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-actinin 1
Institute of Clinical and Molecular Virology, University of Erlangen-Nuernberg, Schlossgarten 4, D-91054 Erlangen, Germany
Correspondence
Sabine M. Lang
sabine.lang{at}yale.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx. Interestingly, deletion of the proline-rich C-terminal domain (aa 101–112) of Vpx, which is important for nuclear localization, resulted in loss of interaction with -actinin 1. These findings suggest that the interaction with -actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.
Present address: Department of Pediatrics and Adolescent Medicine, University of Erlangen-Nuernberg, Loschgestr. 15, D-91054 Erlangen, Germany. 
Present address: Institute for Microbiology, Biochemistry and Genetics, Department of Biotechnology, Henkestrasse 91, D-91052 Erlangen, Germany.
Present address: Yale University School of Medicine, Department of Pathology, PO Box 208023, 310 Cedar St, New Haven, CT 06520-8023, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), encode several accessory and/or regulatory genes in addition to the structural gag, pol and env genes that are present in all retroviruses (Cullen, 1998; Trono, 1998). Human immunodeficiency viruses and the different SIVs share significant genetic homology. HIV-2 and the closely related simian immunodeficiency viruses from macaques, sooty mangabeys and red-capped mangabeys (Beer et al., 2001) contain a vpx gene, as well as the evolutionarily related vpr gene. Due to the sequence similarity of Vpx and Vpr, it has been discussed that the Vpx protein arose from a gene-duplication event (Tristem et al., 1990, 1992). However, vpx is absent from HIV-1 and most other known SIVs (Takemura & Hayami, 2004). It has been shown that SIVsm Vpr and Vpx proteins have distinct, non-complementary functions (Fletcher et al., 1996). Whilst Vpr induces cell-cycle arrest at the G2/M stage of the cell cycle (Fletcher et al., 1996; Mueller & Lang, 2002; Stivahtis et al., 1997; Zhu et al., 2001), Vpx is involved in nuclear import of the viral pre-integration complex (PIC) (Depienne et al., 2000; Fletcher et al., 1996; Pancio et al., 2000). Animal studies showed that both proteins contribute to viral pathogenicity, as rhesus monkeys infected with SIVmac
vpx virus or with vprvpx double-mutant SIV had lower virus loads than animals infected with wild-type virus and progression to AIDS was delayed or absent (Gibbs et al., 1995). Additionally, Hirsch et al. (1998) showed that Vpx is essential for efficient dissemination and spread of SIVsm following mucosal and intravenous infection of macaques. Recent data indicate that Vpx is critical for upregulation of HIV-2 replication in natural target cells by enhancing nuclear import of the viral genome (Ueno et al., 2003). In conclusion, these results indicate that Vpx is important for virus load and disease progression in lentiviruses of the HIV-2/SIVmac lineage.
Vpx is a 12 kDa, 112 aa protein that is highly conserved between SIVmac and HIV-2 (Henderson et al., 1988; Kappes et al., 1988; Yu et al., 1988) (Fig. 1). Interaction of Vpx with the C-terminal proline-rich portion of the Gag precursor allows packaging of an amount comparable to that of Gag proteins into virus particles (Henderson et al., 1988; Pancio & Ratner, 1998; Selig et al., 1999; Wu et al., 1994). The presence of Vpx in the virion suggests an important function in the early life cycle of the virus. One function that has already been demonstrated for SIVsm Vpx and HIV-2 Vpx is to direct the nuclear import of the PIC in quiescent cells (Hirsch et al., 1998; Pancio et al., 2000), a prerequisite for the integration of viral DNA into the host genome (Bukrinsky et al., 1993a; Emerman, 1996; Gallay et al., 1997). The observation that SIVmac/SIVsm lacking Vpx has a significantly reduced ability to replicate in terminally differentiated macaque macrophages (Fletcher et al., 1996; Gibbs et al., 1994) and memory T cells (Hirsch et al., 1998) presumably results from this deficit in PIC transport.
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) but that, when expressed in the absence of other viral proteins, it localizes to the cytoplasm, as well as to the nucleus (Di Marzio et al., 1995; Kappes et al., 1993; Pancio et al., 2000).
