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. Author manuscript; available in PMC: 2008 Nov 10.
Published in final edited form as: Virology. 2007 Jul 19;368(1):1–6. doi: 10.1016/j.virol.2007.06.022

Antiviral Activity of a Rac GEF inhibitor Characterized with a Sensitive HIV/SIV Fusion Assay

Suzanne Pontow 1, Brooke Harmon 1, Nancy Campbell 1, Lee Ratner 1
PMCID: PMC2174213  NIHMSID: NIHMS33610  PMID: 17640696

Abstract

A virus-dependent fusion assay was utilized to examine the activity of a panel of HIV-1, -2, and SIV isolates, of distinct coreceptor phenotypes. This assay allowed identification of entry inhibitors, and characterization of an antagonist of a Rac guanine nucleotide exchange factor, as an inhibitor of HIV-mediated fusion.

Keywords: HIV-1, envelope, Rac, fusion

HIV infection is initiated by binding of envelope surface protein (SU or gp120) to CD4 and coreceptor, usually either chemokine receptor, CCR5 or CXCR4 (Pierson and Doms, 2003). This results in conformational changes in transmembrane envelope protein (TM or gp41), resulting in insertion of the fusion peptide into the cell membrane, and subsequent fusion of viral and cell membranes. HIV envelope acts as a chemokine mimic, stimulating responses such as chemotaxis, gene transcription, and phosphorylation (Sodhi, Montaner, and Gutkind, 2004).

One target of this signaling pathway is the actin filament network (Matarrese and Malorni, 2005). Reorganization of the actin cytoskeleton is a critical feature of HIV-induced fusion (Pontow et al., 2004). This is mediated by activation of Rho family GTPases, especially Rac (Burridge and Wennerberg, 2004). Rac regulates diverse cellular processes, including intercellular adhesion, cytoskeletal membrane ruffling and lamellipodia formation, proliferation, and gene transcription. The active, GTP-bound form of Rac is negatively regulated by Rac GTPases (GAPs) and positively regulated by Rac guanine nucleotide exchange factors (GEFs). Tiam1 is a GEF specific for Rac, while others are more promiscuous in activating multiple Rho GTPases.

In order to further elucidate the role of Rac activation in HIV fusion, we made use of a novel virus-dependent fusion assay (Clavel and Charneau, 1994; Esser et al., 1999; Murakami et al., 2004; Pontow et al., 2004). This is based on the ability of virus particles to bridge at least two cells and allow transfer of cytoplasmic contents. In this assay, we use U87 glioma cells expressing CD4 and CCR5 or CXCR4, as well as vaccinia virus expressing T7 polymerase. The second population of U87 glioma cells, with CD4 and CCR5 or CXCR4, is infected with a vaccinia virus with a β-galactosidase gene under the regulation of the T7 promoter. A three hour incubation of these two cell populations in the presence of fusion-competent virus particles allows fusion, quantified by β-galactosidase activity. Sensitivity of the assay was found to be enhanced by serum starvation for 24-48 hrs prior to fusion. We show here that this assay is rapid, flexible, and applicable to a wide range of lentivirus isolates. Moreover, this assay is useful for examining the activity of inhibitors of receptor or co-receptor binding, fusion peptide activity, as well as subsequent fusion activities, including Rac activation.

Results

Comparison of virus-dependent fusion and infection assays and the env-dependent fusion assay

The virus-dependent fusion assay was directly compared to the env-dependent fusion assay (Fig 1). For the env-dependent fusion assay, a macrophage-tropic virus, derived from the YU2 envelope (WT), was compared to one with a mutation in gag, resulting in substitution of L12E within the MA protein, resulting in a defect in envelope incorporation in virus particles (Freed and Martin, 1996; Kaushik and Ratner, 2004). Both proviral clones, expressed similar amount of cell-surface envelope, as demonstrated by the fusion assay (Fig 1, left-hand bars). However, in the virus-dependent fusion assay the WT virus is capable of inducing fusion, whereas, the L12E virus, defective in envelope incorporation, fails to induce fusion activity in this assay (Fig 1, right-hand bars).

Fig 1. Comparison of Env-dependent and virus-dependent fusion assays, using an Env packaging-defective mutant proviral clone (L12E).

Fig 1

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Virus particles from HIV-1 MA mutant L12E have diminished levels of envelope incorporation and demonstrate little virus-dependent fusion activity. In contrast, transfection of these proviral clones into BSC40 cells result in similar levels of Env-induced fusion when cells are mixed with U87-CD4 cells.

