no effect on the interaction. When the LLP2 sequences were deleted (dN2), interaction with p115 was lost. Thus, the carboxy-terminal 98 residues of gp41C, containing the two LLPs and the leucine-zipper motif, are necessary and sufficient to mediate interaction of gp41C with p115.

Figure 3.

A putative leucine-zipper (LZ) motif of gp41C is involved in the interaction with p115.

(a) Mapping of the gp41C domains involved in interaction with the carboxyl terminus of p115-RhoGEF. TM, transmembrane domain; LLP1 and LLP2, lentivirus lytic peptides. The numbers 1–854 indicate residue positions in the HIV-1 gp160 env polypeptide. The residues after the TM domain (704–854) are referred to as gp41C and numbered 1–151. The fragments gp41C (1–151), dC1 (1–47), dC2 (1–70), dC3 (1–90), dC4 (1–97), dC5 (1–108), dC6 (1–139), dN1 (53–151), and dN2 (83–151) were fused to the Gal4 DNA-binding domain. Binding to g117 (+ or −) was determined by growth on His⁻ plates and expression of lacZ, as in Figure 1. (b) Mutations defined by the reverse yeast-two-hybrid screen are localized around the LZ region. The amphipathic α-helix region containing the LZ motif is shown in the single-letter amino acid code and the leucines comprising the LZ motif (LX₆LX₆LX₆L, where X is any amino acid) are in bold. The mutations defined by the reverse two-hybrid screen leading to loss of interaction with g117 are marked.

To define specific residues in gp41C involved in direct interaction with p115, a modified ‘reverse’ two-hybrid assay (see Supplementary material) was performed. Four out of five mutants identified carried mutations in the amphipathic helix region of gp41C that encodes the putative leucine-zipper motif (Figure 3b). The fifth mutation was in the LLP1 region (data not shown). The four leucine-zipper mutations (tryptophan to serine or arginine and serine to arginine) resulted in an increase in charge on the hydrophobic side of the amphipathic helix. To further confirm that the leucine-zipper motif is involved, we introduced an arginine replacing the second leucine in the leucine-zipper motif (L95R), which also led to loss of interaction with p115.

Loss of interaction between gp41C and p115-RhoGEF impaired HIV-1 replication

To prove genetically the importance of the interaction between gp41C and p115, the L95R mutation was introduced into the HIV_NL4–3 genome. The HIV-1 NL4(L95R) mutant produced wild-type levels of infectious virions when transfected into 293T cells (data not shown). When human leukemia T cells (SupT1, H9, and Jurkat cells) were infected, the L95R mutant had impaired replication (Figure 4). Therefore, the interaction of gp41C with p115 correlated with enhanced HIV-1 replication in human T-cell lines. This interaction provides a new host target (p115-RhoGEF) and a new viral target (gp41C) for the development of anti-HIV therapies.

Figure 4.

The interaction between gp41C and p115-RhoGEF correlates with HIV-1 replication in human T-cell lines. SupT1, H9 or Jurkat cells were infected with an equal number of infectious units of NL4-3 or NL4(L95R) mutant viruses. Viral replication (as measured by reverse transcriptase activity) was monitored every 3 days for 18 days after infection. The experiments were repeated three times with similar results.