Moreover, Pancio et al. (2000) reported that deletion of the proline-rich C terminus (aa 102–112) of the HIV-2ROD Vpx protein abrogates nuclear localization and attenuates HIV-2 replication in macrophages. These data suggest that deletion of the C-terminal proline-rich domain of Vpx is linked to functional loss of Vpx-mediated PIC transport to the nucleus. Other studies showed that the proline-rich C terminus of Vpx is not sufficient for nuclear localization (Belshan & Ratner, 2003; Mahalingam et al., 2001). As Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs), it may contain a novel NLS domain. Recently, Belshan & Ratner (2003) described aa 65–72 of Vpx as the minimal transferable region of Vpx that conferred nuclear import. Similar data were obtained by Mahalingam and coworkers, who identified a conserved domain within the C-terminal part of Vpx that is sufficient to mediate the transport of heterologous proteins, such as GFP and 
-galactosidase (-Gal), into the nucleus (Kumar et al., 2003; Mahalingam et al., 2001). Alternatively, Vpx may gain access to the nucleus by interacting with another NLS-containing protein and thus exploiting cellular nuclear import pathways. Similar interactions have been described for a number of other viral proteins (La Boissière et al., 1999; Weil et al., 1999). Numerous studies have shown that the cytoskeleton plays an important role in intracellular transport processes (Bearer & Satpute-Krishnan, 2002; Cudmore et al., 1995; McDonald et al., 2002; Sodeik, 2000; Sodeik et al., 1997; Suomalainen et al., 1999). In the present study, we report that SIVmac239 Vpx and HIV-2ROD Vpx interact with -actinin 1 (Millake et al., 1989), a cytoskeletal protein that belongs to the spectrin gene superfamily (Dixson et al., 2003). -Actinin 1 is an F-actin bundling protein and participates in the organization of the cytoskeleton (Kuhlman et al., 1994; Wachsstock et al., 1987). Several studies have provided evidence that the cytoskeleton may be involved in the assembly and budding of retroviruses. For example, it has been shown that HIV-1 Gag can bind F-actin (Liu et al., 1999; Rey et al., 1996); this specific cytoskeletal protein has been found in virions (Ott et al., 1996).
Our results indicate that the C terminus of Vpx, which mediates the nuclear localization of HIV-2 Vpx (Pancio et al., 2000), is essential for the interaction of SIVmac239 Vpx with -actinin 1. As the interaction with -actinin 1 is conserved between SIVmac239 Vpx and HIV-2ROD Vpx, it is likely that this interaction may play an important role in the transport of Vpx, and thus the transport of the PIC, into the nucleus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) by using the restriction sites for BamHI and EcoRI (underlined) that were introduced by the PCR primers and the correct sequence of the wild-type vpx gene was verified. The plasmids obtained, pCMV6M 239vpx and pCMV6M RODvpx, resulted in the expression of N-terminally Myc epitope-tagged (EQKLISEEDL) Vpx proteins lacking the first methionine residue of wild-type Vpx.
Yeast two-hybrid screen and interaction analyses.
Yeast two-hybrid screening was performed according to the protocol suggested by the Matchmaker two-hybrid system (Clontech). The yeast strain Saccharomyces cerevisiae CG-1945 was transformed sequentially with either the SIVmac239vpx or HIV-2RODvpx hybrid expression plasmids and the Jurkat cDNA library. Transformants were plated onto synthetic complete medium without tryptophan (Trp), leucine (Leu) and histidine (His) in the presence of 15 mM 3-amino-1,2,4-triazole.
His+ colonies were tested for -Gal activity by filter-lift assays according to the manufacturer's instructions (Clontech). Nucleic acids were extracted from lacZ+ yeast colonies and transformed into Escherichia coli strain KC8. Plasmids from the segregates (Leu+ Amp+) containing only the pACT2 Jurkat cDNA plasmids were isolated and sequenced. For quantitative -Gal assays, bait and target plasmids were transformed into S. cerevisiae strain Y190. Liquid -Gal assays were performed by using o-nitrophenyl--D-galactopyranoside (ONPG) according to the manufacturer's instructions (Clontech) with slight modifications: cells were lysed by adding SDS and chloroform to final concentrations of 0·006 % (w/v) and 0·06 % (v/v) respectively, instead of lysing by freeze–thaw cycles.
Tissue culture and transfection.