The virus-dependent fusion and infection assays were also compared with isogenic viruses that differed only in the sequence of their V3 envelope domain (Fig 2) (Hung, Heyden, and Ratner, 1999). Virus, p2027 includes the V3 loop from R5 strain SF162. In contrast, virus IDI has a V3 loop derived from X4 strain HXB2, with the exception of substitutions at positions 27, 29, and 30 of the V3 loop that are found in SF162. Virus EIDI is identical to virus IDI with the exception of an additional substitution at position 25. Twenty or 50 ng of virus was tested in the virus-dependent fusion assay, as described above. In contrast, 10 or 50 ng of virus was tested for infection of Magi.CD4.CCR5 cells (Pirounaki et al., 2000). The viruses exhibited dose-dependent levels of infection and fusion in these assays, and the results were quite similar.

Fig 2. Virus-dependent fusion assay results are comparable to levels of infection of HeLa.CD4.CCR5 cells containing an LTR-lacz reporter using viruses with Env V3 mutations.

Fig 2

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These viruses include amino acid substitutions in the V3 envelope domain that affect their efficiency of use of CCR5 (Hung, Heyden, and Ratner, 1999).

The virus-dependent fusion and infection assays were also tested with a panel of 40 primary HIV isolates with differences in coreceptor tropism, as well as viruses derived from 14 HIV-1 molecular clones, 2 HIV-2 molecular clones, and 2 SIV molecular clones (Table 1, Fig 3). For this purpose, fusion assays were performed with U87.CD4.CCR5 and U87.CD4.CXCR4 cells, whereas infection assays were performed with Magi.CD4 or Magi.CD4.CCR5 cells (which also express CXCR4). In each case, the virus-dependent fusion and infection assays gave comparable results, demonstrating the utility of this assay to screen a wide variety of laboratory and primary isolates.

Table 1. Panel of Isolates Tested with Virus-Dependent Fusion Assay and Magi Assay.

Virus or Panel Coreceptor Usage Novel Properties
Primary Isolates
BaL, SF162 R5
LAI, 8 primary isolates X4 Isolates from diverse clades from NIH AIDS Repository
89.6, MN R5 X4
Sequential Isolates R5→ X4 27 longitudinal isolates from 6 HIV-infected MACS subjects
Molecular Clones
ADA, BaL, SF162, YU2 (V3 mutant HXB2 chimeras) Variable R5 Env chimeras and mutants that express V3 loop or minimal sequence determinants for CCR5 usage in HXB2 backbone
HXB2 X4
HIV-2 (ES, ROD10) R5 X4
SIV (mne, pbj) R5 X4
Luciferase Reporters
ADA, SF162, YU2 chimeras R5 Multiple mutants with minimal sequence determinants for R5 use
HXB2 X4
SF2, 89.6 R5 X4
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Fig 3. Virus dependent fusion assay and infection results with HIV-2 and SIVmne.

Fig 3

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Different amounts of virus produced from infectious molecular clones by transfection of 293T cells, were used in fusion assays with U87.CD4.CCR5 cells or infection assays with Magi.CD4.CCR5 cells.

Use of virus-dependent fusion assay for studies of HIV entry inhibitors

The virus-dependent fusion and infection assays were compared in studies of entry inhibitors that specifically block SU-mediated CD4 binding (Sim2) or CCR5 binding (2D7), or TM-mediated fusion (T20, Fig 4). U87.CD4.CCR5 cells were pre-incubated with antibody or drug, at the indicated concentrations for 12 hrs. For fusion assays, the cells were also infected with either vaccinia viruses expressing β-galactosidase or T7 polymerase, as described above. The cells were then exposed to 100 ng of HIV-luc virus (YU2-derived), for either 3 hrs for the fusion assay or 24 hrs for the infection assay (Pontow and Ratner, 2001). Fusion activity was determined by β-galactosidase activity, as described above. Infectious virus was quantified by luciferase activity. In each case, a similar concentration-dependence was seen for inhibition, although the infection assay was slightly less sensitive than the fusion assay at high concentrations of the anti-CCR5 antibody (2D7, Fig 4a). Similar assays were carried out with two different preparations of T-20, a TM-fusion inhibitor, and inhibitory concentrations identified in the fusion assay were similar to those reported previously using infection assays (Fig 4b) (Derdeyn et al., 2001).

Fig 4. Inhibition of virus-dependent fusion with antibodies or peptide inhibitors.

Fig 4

Fig 4

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a) Antibody neutralization studies were performed with anti-CCR5 antibody 2D7 or anti-CD4 antibody Sim2. Virus-dependent fusion assays (top) are compared to infection assays performed for 24 hrs with U87.CD4.CCR5 cells (bottom). b) Dose-dependent inhibition of virus-dependent fusion with T-20 was examined. Two different sources of T-20 were obtained from the NIH AIDS Research and Reference Repository, derived originally from DAIDS (with free N and C-terminal amino acids) and Trimeris (N-acetylated derivative). RLU, relative light units.

