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Vol. 15, Issue 12, 5197-5207, December 2004

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Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

Laboratory of Virology and Pathogenesis, AIDS Research Center, National Institute of Infectious Diseases, Tokyo 208-0011, Japan

Submitted April 2, 2004; Revised September 6, 2004; Accepted September 13, 2004
Monitoring Editor: Thomas Pollard

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Characterizing cellular factors involved in the life cycle of human immunodeficiency virus type 1 (HIV-1) is an initial step toward controlling replication of HIV-1. Actin polymerization mediated by the Arp2/3 complex has been found to play a critical role in some pathogens' intracellular motility. We have asked whether this complex also contributes to the viral life cycles including that of HIV-1. We have used both the acidic domains from actin-related protein (Arp) 2/3 complex-binding proteins such as the Wiscott-Aldrich syndrome protein (N-WASP) or cortactin, and siRNA directing toward Arp2 to inhibit viral infection. HIV-1, simian immunodeficiency virus (SIV), and intracellular mature vaccinia virus (IMV) were sensitive to inhibition of the Arp2/3 complex, whereas MLV, HSV-1, and adenovirus were not. Interestingly, pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) overcame this inhibition. Constitutive inhibition of the Arp2/3 complex in the T-cell line H9 also blocked replication of HIV-1. These data suggested the existence of an Arp2/3 complex-dependent event during the early phase of the life cycles of both primate lentiviruses and IMV. Inhibiting the HIV-1's ability to activate Arp2/3 complex could be a potential chemotherapeutic intervention for acquired immunodeficiency syndrome (AIDS).

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
When human immunodeficiency virus type 1 (HIV-1) enters cells, the envelope glycoprotein gp120 binds to CD4 and subsequently CXCR4 or CCR5 and initiates membrane fusion at the cell surface. After the membrane fusion the reverse transcription takes place while the viral core components migrate toward cell nucleus where the proviral DNA integrates into the host cell chromosome. However, the protein-protein interactions during these processes of disassembly/uncoating are the least understood among the whole viral life cycle. Despite historical suggestions that actin plays a role in the early phase of HIV-1 infection, its role remains largely unclear. Early studies used chemical inhibitors of actin, which were broadly active on cell physiology or "nonspecific" (Cudmore et al., 1997; Bukrinskaya et al., 1998; Iyengar et al., 1998). To test for a specific role of actin in the early phase of HIV-1's life cycle, we focused on regulators of actin polymerization. It has now been shown that some bacteria and viruses use cellular actin polymerization to propel themselves within cells (Gruenheid and Finlay, 2003). The key host proteins in these reactions are actin-related protein (Arp) 2/3 complex and its regulators. We hypothesized that Arp2/3 complex-dependent actin nucleation might be required for efficient infection by primate lentiviruses including HIV-1.

The Arp2/3 complex is a seven-subunit protein complex highly conserved among eukaryotes that nucleates actin filaments from the sides of existing filaments (Higgs and Pollard, 2001; Pantaloni et al., 2001). The Arp2/3 complex distributes throughout the cell but is enriched especially at the cortical layer underneath the plasma membrane through which viruses have to pass to infect cells (Flanagan et al., 2001). The Arp2/3 complex is regulated by both Wiscott-Aldrich syndrome protein (WASP) family of proteins and cortactin (Weaver et al., 2003). The carboxy terminal domain of WASP is called VCA domain (verprolin homology, cofilin homology and acidic subdomains) and is also named the WA domain. Intensive studies had revealed that VCA's ability to bind monomer actin through its V subdomain is critical for actin nucleation (Miki and Takenawa, 1998). The CA subdomain confers to N-WASP its binding ability to the Arp2/3 complex as evidenced by physicochemical assays (Machesky and Insall, 1998; Marchand et al., 2001) and x-ray crystallography and cross-linking experiments (Gournier et al., 2001; Robinson et al., 2001; Zalevsky et al., 2001). Actin polymerization, nucleation, and branching are enhanced in the presence of VCA protein in vitro (Higgs et al., 1999; Machesky et al., 1999; Rohatgi et al., 1999). Expression of the VCA protein sequesters the Arp2/3 complex and displaces it from physiological regulation in vivo (Machesky and Insall, 1998; Machesky et al., 1999; Rozelle et al., 2000; Castellano et al., 2001; Harlander et al., 2003). By expressing in tissue culture cells, the VCA protein has been used successfully as an inhibitor of Arp2/3 complex to study the role of Arp2/3 complex in many biologic processes (Zhang et al., 1999; Krause et al., 2000; May et al., 2000; Moreau et al., 2000; Rozelle et al., 2000; McGee et al., 2001; Zhang et al., 2002).

Another Arp2/3 complex regulator is cortactin, a filamentous actin-associated protein originally identified as a substrate of Src (Weed and Parsons, 2001) that is also implicated in the phagocytosis of several invasive bacteria (Dehio et al., 1995; Fawaz et al., 1997; Cantarelli et al., 2000). Cortactin binds directly to the Arp2/3 complex through its aminoterminal acidic domain, NTA, and activates it (Weed et al., 2000; Uruno et al., 2001; Weaver et al., 2001). The NTA protein, like VCA, can serve as an inhibitor of Arp2/3 complex.

