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. 1999 Nov 4;9(21):1271-4.
doi: 10.1016/s0960-9822(99)80511-9.

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

H Zhang  1 , L Wang, S Kao, I P Whitehead, M J Hart, B Liu, K Duus, K Burridge, C J Der, L Su
Affiliations

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

H Zhang et al. Curr Biol. .

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] [3] [4] [5] [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.

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Figures

Figure 1
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).
Figure 2
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.
Figure 3
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.
Figure 4
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.
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