COS7 cells were grown in Dulbecco's modified Eagle medium supplemented with 10 % fetal calf serum (FCS), glutamine (0·35 g l–1), streptomycin (0·12 g l–1) and penicillin (0·122 g l–1). Transfection of cells used for transient-expression experiments was carried out according to a diethylaminoethyl Dextran protocol (Aruffo & Seed, 1987). Cells were harvested 48 h after transfection, washed once with PBS and used immediately or frozen for later use. COS7 cells used for immunofluorescence studies were transfected with Lipofectamine reagent according to the protocol suggested by GibcoBRL. Cells were harvested 48 h after transfection and were stained immediately. The hybridoma cell line Myc 1-9E10.2 (ATCC) was propagated in RPMI 1640 medium supplemented with 10 % FCS, glutamine, streptomycin and penicillin. For antibody production, cells were transferred to medium supplemented with 5 % FCS and cultivated for 5 days. The supernatant was cleared from cells and debris by centrifugation and buffered with 20 mM Tris, pH 8·0.
Immunoprecipitation and Western blot analysis.
Transfected COS7 cells were lysed in NP-40 buffer (0·5 % Nonidet NP-40, 0·15 M NaCl and 50 mM HEPES, pH 7·5) that contained phosphatase and protease inhibitors (1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 5 µg leupeptin ml–1 and 28 µg aprotinin ml–1). Insoluble components were removed from lysates by centrifugation at 18 000 g at 4 °C for 30 min. Immunoprecipitations were performed with either 1–2 µg anti-Vpx or anti-HA antibody or a suitable amount of Myc 9E10 hybridoma supernatant. Immune complexes were recovered by adsorption to protein A–Sepharose (Amersham Biosciences) and were washed three times with lysis buffer and once with 10 mM Tris/HCl, pH 7·5. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to an Immobilon-P membrane filter (Millipore) by using a Hoefer semi-dry unit. Membrane filters were blocked for 1 h either with PBS, 0·4 % Tween 20, 5 % non-fat dry milk or with PBS, 0·4 % Tween 20, 5 % FCS. Antibodies were diluted according to the manufacturer's recommendations. Anti-HA antibodies were pre-adsorbed onto COS7 cells for at least 1 h prior to use. Filters were incubated with the appropriate primary antibody for 1 h at room temperature or at 4 °C overnight. Subsequently, filters were washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30–45 min at room temperature. Proteins were detected by enhanced chemiluminescence (Amersham Biosciences).
Indirect immunofluorescence.
Transfected cells were fixed with 3 % paraformaldehyde in PBS for 30 min at room temperature and subsequently treated with PBS containing 0·1 % Triton X-100 to permeabilize cell membranes. Cells were washed with PBS and blocked with PBS containing 1 % BSA for 30 min. Antibodies were diluted in PBS according to the manufacturer's recommendations. Cells were incubated with the appropriate antibody for 30 min at room temperature. Nuclei were stained with DAPI (3 µg ml–1; Roche) before embedding. Immunofluorescence images were analysed with an Axioplan 2 microscope (Zeiss) or with a confocal microscope (Leica).
Antibodies and antisera.
Anti-Vpx antisera were generated by immunization of rabbits with purified glutathione S-transferase (GST)–SIVmac239 Vpx fusion protein. Obtained antibodies showed comparable reactivity against SIVmac239 Vpx and HIV-2ROD Vpx proteins. An anti--actinin mAb (clone BM-75.2) was purchased from Sigma. HA epitope-specific antibodies were obtained from BAbCo and Myc epitope-specific antibodies were produced by using the hybridoma cell line Myc 1-9E10.2. HRP-conjugated secondary antibodies were purchased from Santa Cruz or DAKO. Anti-Vpx antibodies were used at a 1 : 1000 dilution, whereas anti-Myc supernatant was used at a 1 : 50 dilution. For immunofluorescence studies, anti-Vpx polyclonal antibodies and Alexa Fluor 488-conjugated anti-HA mAbs (Molecular Probes), in combination with Texas red-conjugated anti-rabbit antibodies (Molecular Probes), were used in order to visualize the proteins by confocal microscopy.
Construction of SIVmac239 vpx deletion variants.