We explored the possible involvement of Arp2/3 complex in the early phase of life cycle of primate lentiviruses. In parallel, we tested different virus species including adenovirus, herpes simplex virus type 1 (HSV-1), Moloney murine leukemia virus (MLV), and intracellular mature vaccinia virus (IMV), all of which were reported to use the actin cytoskeleton to infect cells; however, the physical properties and mechanisms of their entry vary (Rosenthal et al., 1985; Kizhatil and Albritton, 1997; Bukrinskaya et al., 1998; Iyengar et al., 1998; Li et al., 1998). We also tested whether changing retroviral envelopes, which forces viruses to enter through different routes, affected the efficiencies of viral entry.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cells and Viruses
Human embryonic kidney (HEK) 293 cells and Chinese hamster ovary (CHO)-K1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma, St. Louis, MO) supplemented with 10% FBS (Hyclone, Logan, UT), penicillin, and streptomycin (Invitrogen, Carlsbad, CA). H9 cells were maintained in RPMI1640 (Sigma) supplemented with 10% FBS, penicillin, and streptomycin. All the mammalian cell lines were incubated at 37°C in the humidified 5% CO₂ atmosphere. Replication incompetent HIV-1 (HXB2 vpr, rev, env, nef) was produced by transfecting the proviral DNA carrying renilla luciferase in place of nef open reading frame into 293 T-cells along with the expression plasmid for env, tat, rev and nef (pIIIex). When pseudotyping HIV-1, rev expressing plasmid and either ecotropic MLV envelope (Ragheb and Anderson, 1994) or vesicular stomatitis virus G (VSV-G) expressing plasmid (Clontech, Palo Alto, CA) were cotransfected with the proviral DNA of HIV-1. HXB2 was used for the replication competent HIV-1. The virus was prepared by transfecting the proviral DNA into 293 T-cells, and the supernatants were collected at 2 d posttransfection. SIV (nef) encoding firefly luciferase in place of nef open reading frame was created based on the IL-2–carrying molecular clone of SIVmac239 (Gundlach et al., 1997; kindly provided by Dr. K. Mori). SIV was prepared by transfecting the proviral DNA into COS7 cells, and the supernatants were collected. MLV was produced by transfecting pCMMP LacZIRESGFP, pCMMP eGFP (generous gift from Dr. J. Young), pCMMP GFP-VCA, or pQcLIN (Clontech) into 293 cells along with gag/pol and either ecotropic, amphotropic env, or VSV-G–expressing plasmids (Ragheb and Anderson, 1994). HSV-1 (KOS)Rid1/tk12 encoding beta-galactosidase under the regulation of the immediate early promoter ICP4, vaccinia virus encoding T7 RNA polymerase (vTF7.3), and adenovirus type 5 expressing T7 RNA polymerase were generous gifts from Drs. Spear (Dean et al., 1994), Moss (Fuerst et al., 1986), and Ishii (Aoki et al., 1998), respectively, and were prepared according to the previous publications. Adenovirus, HSV-1 and IMV were titrated by the plaque assay using HEK293 or Hela cells; MLV by counting reporter-positive HEK293 cells after 2 to 3 d postinfection, and HIV-1, VSV-G HIV-1, and SIV by using the indicator cells that expressed beta-galactosidase or GFP upon infection. The p24 concentration of eco HIV-1 was adjusted to that of HIV-1 (100 ng/ml). The neutralization assay using the antibody 2D5 that inhibits infection of IMV but not that of EEV (Ichihashi and Oie, 1996) revealed that 99.4% of our vaccinia virus preparation contained intracellular mature virus (IMV).

Plasmids
The VCA domain (amino acid 392–505) of N-WASP was amplified by PCR from N-WASP cDNA kindly provided by Dr. Takenawa (Miki et al., 1996) with the following primers: VCA sense 5'-CAATTGCCTTCTGATGGGGACCATCAGG-3' and 5'-AAGCTTCAGTCTTCCCACTCATCATC-3'. The PCR product was subcloned into pCR4 Blunt TOPO (Invitrogen), sequenced, digested with MfeI and EcoRI, and cloned into EcoRI site of pEGFP-C2 (Clontech), generating pGFP-VCA. Following two primers, 5'-AGATCTTAGTGGCTGATGGCCAAGAGTCCACACC-3' and 5'CAATTGTCAGTCTTCCCACTCATCATC-3', were used to amplify the CA domain (amino acid 470–505) of N-WASP. The PCR fragment was cloned into BglII-EcoRI sites of pEGFP-C2, giving rise to pGFP-CA. GFP-A expression plasmid was generated by annealing following two oligonucleotides and cloned it into the BglII-EcoRI sites of pEGFP-C2: 5'-GATCGATGAAGATGAAGATGAAGATGATGAAGAAGATTTTGAGGATGATGATGAGTGGGAAGACTGA-3' and 5'-AATTTCAGTCTTCCCACTCATCATCATCCTCAAAATCTTCTTCATCATCTTCATCTTCATCTTCATC-3'. Following oligonucleotides were annealed and ligated into the BglII site of pGFP-VCA giving rise to pGFP-VCA*: 5'-GATCTAGATAACTGATGCGGCCGCCG-3' and 5'-GATCCGGCGGCCGCATCAGTTATCTA-3'. The NTA domain (amino acid 1–84) of cortactin and Arp2 were amplified by PCR from 293T cDNA with following primers: NTA sense 5'-GGATCCTCGAGATGTGGAAAGCTTCAGCAGGCCAC-3', NTA antisense 5'-CAATTGTCAATAGCCATGGGAAGCTTTTGGTCC-3'; Arp2 sense 5'-GGATCCTCGAGATGGACAGCCAGGGCAGGAAGG-3', Arp2 antisense 5'-CAATTGTTATCGAACAGTCACACCAAGTTTC-3'. PCR fragments were cloned into pCR4 Blunt TOPO, and the BamHI-MfeI fragments were cloned into BglII-EcoRI sites of pEGFP-C2, generating pGFP-NTA and pGFP-Arp2. pCMMP GFP-VCA was generated by digesting pCMMP GFP with Age I and BamHI, ligated with Age I-BamHI fragment from pGFPVCA. Transfection efficiencies were measured by phRL/CMV (Promega, Madison, WI) or pHIV-1 LTR-GFP-Luciferase. F10, the ecotropic MLV receptor from rat, expression plasmid was created by digesting pcDNA F10-ecoR (Takase-Yoden and Watanabe, 1999) with NaeI followed by the self-ligation. The human nectin 1 alpha (HigR) expression plasmid was generated by inserting EcoRI fragment of pEF-BOS human nectin 1 alpha 3xFLAG (Sakisaka et al., 2001) into EcoRI site of pcDNA3 (Invitrogen). The XhoI-NotI fragment of either pBCMGSneoCD4 or pBCMGSneoCCR5 (a generous gift from Dr. Yamashita) was cloned into XhoI-NotI sites of pcDNA3 (Invitrogen), generating pCD4 and pCCR5, respectively. The SnaBI-NotI fragment from pCCR5 was cloned into pMACS4ires (Miltenyi Biotec, Bergisch Gladbach, Germany), generating pCCR5 IRES CD4. The CD4 lacking the cytoplasmic tail was amplified by the following primers: 5'-GGATCCCGGGCCACCATGAACCGGGGAGTCCCTTTTAGGC-3'and 5'GAATTCGTGCCGGCACCTGACACAGAAGAAGATGCC-3'. The XmaI-EcoRI fragment was cloned into the AgeI-EcoRI sites of pEGFP-C2, generating pCD4cyt. The T7 RNA polymerase (T7 RNAP) expression plasmid pCMMP T7RNAP IRES GFP was created by inserting XmaIEcoRI fragment from pVR1-T7 into AgeI-MfeI sites of pCMMP IRES GFP (Aoki et al., 1998). The T7RNAP reporter plasmid pTM3Luci was described previously (Aoki et al., 1998). The following pairs of oligonucleotides were annealed and cloned into ApaI-EcoRI sites of pSilencer 1.0-U6 (Ambion, Austin, TX) to generate siRNA expressing vectors directing against GFP and Arp2: GFP sense 5'-GCTGACCCTGAAGTTCATCTTCAAGAGAGATGAACTTCAGGGTCAGCTTTTTT-3' and GFP antisense 5'-AATTAAAAAAGCTGACCCTGAAGTTCATCTCTCTTGAAGATGAACTTCAGGGTCAGCGGCC-3'; Arp2 sense 5'-CAGCTTTACTTAGAACGAGTTCAAGAGACTCGTTCTAAGTAAAGCTGTTTTTT-3' and Arp2 antisense 5'-AATTAAAAAACAGCTTTACTTAGAACGAGTCTCTTGAACTCGTTCTAAGTAAAGCTGGGCC-3'.