Identification of a Rac GEF inhibitor that blocks HIV entry

The virus-dependent fusion assay was utilized to identify novel HIV entry inhibitors. Based on previous observations of a role of Rac activation during HIV fusion, we examined an inhibitor of the Rac 1 GEFs, Trio and Tiam, NSC23766 (Gao et al., 2004). This drug blocked HIV fusion, as measured by the Env-dependent fusion assay (Fig 5a,b), infection (Fig 5c), or virus-dependent fusion (Fig 5d). The concentration range of HIV inhibitory activity was similar to that required to block Rac-1 activation (not shown).

Fig 5. Dose dependent inhibition of R5-Env dependent fusion or infection of U87.CD4.CCR5 cells using a Rac GEF inhibitor.

Fig 5

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a) Rac GEF inhibitor was added at the time of cell mixing or b) target U87 and/or HIV-1 ADA Env-expressing BSC40 cells were pretreated for 1 hr, washed, and mixed prior to measuring fusion activity by β-galactosidase activity. c) Cells were infected with 10 ng p24 of HIVluc derived from HIV-1 ADA, with drug added at the time of virus addition. Luciferase activity was determined 16 hr post-infection, expressed as relative light units (RLU). d) Rac GEF inhibitor was added at the time of virus in a virus-dependent fusion assay. Experiments were repeated 3 times, and representative data shown.

In order to examine the specificity of the effects of the Rac GEF inhibitor, a constitutively active Rac mutant, RacV12, was used to overcome the inhibitor effects by activating the signaling pathway downstream of the Rac GEFs. RacV12 overcame the effects on Rac activation as measured by the amount of Rac-GTP in cell lysates resulting from HIV Env-mediated fusion (Fig 6a). No effects of RacV12 were seen on Rho activation resulting from HIV Env mediated fusion (not shown). Moreover, RacV12 overcame the effects on Env-mediated fusion of the Rac GEF inhibitor (Fig 6b).

Fig 6. Constitutively active RacV12 overcomes the effects of the Rac GEF inhibitor.

Fig 6

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a) Effects of RacV12 on the ability of the Rac GEF inhibitor to block the ability of HIV ADA mediated fusion to activate Rac activation, as measured by the level of Rac-GTP in the cells. b) Effects of RacV12 on the ability of the Rac GEF inhibitor to block HIV-1 ADA Env-dependent fusion.

Conclusions

The virus-dependent fusion assay provides a rapid, sensitive, flexible method to examine the biological activity of laboratory or natural isolates of HIV-1, -2, or SIV, and may have applications to many other viruses that mediate pH-independent fusion. It compares favorably with other assays of virus, such as one that depends upon transfer of a virus core containing a Vpr-β-lactamase fusion protein into a target cell, which requires a longer incubation time and FACS analyses (Cavrois et al., 2004; Cavrois, Noronha, and Greene, 2002). It is likely that the virus-dependent fusion assay can also examine pseudotyped HIV particles, to identify inhibitors of other viral glycoproteins, as shown with the β-lactamase assay (Yonezawa, Cavrois, and Greene, 2005). This assay has advantages over various env-dependent fusion assays, since it utilizes relevant levels of virus-associated glycoprotein and does not require multiple receptor contacts for initiating the activity (Saeed, Kolokoltsov, and Davey, 2006).

The rapidity of the virus-dependent assay is ideal for screening panels of potential HIV entry inhibitors, and is readily adaptable for high throughput screens. The assay is effective at identifying inhibitory small molecules and antibodies. It may have applications for studies of neutralizing antibodies directed against HIV-1. Lastly, differences in results of virus-dependent fusion and infection assays may provide new insights into biological differences between different virus isolates.

Rac activation is critical in other aspects of HIV biology besides entry. HIV-1 Nef binds the DOCK-ELMO complex and p21-activated kinase 2 complex to initiate Rac activation, inhibit lymphocyte chemotaxis, and induce merlin phosphorylation (Janardhan et al., 2004; Lakhe-Reddy et al., 2006). Rac activation is also important for other viruses. Rho and cdc42 mediate adenovirus endocytosis (Li et al., 1998). Early steps in herpes simplex virus type 1 infection are dependent upon regulated Rac and Cdc42 signalling (Hoppe et al., 2006). In contrast, Rho activation facilities Kaposi's sarcoma herpesvirus entry (Veettil et al., 2006).

The activity of the inhibitor of Rac GEF, Trio, further substantiates the role of Rac activation in the fusion process. Additional studies of downstream mediators of Rac activity in HIV fusion have shown no effects of chemical inhibitors of NADPH oxidase, MEK, PI-3 kinase, or myosin light chain kinase, or dominant-negative inhibitors of Pak-2 or PI-3 kinase (our unpublished findings). It is quite possible that Rac activation through the WAVE, Arp 2/3 complex is important for HIV fusion. Further studies of the entire signaling pathway involved in HIV-fusion should be informative in developing new strategies to block infection.