In order to map the protein-interaction domains within SIVmac239 Vpx, several N- and C-terminal deletion mutants (designated N1, N2, N3, C1, C2 and C3) were created. Plasmid pCMV6M 239vpx, containing the wild-type ORF of SIVmac239 vpx, was used as the template for PCR amplification of the truncated vpx fragments. Fragments N1, N2 and N3 were amplified by using the 5' primers N1 (5'-CGGGATCCAGTGGAGAAGAGACAATAGGAG-3', positions 6004–6025), N2 (5'-CGGGATCCAACAGAACAGTAGAGGAGATA-3', positions 6143–6163), N3 (5'-CGGGATCCGCGGTAAACCACCTACCAAG-3', positions 6173–6192) and the 3' primer 239vpx-3'. Fragments C1, C2 and C3 were amplified by using the 5' primer 239vpx-5' and the 3' primers C1 (5'-CGGAATTCTTATGGTCTCCATCCCCCTGC-3', positions 6370–6353), C2 (5'-CGGAATTCTTAACAGCCTTTCTTGCAATGCAT-3', positions 6328–6307) or C3 (5'-CGGAATTCTATATTAAACACAAGTATCTGTATTT-3', positions 6292–6268). Unique restriction sites for BamHI and EcoRI (underlined) were introduced by the PCR primers. PCR products were cloned into the prokaryotic expression vector pGex2TK (Amersham Biosciences) and the correct sequence of the mutant vpx genes was verified. Obtained plasmids resulted in the expression of N-terminally GST-tagged Vpx proteins.
Expression and purification of recombinant GST fusion proteins.
E. coli XL2 Blue cells (Stratagene) were transformed with the bacterial expression vector pGEX-2TK, pGEX-2TK 239vpx or plasmids encoding the vpx deletion variants. GST–Vpx fusion proteins were expressed in E. coli XL2 Blue cells following induction by using IPTG (1 mM final concentration) and purified by binding to glutathione–Sepharose beads (Amersham Biosciences) as described by Smith & Johnson (1988).
In vitro binding assay.
Cells expressing the cytoskeletal protein -actinin 1 were lysed in NP-40 buffer containing phosphatase and protease inhibitors. Cleared cell lysates were mixed with 2–5 µg GST–Vpx fusion proteins or GST alone, bound to 25–50 µl glutathione–Sepharose beads and incubated for 2–4 h at 4 °C. The affinity-purified proteins were washed with NP-40 buffer. For expression controls, GST–Vpx wild-type and mutant proteins were separated by SDS-PAGE and stained with Coomassie blue.
DNA sequencing.
Sequence analyses were performed with a Big Dye Terminator cycle sequencing ready reaction kit from Perkin Elmer. The procedure was carried out according to the manufacturer's recommendations. All analyses were carried out on an ABI Prism 377 DNA sequencer (Applied Biosystems).
Bioinformatics.
DNA sequence analysis, including multiple alignments and predicted translations, was performed with the Wisconsin Package version 10.1 from GCG (Genetics Computer Group) (Devereux et al., 1984). BLAST searches were carried out with the ncbi MEGABLAST system.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-actinin 1 as a cellular interaction partner of SIVmac239 and HIV-2ROD Vpx by yeast two-hybrid experiments-Gal expression of the yeast cells (CG-1945e) transformed with the Gal4–Vpx constructs was tested by filter-lift experiments. No -Gal expression could be detected with this combination. A Jurkat cDNA library fused to the Gal4 activation domain was introduced into yeast cells that had previously been transformed with Gal4–Vpx expression plasmids. Transformants harbouring interacting proteins were selected in the absence of histidine. For 239Vpx, 3·0x105 transformants were plated onto selective synthetic medium, whilst for RODVpx, 4·3x106 transformants were plated. His+ transformants were screened for their ability to produce -Gal by using a filter-lift assay (data not shown). Out of 256 HIS3-positive clones that were identified in the SIVmac239 Vpx screen, 36 tested positive for -Gal activity. In the HIV-2ROD Vpx screen, 53 His+ clones were identified; 45 of these were able to produce -Gal (data not shown). Sequence analysis and a search for homologies in the NCBI database revealed that two clones that were identified in the 239Vpx screen showed 99 % nucleotide sequence identity with the human -actinin 1 protein, encoded by the ACTN1 gene. Comparison of the amino acid sequence encoded by these two clones showed that one clone encoded aa 1–892 of -actinin 1 (full-length -actinin 1) and the other encoded aa 316–892 of -actinin 1. From the 45 clones isolated in the HIV-2ROD Vpx screen, one encoded aa 1–892 of -actinin 1, two clones encoded aa 316–892 of -actinin 1 and three clones encoded aa 346–892. To summarize, two independent clones of -actinin 1 were identified in the SIVmac239 screen and three independent clones were identified in the HIV-2ROD screen, indicating sufficient complexity of the cDNA library and the specificity of the interaction with 239Vpx and HIV-2ROD Vpx. In order to quantify the interaction of the different -actinin 1 clones and the two different Vpx proteins, liquid -Gal assays were performed by using the yeast strain Y190. All -actinin 1 clones, full-length as well as the N-terminal deletion variants, showed a strong, specific interaction with SIVmac239 Vpx and HIV-2ROD Vpx (Fig. 2).