Transfection, Magnetic Selection, and Infection
Plasmid DNAs were transfected into cells by using either lipofectamin/lipofectamin plus reagent (Invitrogen) or X-tremeGENE siRNA transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. The latter reagent, with which the transfection efficiencies reached >90% in 293 cells, was specifically used for preparing samples to carry out Western blot analysis demonstrating the reduction of Arp2 levels. In brief, cells were fed in 48-well plates 1–2 d before transfection. Cells were approximately in 50–60% confluency at transfection. After transfection, cells were trypsinized and plated onto 96-well plates. Transfected cells were magnetically selected by using MACS system directing toward CD4 (Miltenyi Biotec). Cells were infected with viruses of 0.1–0.5 multiplicity of infection by incubating in the virus-containing culture medium at 37°C for 1–4 h.

Cell-to-cell Fusion Assay
The fusion assay was based on the T7RNAP transcription-dependent reporter assay originally described by Nussbaum et al. (1994), modified versions by Sakamoto et al. (2003). In brief, the T7RNAP "donor" cells were generated by transfecting 293 cells with both T7RNAP and HIV-1's Env expression vectors. The T7RNAP "acceptor" cells were generated by transfecting 293 cells with the T7RNAP reporter (pT7-IRES-Luciferase) along with either CD4 or CD4cyt, GFP or GFP-VCA, and the renilla luciferase expression vectors. At 48 h posttransfection, these cells were cocultivated for 24 h and lysed in the passive lysis buffer to carry out the dual luciferase assay (Promega).

Detection of Fluorescent Signals
The transfected 293 cells were fixed by 4% formaldehyde in PBS at 48 h posttransfection and imaged by using confocal microscopy META 510 (Carl Zeiss, Jene, Germany). For the images showing pseudopodial extensions, images of different focal planes were projected to generate a single image. The transfected H9 cells were imaged similarly at 48 h posttransfection without fixation. The light transmission image was merged with the green fluorescent signal. Alternatively, the transfected cells were analyzed by the flow cytometry (FACS calibur, Becton Dickinson, San Jose, CA).

Western Blot Analysis
Cells were washed with PBS and lysed in a buffer containing 4% SDS, 100 mM Tris-HCl (pH 6.8), 12% 2-ME, 20% glycerol, and bromophenol blue. Samples were boiled for 10 min. Protein lysates approximately equivalent to 5 x 10⁴ cells were separated in SDS-PAGE (Perfect NT Gel, DRC, Tokyo, Japan), transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P^SQ, Millipore, Bedford, MA), and blocked with 5% dried nonfat milk (Yuki-Jirushi, Tokyo, Japan) in PBS. For the primary antibody, we used anti-Arp2 antibody H-84 (Santa Cruz Biotechnology, Santa Cruz, CA), antiactin antibody MAB1501 (Chemicon, Temecula, CA), or either an mAb or a polyclonal rabbit antiserum against GFP (Clontech). For the secondary antibody, either a biotinylated anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ) or a biotinylated anti-rabbit antibody was used. For the tertiary probe, a horseradish peroxidase (HRP)-conjugated streptoavidin (Amersham Pharmacia Biotech) was used. Signals were developed by incubating blots with the chemilumenescent HRP substrate (Amersham Pharmacia Biotech) and detected by using LumiImager F1 (Boehringer Mannheim, Mannheim, Germany).

Reporter Assays
Cells were lysed in the Passive Lysis Buffer (Promega) and the dual luciferase assay was performed to measure both firefly and renilla luciferase activities according to the manufacturer's protocol (Promega). The beta-galactosidase activity was measured by using the LumiGal assay kit according to the manufacturer's protocol (Clontech). The chemiluminescence was detected by Lmax (Nihon Molecular Devices, Tokyo, Japan).

ELISA
The amount of p24 antigen of HIV-1 in the culture supernatants was quantified by using Retro TEK p24 antigen ELISA kit according to the manufacturer's protocol (Zepto Metrix, Buffalo, NY). The signals were measured by Vmax ELISA reader (Nihon Molecular Devices).