Materials and methods

Inhibitors

The CCR5 and CD4 monoclonal antibodies 2D7 and Sim.2, and T-20 drugs were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (Derdeyn et al., 2001; Pravecz and Norcross, 1993; Wu et al., 1997).

Viruses

Recombinant vaccinia viruses expressing β-galactosidase (vCB21R), T7 polymerase (vPT7-3), and CD4 (vCB-3) were obtained from the AIDS Research and Reference Reagent Program, vaccinia viruses encoding an uncleaved HIV envelope (UNC; vCB-16), ADA envelope (vCB-39), and HXB2 envelope (vSC60) as well as wild type vaccinia virus (vWT) were gifts from Dr E Berger, vaccinia viruses encoding the YU2 envelope (vSP-5) and constitutively active Rac GTPase (vRacV12) were gifts from Drs C Broder and S Wei, respectively. HIV virus stocks were prepared by lipofection of plasmid DNA encoding full-length proviral molecular clones, with the Env gene of the R5 YU2, ADA, or SF162 strains or the X4 HXB2 strain in the HIV NL4-3 backbone (Pontow and Ratner, 2001; Pontow et al., 2004), or with a mutation in the Gag gene at residue 12 (L12E; our unpublished data). HIV Luc viruses encode the firefly luciferase gene in place of Nef. Transfected 293T cell supernatants were harvested after 48 hrs, filtered, and tittered for p24 antigen content by ELISA. Twenty-seven low-passage isolates obtained at serial visits from participants in the Baltimore site of the Multicenter AIDS Cohort Study (MACS) are also included (Kaslow et al., 1987).

Fusion and infection assays

The HIV-1 envelope-mediated fusion assay was modified from that developed by Dr Berger (Nussbaum, Broder, and Berger, 1994). The U87 cell lines were serum-starved for 36 hrs, then infected overnight at 37°C with vCB21R at MOI=10, and in some cases recombinant vaccinia virus encoding the constitutively active GTPase RacV12 mutant (vRacV12). Fusion partner BSC40 cells were infected overnight with vPT7-3 and vCB-39 encoding the ADA envelope. Alternatively, in some experiments 106 BSC40 cells were transfected with 5-10 ug of proviral plasmids and infected after 1 hr with vPT7-3. Cells were then lightly trypsinized, washed, and incubated with or without the Rac GEF inhibitor for 1 hr at 37°C. Cells (105) of each type are then mixed 1:1 in triplicate wells, incubated for 3 hrs at 37°C, and fusion stopped by the addition of NP-40 to a final concentration of 1% and freeze thawing at -20°C. β galactosidase activity was determined using chlorophenol red-β-d-galactopyoranoside (CPRG, Calbiochem) and the absorbance of each sample determined at 579 nm (Pontow et al., 2004).

The virus-dependent fusion assay was performed with free virus particles obtained 48 hrs after transfection of 293T cells (Pontow et al., 2004). Virus was incubated in the presence of 20 ug/ml DEAE-dextran with 105 U87.CD4.CCR5 cells infected for 18 hrs with vPT7-3 and 105 U87.CD4.CCR5 cells infected for 18 hrs with vCB21R. Assays were performed in triplicate wells. Fusion activity was quantified as described above.

Infection assays were performed with Magi.CD4 or Magi.CD4.CCR5 cells and number of blue foci determined days post-infection (Pirounaki et al., 2000). Alternatively, for some assays, viruses with luciferase expressed in place of Nef were utilized, and luciferase activity measured 1-2 days after infection.

Rac and Rho Activation Assays

These assays were performed with 2×106 serum starved U87.CD4.CCR5 cell mixed at a ratio of 1:1 with BSC40 cells infected with vaccinia virus expressing Env or vWT. In some cases, one population of U87.CD4.CCR5 cells was infected with vRacV12. Rac GEF inhibitor was added at the indicated concentrations to the target cells 1 hr prior to mixing and again at the time of mixing. Reactions were incubated at 37°C for 30 min, washed two times with ice cold PBS, and cells lysed. Lysates were snap frozen and later equal amounts of protein per well were analyzed using a G-LISA Rac activation or Rho-A activation assay kit according to the manufacturer's instructions (Cytoskeleton, Denver, CO).

Acknowledgments

We thank Drs. Joseph Margolick and Homayoon Farzadegan for providing primary isolates from men participating in the Baltimore site of the Multicenter AIDS Cohort Study” and acknowledge grants UO1-AI-35042 and 5-MO1-RR-00722 (GCRC). This work was supported by PHS grant AI24745.

Footnotes

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