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-actinin 1 with SIVmac239 Vpx and HIV-2ROD Vpx-actinin 1 with SIVmac239 Vpx and HIV-2ROD Vpx, we performed binding assays in a mammalian system. For this purpose, pCMV6M 239vpx and pCMV6M RODvpx and the N-terminally HA-tagged -actinin 1 (aa 346–892) were expressed in COS7 cells. Expression of the N-terminally Myc-tagged Vpx proteins and of HA–-actinin 1 was confirmed by Western blot (Fig. 3b, c, e, f). For immunoprecipitation, whole-cell lysates were incubated with anti-Myc mAbs and precipitated proteins were separated by SDS-PAGE. The presence of HA–-actinin 1 in the Myc–Vpx immune complexes was detected by immunoblot using anti-HA antibodies. Specific coimmunoprecipitation of HA–-actinin 1 was detected readily with Vpx from SIVmac239 and HIV-2ROD, but not with mock-transfected cells (Fig. 3a, d). Comparable results were obtained by using full-length -actinin 1 (Fig. 3g–l) and by immunoprecipitation for -actinin 1 using anti-HA antibodies followed by Western blotting for Vpx (data not shown). These results indicate that HA–-actinin 1 binds strongly to 239Vpx, as well as to RODVpx.
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-actinin 1 in eukaryotic cells-actinin 1, we analysed the subcellular distribution of 239Vpx alone. In all examined cells, 239Vpx and RODVpx were detected in the nucleus or localized to the cytoplasm (Fig. 4a–d). COS7 cells that were transfected with the different HA–-actinin 1 plasmids (encoding aa 346–892 and full-length -actinin 1) showed an even distribution of HA–-actinin 1 throughout the nucleus and the cytoplasm after staining with FITC-conjugated anti-HA antibodies (Fig. 4e and data not shown). Specificity of the antibodies used was shown by staining cells that were transfected with empty expression vectors (Fig. 4f). In order to test whether 239Vpx and RODVpx affect the subcellular distribution of -actinin 1, COS7 cells were cotransfected with HA–-actinin 1 (aa 346–892) and the Myc-tagged Vpx proteins. In contrast to cells that were transfected with the HA–-actinin 1 expression plasmids alone, the cytoplasmic distribution of HA–-actinin 1 in the presence of 239Vpx or RODVpx either decreased, whereas the accumulation of HA–-actinin 1 in the nucleus became more pronounced (Fig. 4g, i, j), or both proteins (239Vpx/RODVpx and HA–-actinin 1) accumulated at the nuclear membrane (Fig. 4h). Superimposition of the two confocal images clearly shows a high degree of colocalization and that 239Vpx and RODVpx influence the subcellular distribution of HA–-actinin 1. Comparable results were obtained when the HA–-actinin 1 full-length expression plasmids were used (data not shown).
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-actinin 1-actinin 1 within Vpx, three N-terminal and three C-terminal deletion mutants of SIVmac239 vpx were generated (Fig. 5). The deletions are based on amino acid alignments between the highly homologous Vpx proteins of SIVmac239 and HIV-2ROD (Fig. 1
) and earlier studies that defined three distinct structural domains within Vpx, two putative -helices (I and II) and one proline-rich domain at the C terminus. All deletion mutants were subcloned into the yeast expression vector pGBT9 and expressed as hybrid proteins with the Gal4 DNA-binding domain. Expression of the deletion variants of 239Vpx in yeast was verified by Western blot analysis (data not shown). In order to perform interaction studies, SIVmac239 vpx deletion variants and -actinin 1 (aa 346–892) were introduced in the yeast strain Y190. Subsequently, liquid -Gal assays were performed on Leu+ Trp+ transformants (Fig. 6a). None of the C-terminal deletion variants showed an interaction with HA–-actinin 1. Deletion of the C-terminal 11 aa, containing the proline-rich motif, was sufficient to abolish binding of 239Vpx to HA–-actinin 1. In contrast, all three N-terminal deletion mutants exhibited a strong interaction with HA–-actinin 1, whereas 239Vpx N2 and 239Vpx N3 showed a slightly stronger interaction with -actinin 1 than the wild-type protein (Fig. 6a). None of the deletion mutants had an activating effect in the absence of -actinin 1 (Fig. 6b). These results indicate that aa 102–112 of Vpx are required for binding to -actinin 1.