Statistical Analysis
The significance of differences was tested by one-way analysis of variance (ANOVA) and Student's t test. P values < 0.05 were considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
GFP-VCA Inhibits Infection of Primate Lentiviruses
We first tested whether the viral infection was affected by expressing a potential inhibitor of the Arp2/3 complex GFPVCA, the VCA domain of N-WASP fused to GFP (Figure 1A). The expression of GFP-VCA protein was verified by Western blot analysis (Figure 1A). The expression levels of GFP-VCA were similar to those of GFP as measured by the flow cytometric analysis at 48 h posttransfection (Figure 1B). GFP-VCA preferentially distributed to the cytoplasm and inhibited the pseudopodial extension compared with GFP alone in agreement with the previous report (Rozelle et al., 2000; Figure 1C). To exclude the possibility that GFP fusion proteins negatively affects the cell surface expression of membrane proteins, we confirmed that the distribution and the levels of transiently and constitutively expressed CD4 and CXCR4 on the cell surface were similar in both GFPand GFP-VCA–expressing cells as assessed by both confocal microscopy and the flow cytometric analysis (unpublished data).

View larger version (27K): [in this window] [in a new window]   Figure 1. Inhibiting the Arp2/3 complex limited the entry of primate lentiviruses. (A) The carboxy-terminus of N-WASP, VCA domain, was fused to GFP to give rise GFP-VCA. Western blot analysis detected the 41-kDa band, the predicted mol wt for GFP-VCA. (B) The green fluorescence profiles of 293 cells transfected with either GFP or GFP-VCA expression vector were similar to each other as measured by the flow cytometric analysis. (C) GFP-VCA preferentially distributed evenly throughout the cytoplasm. The pseudopodial extension was less active when GFP-VCA was expressed in 293 cells compared with GFP (magnification, x200; inset, x400). (D) The experimental procedure was drawn schematically. (E) The relative infection efficiencies of HIV-1, HIV-1 pseudotyped with ecotropic MLV envelope (eco HIV-1), and SIV into cells expressing GFP-VCA were significantly decreased, whereas those of ecotropic MLV (eco MLV) and HSV-1 were not (asterisk, p < 0.01). The data represent the average and SD of indicated number of independent experiments.

To evaluate the contribution of the Arp2/3 complex on the viral infection, we used a transient transfection/infection system in which target cells were transfected with a mixture of plasmid DNA expressing 1) an Arp2/3 complex inhibitor GFP-VCA, 2) a viral receptor, and 3) a reporter gene to normalize transfection efficiencies (depicted in Figure 1D). Because of the nature of cotransfection, the majority of cells uptook the expression vector for GFP-VCA were transfected with the other plasmids because the mixture of DNA contained fivefold excess amount of the GFP-VCA expressing plasmid. At 48 h posttransfection, a portion of transfected cells were collected to measure the transfection efficiencies and others were challenged by a virus expressing a reporter gene to monitor the efficiency of infection. Various times after infection, cells were lysed and the virally encoded reporter gene activities were measured, which was divided by the transfection efficiencies to normalize. Infection of viruses was restricted to the transfected cells by using viral receptors: human CD4 for HIV-1, human CD4 and CCR5 for SIV, F10 for ecotropic MLV envelope-pseudotyped HIV-1 (eco HIV-1) and ecotropic MLV (eco MLV), human nectin-1 alpha (HigR) for HSV-1. Viruses, receptors, and the combination of reporter genes for each virus were summarized in Table 1. Primarily we targeted HEK293 cells because actin cytoskeletal system is shared among eukaryotes. To monitor HSV-1 infection, CHO-K1 cells were used that lack entry molecules for HSV-1. To monitor the efficiencies of singleround viral infection, we used replication-incompetent HIV-1 and MLV. Alternatively, cells infected with replication-competent viruses were lysed at time points before viruses entered the second replication cycle, except SIV, which required 48 h to give sufficient signal to be detected. Importantly, all the viruses tested in this study were reported to utilize actin cytoskeleton to infect cells as examined by using chemical inhibitors against actin such as cytochalasin (CC; Rosenthal et al., 1985; Kizhatil and Albritton, 1997; Bukrinskaya et al., 1998; Iyengar et al., 1998; Li et al., 1998). The infection efficiencies of viruses into cells expressing GFP were set at 100% throughout the study unless stated and the infection efficiencies into GFP-VCA–expressing cells relative to GFP-expressing cells were calculated. For example, when eco HIV-1 was tested, the firefly luciferase activities representing the transfection efficiencies for GFPand GFP-VCA–transfected cells were 2,065 and 1,854 relative light unit (RLU), respectively, where the background signal was 3 RLU. The renilla luciferease activities reflecting the infection efficiencies into GFPand GFP-VCA–transfected cells were 4,018 and 1,254 RLU, respectively, where the background was 15 RLU. In this case, the relative infection efficiency of eco HIV-1 into GFP-VCA expressing cells was 34.4%. The relative infection efficiency was introduced because we were able to integrate data from independent experiments. Results from the indicated number of independent experiments were summarized in Figure 1E. Expression of the GFP-VCA significantly reduced the relative infection efficiencies of HIV-1, eco HIV-1, and SIV (30.3, 35.8, and 22.7%, respectively; p < 0.01; Figure 1E), whereas those of eco MLV and HSV-1 (98.5 and 103.5%, respectively; Figure 1E) did not. A 10-fold higher or lower titer of HSV-1 did not affect the results (unpublished data). Consistent with this, the average signal-to-noise ratio did not correlate with the efficiency of inhibition (Figure 1E and Table 1). The GFP-VCA did not inhibit eco MLV but did inhibit the infection of eco HIV-1, suggesting that GFP-VCA's ability to inhibit the infection of eco HIV-1 was eco env-independent. It also suggested that the cell surface expression of the F10 was not affected by GFP-VCA expression. In addition, our SIV clone did not encode nef and HIV-1 vpr, demonstrating that both nef and vpr were not necessary for primate lentiviruses (HIV-1 and SIV) to infect GFP-VCA–expressing cells efficiently. These results suggested that inhibiting the Arp2/3 complex by GFP-VCA negatively affected the infection of primate lentiviruses.

View larger version (28K): [in this window] [in a new window]   Figure 4. Inhibitory effects of GFP-VCA derivatives, GFP-NTA, and siRNA against Arp2 on the infection of both HIV-1 and IMV. (A) Schematic drawing of GFP-VCA derivatives and GFP-NTA. (B) Western blot analysis detected 30-, 41-, 32-, 30-, 28-, and 37-kDa bands, each predicted mol wt for GFP, GFP-VCA, -CA, -A, VCA*, and -NTA, respectively. (C) The relative infection efficiencies of HIV-1 () and IMV () were significantly inhibited when cells expressed GFP-CA and GFP-NTA (asterisk, p <0.01; double asterisks, p <0.05). Expression of GFP-A significantly reduced the relative infection efficiency for HIV-1 but not for IMV. GFP-VCA* did not detectably inhibited infection of both viruses. The data represent the average and SD of more than three independent experiments. (D) The siRNA directed against Arp2 down-modulated expression of Arp2 by 73.5% on the average, whereas the expression of siRNA against GFP did not as demonstrated by Western blot analysis in which 293 cell lysates corresponding to the 5 x 104 or 2 x 105 were analyzed at 2 d posttransfection. (E) Introducing siRNA against Arp2 significantly reduced the relative infection efficiencies of HIV-1 and HIV-1 pseudotyped with ecotropic MLV envelope (eco HIV-1; asterisk, p < 0.01) but not ecotropic MLV (eco MLV), HSV-1, adenovirus, and IMV. The data represent the average and SD of indicated number of independent experiments.

View larger version (18K): [in this window] [in a new window]   Figure 2. GFP-VCA does not inhibit the infection efficiency of HIV-1 pseudotyped with VSV-G or the membrane fusion induced by the HIV-1's Env-CD4 interaction. (A) The experimental procedure using the magnetic selection was drawn schematically. (B) The relative infection efficiencies of HIV-1 and HIV-1 pseudotyped with ecotropic MLV envelope (eco HIV-1) were significantly decreased (asterisk, p < 0.01). In contrast, the VSV-G–pseudotyped HIV-1 (VSV-G HIV-1) entered GFP-VCA–expressing cells as efficient as GFP-expressing cells similar to amphotropic MLV (amhpo MLV) and VSV-G–pseudotyped MLV (VSV-G MLV). (C) The experimental procedure for the cell-to-cell fusion assay was drawn schematically. (D) GFP-VCA did not negatively affect the cell-to-cell fusion mediated by HIV-1's Env and either the full-length CD4 or the CD4 without the cytoplasmic tail (CD4cyt) compared with GFP. The data represent the average and SD of indicated number of independent experiments.

The Efficiency of the Membrane Fusion Is Not Negatively Affected by GFP-VCA
The membrane fusion is the critical event when enveloped viruses infect cells. We next asked if the expression of GFP-VCA inhibited the membrane fusion through HIV-1's Envreceptor interaction. We carried out the cell-to-cell fusion assay in which Env-positive cells expressing T7 RNA polymerase (T7RNAP) were fused to the CD4-positive cells carrying the T7RNAP promoter-driven firefly luciferase reporter plasmid (depicted in Figure 2C). The efficiency of the cell-to-cell fusion was measured by the firefly luciferase activity divided by the renilla luciferase activity representing the transfection efficiency. The ratio of the firefly luciferase to renilla luciferase activities in GFP-expressing cells was set at 100%, and the relative efficiencies of cell-to-cell fusion of GFP-VCA–expressing cells were calculated. It revealed that the efficiencies of cell-to-cell fusion initiated by the interaction between HIV-1's Env and CD4 were not inhibited when GFP-VCA was expressed in CD4-expressing cells compared with GFP (114%, Figure 2D).

To eliminate a possibility that the GFP-VCA inhibited the expression/distribution of CD4 on the cell surface, we examined whether the CD4 lacking the cytoplasmic domain (CD4cyt) was able to support the membrane fusion in the presence of GFP-VCA. Because GFP-VCA distributed throughout the cytoplasm, it was unlikely that the motility of CD4cyt on the cell surface was restricted by GFP-VCA. It was found that CD4cyt induced the cell-to-cell fusion in the presence of GFP-VCA at efficiencies almost equal to the full length CD4 (113%, Figure 2D). In support of this, enveloped viruses (HSV-1, MLV, and VSV-G HIV-1) were able to infect GFP-VCA–positive cells as efficient as GFP-positive cells (Figures 1E and 2B). In particular, when HSV-1 and ampho MLV enter cells, like HIV-1, the membrane fusion takes place at the cell surface (Fuller and Spear, 1987; McClure et al., 1990; Wittels and Spear, 1991; Nussbaum et al., 1993). These data indicated that the virus-cell membrane fusion was not inhibited by GFP-VCA. According to the data presented hereby, it seemed likely that GFP-VCA did not negatively affect the expression of receptors or viral attachment to receptors, or the virus-cell membrane fusion. Because VSV-G HIV-1–infected cells in the presence of GFPVCA, we assume that GFP-VCA inhibits HIV-1's life cycle after the membrane fusion before or at the reverse transcription, especially when HIV-1 enters cells through the membrane fusion at cell surface where the viral core is placed in the cortical compartment.