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). When purified GST proteins were mixed with COS7 cell lysates containing HA-tagged -actinin 1, GST-239Vpx wild-type fusion protein bound efficiently to HA–-actinin 1, whereas GST protein alone did not (Fig. 6c). In accordance with the results from the yeast system, all three N-terminal deletion mutants showed a strong interaction with HA–-actinin 1, whereas none of the C-terminal deletion variants bound to HA–-actinin 1. This proves that aa 102–112 are necessary for the interaction of 239Vpx and HA–-actinin 1 and that deletion of the proline-rich motif is sufficient to abolish binding of 239Vpx to -actinin 1 (Fig. 6c). In contrast to the ONPG assays, the in vitro GST-binding assays did not indicate an increased binding affinity for 239Vpx N2 or 239Vpx N3, as observed in the yeast system. In summary, these data show that HA–-actinin 1 interacts with the Vpx proteins of SIVmac239 and HIV-2ROD and that the interaction of SIVmac239 and -actinin 1 can be mapped to the C terminus of 239Vpx. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sodeik, 2000). Bacterial actin-based motility is one of the best-documented examples of exploitation of mammalian cell machinery by bacterial pathogens such as Listeria and Shigella (Cossart, 2000). Furthermore, cytoskeleton-dependent transport has been shown for a number of different viruses. For example, it has been demonstrated that microtubules play a prominent role in efficient nuclear targeting during the entry of herpesviruses (Döhner et al., 2002; Mabit et al., 2002; Sodeik et al., 1997) and adenoviruses (Mabit et al., 2002; Suomalainen et al., 1999). A recent study showed that, after viral entry, HIV particles move along the cytoskeleton to transport the viral PIC to the nucleus (McDonald et al., 2002). This finding suggests a vital role for interaction of components of the viral PIC with proteins of the cytoskeleton. The PIC is a nucleoprotein complex that comprises a variety of viral components, including dsDNA and reverse transcriptase, matrix, nucleocapsid, integrase and Vpr (HIV-1) or Vpx (HIV-2, SIV) proteins (Bukrinsky et al., 1993b; Farnet & Haseltine, 1991; Fletcher et al., 1996; Fouchier & Malim, 1999; Hansen & Bushman, 1997; Heinzinger et al., 1994; Miller et al., 1997). These proteins have been demonstrated to be crucial regulators of nuclear PIC import, but it remains unclear as to the exact contribution that each protein makes. In the present study, we show that SIVmac239 and HIV-2ROD Vpx proteins interact with -actinin 1 (Millake et al., 1989), one of four different isoforms of a cytoskeletal protein that belongs to the spectrin superfamily (Dixson et al., 2003). -Actinin 1 and its isoforms interact with a large number of different cellular proteins, such as the cytoplasmic domains of several surface receptors (Carpén et al., 1992; Heiska et al., 1996; Pavalko & LaRoche, 1993; Pavalko et al., 1995; Wyszynski et al., 1997) and cell–cell adherence junction proteins (Craig & Pardo, 1979; Louis et al., 1997; Schmeichel & Beckerle, 1994; Yamada & Geiger, 1997). Most importantly, they are involved in cytoskeletal reorganization (Critchley & Flood, 1999; Pavalko & LaRoche, 1993), bundling and cross-linking of actin filaments (Lazarides & Burridge, 1975; Podlubnaya et al., 1975) and binding of actin to the plasma membrane (Takubo et al., 1999).
It has been shown previously that Vpx augments HIV-2 replication in natural target cells by enhancing nuclear import of the viral genome (Ueno et al., 2003) and is essential for PIC transport and replication in non-dividing macrophages (Pancio et al., 2000). In order to elucidate the function of Vpx in the context of nuclear PIC transport, we performed yeast two-hybrid experiments to identify cellular targets of Vpx. Here, we show the interaction of Vpx with -actinin 1 in yeast cells, as well as in mammalian cells. So far, this interaction has not been confirmed to occur in HIV-infectable cells. However, -actinin 1 is expressed in T lymphocytes, indicating that the interaction with Vpx can occur in vivo (Egerton et al., 1996).