GFP-VCA Inhibits Infection of Intracellular Mature Vaccinia Virus but Not Adenovirus
To test whether the inhibition of viral entry by GFP-VCA was limited to the primate lentiviruses, we examined both intracellular mature vaccinia virus (IMV) and adenovirus. IMV enters cells via the membrane fusion at cell surface, which is accompanied by a drastic actin cytoskeletal reorganization (reviewed by Smith et al., 2002). Adenovirus infects cells through clathrin-dependent endocytosis (Wang et al., 1998). Adenovirus was the only envelope-free virus studied in this article. These viruses encoded T7 RNA polymerase as a reporter (Table 3). To detect the infection signal from transfected/infected cells, we introduced the T7 promoterdriven luciferase reporter into 293 cells (Figure 3A). For example, when IMV was tested, the renilla luciferase activities representing the transfection efficiencies for GFPand GFP-VCA–transfected cells were 485 and 556 RLU, respectively, where the background signal was 8 RLU. The firefly luciferease activities reflecting the infection efficiencies of virus into GFPand GFP-VCA–transfected cells were 29,020 and 10,044 RLU, respectively, where the background was 23 RLU. In this case, the relative infection efficiency of IMV into GFP-VCA–expressing cells was 30.3%. Results from a number of independent experiments were summarized in Figure 3B. The relative infection efficiency of IMV was significantly reduced (30.6%, p <0.01; Figure 3B), whereas adenovirusinfected GFP-VCA–expressing cells as efficiently as GFPexpressing cells (98.3%, Figure 3B). IMV gave the highest signal-to-noise ratio throughout the study, yet its infection was blocked efficiently by GFP-VCA (Table 3). Considering all the signal-to-noise data, it was suggested that the signalto-noise ratio did not positively correlate with the magnitude of inhibition of viral infection. Ten-fold higher or lower titer of either IMV or adenovirus did not affect the results (unpublished data). These data suggested that inhibiting the Arp2/3 complex by GFP-VCA negatively affected infection of IMV as well as primate lentiviruses. We focused on HIV-1 and IMV for the further studies.

View larger version (22K): [in this window] [in a new window]   Figure 3. Limiting infection of IMV but not adenovirus by inhibiting the Arp2/3 complex. (A) The experimental procedure was drawn schematically. (B) The significant decrease of relative infection efficiency of IMV, but not adenovirus, was observed (asterisk, p < 0.01). The data represent the average and SD of indicated number of independent experiments.

GFP-VCA's Ability to Nucleate Actin Filament Is Not Necessary to Inhibit Viral Entry
We attempted to locate the domain within VCA responsible for the inhibition of HIV-1 and IMV infection. We generated a series of truncated GFP-VCA mutants (Figure 4A). Their functions were also summarized according to the previous reports (Figure 4A; Takenawa and Miki, 2001; Weaver et al., 2003). Expression of each mutant was verified in Western blot analysis (Figure 4B). Introducing a stop codon into GFP-VCA after the GFP open reading frame allowed VCAencoding RNA to be expressed but not VCA protein (GFPVCA*). Expression of GFP-VCA* did not inhibit infection of both HIV-1 and IMV (82.9 and 112.6%, respectively; Figure 4C), suggesting that the inhibition of viral entry attributed to the VCA protein, not the VCA-encoding RNA (Figure 4C). GFP-CA retained the ability to limit both HIV-1 and IMV infection (46.7 and 46.4%, p < 0.01 and p < 0.05, respectively, Figure 4C). Expression of GFP-A inhibited HIV-1 infection less efficiently than other derivatives (57.7%, p < 0.05; Figure 4C). However, GFP-A was unable to limit IMV infection (110.8%, Figure 4C) although synthetic peptides of A subdomain has been shown to retain the binding affinity to the Arp2/3 complex as high as VCA in vitro (Panchal et al., 2003). These data suggested that the V subdomain was not required for the inhibition of HIV-1 and IMV infection. It seemed likely that C and A subdomains functioned cooperatively in vivo to inhibit infection of both HIV-1 and IMV.

To further verify that the reduction of viral infection efficiencies was due to the inhibition of Arp2/3 complex functions, we have tested whether another potential Arp2/3 complex inhibitor NTA, amino-terminal acidic domain of cortactin (amino acids 1–84), was also able to limit both HIV-1 and IMV infection as did VCA. Cortactin is unrelated to WASP family of proteins but is able to bind the Arp2/3 complex through the NTA domain. Expression of GFP-NTA was verified by Western blot analysis (Figure 4B). As expected, GFP-NTA reduced the infection efficiencies of both HIV-1 and IMV (39 and 50%, p < 0.01 and p < 0.05, respectively; Figure 4C). These data confirmed that inhibiting functions of the Arp2/3 complex was indeed responsible for limiting both HIV-1 and IMV infection. As GFP-NTA lacked the ability to nucleate actin filaments similar to GFP-CA and because of the reported functions of CA subdomains (Figure 4A), it was strongly suggested that, not their abilities to enhance actin nucleation, but GFP fusion proteins' abilities to bind the Arp2/3 complex was primarily important to inhibit viral infection.

In addition, we tested whether down-regulating expression of Arp2 by using siRNA technique inhibited both HIV-1 and IMV infection. The GFP-VCA and -NTA were able to inhibit functions of Arp2/3 complex by binding to it directly, whereas siRNA against Arp2 down-modulated expression of Arp2, therefore decreasing the number of Arp2/3 complex. Because siRNA against Arp2 was not able to inhibit the function of preexisting Arp2/3 complex directly, it was expected that siRNA against Arp2 should be able to limit viral infection, if any, less efficiently than GFPVCA. Transfecting the plasmid vector expressing siRNA directing against Arp2 reduced the expression of endogenously expressed Arp2–26.5% in 293 cells as demonstrated by Western blotting analysis (Figure 4D). Viral infection efficiencies were measured using the experimental setups (Figures 1D and 3A) except the GFP or GFP-VCA expression plasmid was replaced with the siRNA expression vectors against GFP or Arp2. When siRNA directing toward GFP was set as 100%, the relative efficiencies of both HIV-1 and eco HIV-1 infection in cells expressing siRNA against Arp2 became 39.4 and 39.1%, respectively (both, p < 0.01; Figure 4E). Adenovirus infection was slightly inhibited by the siRNA against Arp2 (69.4%, Figure 4E). However, the relative infection efficiencies of eco MLV, HSV-1, and IMV were not significantly reduced by siRNA directed toward Arp2 (91.3, 103.8, and 104.4%, respectively, Figure 4E). Given that the other two Arp2/3 inhibitors were able to limit IMV and HIV-1 infection, it seemed reasonable to speculate that the inhibition of HIV-1 infection by siRNA was due to the down-modulation of newly synthesized Arp2. However, we were unable to limit the infection of IMV by siRNA against Arp2. We assumed that this was either because siRNA was unable to down-regulate expression of Arp2, therefore Arp2/3 complex, at levels sufficient to block the entry of IMV or the number of the preexisting Arp2/3 complex might be sufficient to support IMV infection, or both.