When expressed alone, the localization of Vpx was concordant with its previously reported localization in the cytoplasm (Kappes et al., 1993), nucleus (Depienne et al., 2000; Di Marzio et al., 1995; Mahalingam et al., 2001; Pancio et al., 2000) and nuclear membrane (Mahalingam et al., 2001), but could not be detected at the plasma membrane (Kappes et al., 1993). The lack of plasma-membrane staining is probably due to the absence of Gag proteins in transiently transfected cells. Further investigation is needed to determine whether the different localization patterns of Vpx proteins are relevant for PIC transport or are simply due to overexpression.
Interaction of the three different -actinin 1 clones, one of them being full-length -actinin 1, with Vpx was shown in the yeast two-hybrid screen and confirmed by liquid -Gal assays. As the three different clones showed similar binding behaviour to 239Vpx and RODVpx in yeast cells, we reason that the binding domain of Vpx lies within aa 346–892 of -actinin 1. The interaction of -actinin 1 and the viral Vpx proteins was verified by coimmunoprecipitation assays and a clear colocalization of -actinin 1 and Vpx in intracellular staining experiments. Remarkably, the localization patterns of Vpx and -actinin 1 changed when coexpressed. In addition to the nuclear and cytoplasmic localization, distinct colocalization of both proteins was observed at the nuclear membrane. Takubo et al. (1999) showed that -actinin 1 anchors actin filaments to the cell membrane. In concordance with these findings, it is conceivable that -actinin 1 may be an early binding partner of the PIC following the uncoating process of the virus and may play a role during the transport of the PIC to the nucleus.
The binding domain of -actinin 1 was mapped clearly to the C-terminal proline-rich region of SIVmac239 Vpx. N-terminal Vpx deletions did not interfere with the interaction of Vpx and -actinin 1. Interestingly, a slight increase (1·5–2·4-fold) in binding activity was observed for N-terminal deletion mutants, in comparison to wild-type Vpx, in the yeast system. This may be due to conformational changes of the protein, resulting in a higher binding affinity. So far, there are no data available concerning the three-dimensional structure or possible dimerization of Vpx, but data obtained for the homologous Vpr protein (Henklein et al., 2000; Zhao et al., 1994) indicate that self-association may influence the interaction of Vpx with cellular proteins. However, the mechanism of this effect remains unclear. So far, two new non-canonical NLSs have been identified within Vpx. The C-terminal proline-rich region of Vpx was reported by Pancio et al. (2000) to be important for Vpx-mediated nuclear import of the HIV-2 PIC. In the present study, we have shown that the C-terminal proline-rich domain of SIVmac239 Vpx is essential for its interaction with -actinin 1. As the amino acid sequences of the Vpx proteins are highly conserved and the interaction of -actinin 1 with SIVmac239 Vpx and HIV-2ROD Vpx is comparable, it is likely that binding of -actinin 1 is required for Vpx-mediated nuclear import of the PICs of both viruses.
A second motif conferring nuclear localization has been described within Vpx between aa 60–85 and 65–72 (Belshan & Ratner, 2003; Kumar et al., 2003; Mahalingam et al., 2001). Considering that our data suggest that the C-terminal deletion was sufficient to abolish -actinin 1 binding, these authors' results may define additional structural requirements for Vpx-mediated nuclear import. Our findings imply that Vpx tethers the PIC to the cytoskeleton via -actinin 1 and may therefore play an important role in transport of the viral PIC.
The second NLS, and possibly interaction with additional cellular proteins, may be necessary for transfer of the PIC through the nuclear membrane. Consistent with published data (Pancio et al., 2000), our results provide strong evidence for the existence of an -actinin 1-dependent pathway of Vpx transport and associated transport of the viral PIC.
As nuclear transport of Vpx and transport of the PIC become increasingly complex, further investigations of the interaction between viral and cellular proteins with Vpx and other compounds of the PIC will be required to clarify the mechanism of nuclear import of the viral genome. Precise knowledge of the mechanism by which the PIC is imported into the nucleus could provide new means to intervene in the viral life cycle before integration of the viral genome into the host DNA takes place.
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ACKNOWLEDGEMENTS |
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