HIV-1 Replication Is Negatively Affected When GFP-VCA Is Constitutively Expressed in H9 Cells
Finally, we asked whether the replication of HIV-1 was inhibited in T-cells, one of the natural targets of HIV-1. To do this, we isolated H9 cell clones either expressing GFP or GFP-VCA constitutively by infecting H9 cells with MLV vector followed by the limiting dilution. Introducing expression plasmid for GFP-VCA did not alter the morphology of H9 cells compared with GFP-expressing cells or untransfected cells when observed under the confocal microscopy at 48 h posttransfection (Figure 5A) as well as stable cell clones (unpublished data). H9 GFP-VCA clones proliferated at speed indistinguishable from H9 GFP clones (unpublished data). We verified the expression of GFP and GFP-VCA in isolated clones by Western blot analysis (Figure 5B). However, the average expression levels of GFP-VCA appeared low compared with those of GFP in H9 clones (Figure 5B). We infected six H9-GFP and three H9-GFP-VCA clones with a replication-competent HIV-1 (HXB2) and collected culture supernatants at different time points to monitor the viral replication by measuring the amount of p24 viral antigen. The replication kinetics of HIV-1 in GFP-expressing H9 clones showed a rapid propagation of HIV-1 in culture. In contrast, the amount of p24 in the culture supernatants of H9 GFP-VCA clones did not accumulate, suggesting that the HIV-1 replication was substantially suppressed in H9 GFPVCA clones (Figure 5C). The replication assay was carried out twice consecutively and similar data were obtained. On the average, the peak of HIV-1 replication kinetics in GFPVCA clones delayed a week compared with GFP clones. The maximum amount of p24 in the culture supernatants of H9 GFP-VCA clones did not appear different from those of H9 GFP clones.

View larger version (27K): [in this window] [in a new window]   Figure 5. Slow replication kinetics of HIV-1 in H9 cells expressing GFP-VCA constitutively. (A) The morphology of H9 cells was not drastically altered when GFP-VCA was expressed compared with GFP or untransfected cells. The green fluorescent image was merged with the transmission image (magnification, x630). (B) H9 clones stably expressed GFP or GFP-VCA as demonstrated by Western blot analysis (arrowheads). (C) The amount of p24 antigen in the culture supernatants accumulated rapidly in H9-GFP cell clones (red), whereas H9-GFP-VCA clones did not support the efficient HIV-1 replication (blue). Similar results were obtained by two independent experiments.

We next examined whether the viruses propagated in H9 GFP-VCA clones were mutants capable of replicating in the presence of GFP-VCA. We isolated viral RNA from the culture supernatants of a H9 GFP clone and three H9 GFPVCA clones and sequenced the gag/pro/pol region because the GFP-VCA's ability to limit HIV-1 infection did not depend on Env, Nef, Vpr, and Rev. However, we could not find any nucleic acid alterations when the viral sequences from an H9 GFP-VCA culture were compared with the one from an H9 GFP culture. These data suggested that the levels of GFP-VCA in H9 clones might be insufficient to confer the selective advantage for mutant viruses to take over the wild-type HIV-1. We found that the late phase of HIV-1 viral life cycle was slightly affected by GFP-VCA. Transfecting proviral DNA into 293 T-cells along with the expression vector for GFP-VCA yielded fewer amount of p24 antigen in the culture supernatant (72%) compared with GFP alone. Taken together, the decreased replication kinetics of HIV-1 in H9 GFP-VCA was assumed to be mostly due to the inhibition of the early phase, partly the late phase, of HIV-1's life cycle. These data demonstrated that the constitutive inhibition of the Arp2/3 complex by expressing GFP-VCA limited efficient replication of HIV-1 in T-cells as well as epithelial cell systems.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We have demonstrated that the Arp2/3 complex contributes to the efficient infection of both primate lentiviruses (HIV-1 and SIV) and IMV but not MLV, HSV-1, and adenovirus. Actin cytoskeleton has been shown to play a role in the infection of all the viruses tested in this study according to previous studies using the chemical actin inhibitor (Rosenthal et al., 1985; Kizhatil and Albritton, 1997; Bukrinskaya et al., 1998; Iyengar et al., 1998; Li et al., 1998; Locker et al., 2000). In addition, the Arp2/3 complex-mediated actin nucleation is sensitive to CC (Welch et al., 1998). Therefore, GFP-VCA's ability to limit infection of primate lentiviruses and IMV might be a part of, if not all, the mechanism by which CC reduced the efficiency of viral infection. In other words, primate lentiviruses and IMV might utilize the Arp2/3 complex-dependent actin polymerization system to support their early phase of viral life cycles. In contrast, MLV, HSV-1, and adenovirus might utilize actin to enter cells in an Arp2/3 complex-independent manner. Perhaps, other functional aspects of actin are important for their efficient infection such as the actin cable-dependent trafficking system. Our data clearly demonstrated that the different viruses use actin system differently. It was reported that the infection of HIV-1 was inhibited by CC at least two different levels: 1) by limiting viral receptor/coreceptor clustering upon viral attachment (Iyengar et al., 1998); and 2) by disrupting establishment of active reverse transcription complex (Bukrinskaya et al., 1998). Our findings suggest a possible involvement of the Arp2/3 complex in the latter process or a presence of another actin polymerization-dependent step between two processes.

What is the molecular mechanism by which the Arp2/3 complex supports infection of both primate lentiviruses and IMV? One of the possibilities is that these viruses may activate the Arp2/3 complex and nucleate actin filaments to support their entry. It has been reported that an acidic motif DDW or DEW can be found among cellular Arp2/3 complex-binding proteins including WASP, cortactin, and MyoD as well as Listeria monocytogenes surface protein ActA (Weed and Parsons, 2001). We are unable to find such a binding motif in proteins encoded by both HIV-1 and SIV. Therefore, these viruses may not activate Arp2/3 complex due to a direct interaction between viral gene products and Arp2/3 complex. There are two regulatory pathways known to activate the Arp2/3 complex. One is WASP/WAVE pathway and the other being cortactin pathway. Expression of the dominant-negative derivatives of N-WASP, WAVEs, and cortactin were unable to limit the infection of both HIV-1 and IMV (unpublished observation, consistent with the Locker's finding, Locker et al., 2000). The dominant-negative derivative of cdc42, RhoGTPase family protein that locates upstream of WASP/WAVE pathway, was also unable to reduce the relative infection efficiencies of both viruses (unpublished observation). These data implied that the activation of Arp2/3 complex upon infection of both lentiviruses and IMV might be mediated by novel virus-host interactions. Inhibiting the Arp2/3 complex by GFP-VCA did not drastically reduce the efficiency of HIV-1 production as did that of HIV-1 infection, suggesting that the incoming HIV-1 might have something unique which budding virus lacks. We speculate that viral gene products cleaved by HIV-1's protease may be responsible to induce activation of Arp2/3 complex because viral protease become active when viral particles are released from cells such that the cleaved proteins are present at high concentrations only in mature virus particles. VV encodes envelope protein A36R that binds adaptor proteins Nck, Wip, Grb2 that recruits and activates WASP-Arp2/3 complex system (Frischknecht et al., 1999; Rietdorf et al., 2001; Scaplehorn et al., 2002). These molecular interactions are known to be important for vaccinia virus to bud from infected cells as extracellular enveloped virus (EEV). However, IMV does not have A36R on its envelope. Accordingly, IMV should initiate activation of Arp2/3 complex via A36R-independent mechanisms.

On the basis of our data as well as previous observations, we propose models in which the Arp2/3 complex plays a role in the entry of both primate lentiviruses and IMV. HIV-1 enters cells via the membrane fusion on the cell surface (Stein et al., 1987; Maddon et al., 1988). The viral core complex is released to the cytoplasm immediately after the membrane fusion over the cortical layer. Our model is that the viral core components, independent of Env, Nef, Vpr, and Rev, recruit adaptor proteins and activate the Arp2/3 complex to generate mechanical force by which HIV-1's core complex passes through the cortical layer and migrates toward the nucleus efficiently (Figure 6A). McDonald et al. (2002) had shown that the microtubule system supports long-distance movement of HIV-1's core complex. It is possible that lentiviruses utilize the Arp2/3 complex-mediated active transport system to get access to a subcellular compartment where they meet microtubule system. Consistent with our model, a short-distance rapid movement of HIV-1's core was observed in the time-lapse imaging, which suggested a presence of an actin polymerization-dependent transport (McDonald et al., 2002). On the other hand, IMV enters cells via the membrane fusion at the cell surface as suggested by the intensive studies including electron microscopic studies (reviewed in Smith et al., 2002). IMV infection induces the formation of actin-rich cell surface protrusions partly due to GTPase Rac1 signaling (Locker et al., 2000) and is inhibited by treating cells with CCD (Vanderplasschen et al., 1998; Locker et al., 2000). Taken together, the Arp2/3 complex-mediated actin polymerization may be required for not only the late phase but also the early phase of IMV's life cycle, specifically at the postmembrane fusion processes (Figure 6B). Also, it is necessary to reorganize the cortical actin network to transport the virus-core toward the cytoplasmic subcompartment where the vaccinia virus replicates. The Arp2/3 complex may play a role in the latter process (Figure 6B). In any case, IMV induces relatively global cytoskeletal reorganization in which a substantial number of Arp2/3 complex should participate. This may account for the less efficient block of IMV's infection by both GFP-A and siRNA directing against Arp2 (Figure 4, C and E).

View larger version (27K): [in this window] [in a new window]   Figure 6. Models in which the Arp2/3 complex supports infection of both primate lentiviruses and IMV. (A) Primate lentiviruses enter cells via membrane fusion at cell surface. After membrane fusion, viral gene products might initiate activation of Arp2/3 complexdependent actin polymerization (red) behind the viral core to cross the cortical layer (gray). (B) IMV also enters cells via membrane fusion at the cell surface. At or soon after the attachment, the dynamic actin cytoskeletal reorganization takes place that depends partly on the Arp2/3 complex-mediated actin polymerization (red), which may facilitate viral entry. Alternatively, the Arp2/3 complexmediated actin polymerization (red) powers the viral core (green) to migrate toward the cytoplasmic compartment in which vaccinia virus replicates.

Our finding suggested a new therapeutic target to control HIV-1 replication in AIDS patients. We should be able to limit HIV-1 replication by inhibiting the HIV-1's ability to activate Arp2/3 complex by a small chemical compound. It is underway to determine which viral gene product is responsible to activate Arp2/3 complex.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank Drs. Hironori Sato, Bill Sugden, and Tsutomu Murakami for critical reading of the manuscript and Yuko Futahashi for the technical assistance. We also thank Drs. T. Takenawa, A. Yamashita, J. Young, E. Freed, P. G. Spear, Y. Takai, S. Takase-Yoden, W. Sugiura, and K. Mori for generously sharing reagents and equipments. This work was partly supported by both the Japan Health Science Foundation and a grant from Japanese Ministry of Health, Labor, and Welfare.

	Footnotes

 
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04–04–0279. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–04–0279.

Abbreviations used: Arp2/3, actin-related protein 2/3; CC, cytochalasin; GFP, green fluorescent protein; HIV-1, human immunodeficiency virus type 1; HSV-1, herpes simplex virus type 1; IMV, intracellular mature virus; MLV, murine leukemia virus; RLU, relative light unit; SIV, simian immunodeficiency virus; VSV-G, vesicular stomatitis virus G protein; VV, vaccinia virus; WASP, Wiscott-Aldrich syndrome protein.

^* Corresponding author. E-mail address: ajkomano{at}nih.go.jp.

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