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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
Moreover, Pancio et al. (2000) reported that deletion of the proline-rich C terminus (aa 102112) 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 6572 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.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 freezethaw 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 l1), streptomycin (0·12 g l1) and penicillin (0·122 g l1). 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 ml1 and 28 µg aprotinin ml1). Insoluble components were removed from lysates by centrifugation at 18 000 g at 4 °C for 30 min. Immunoprecipitations were performed with either 12 µg anti-Vpx or anti-HA antibody or a suitable amount of Myc 9E10 hybridoma supernatant. Immune complexes were recovered by adsorption to protein ASepharose (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 3045 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 ml1; 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 60046025),
N2 (5'-CGGGATCCAACAGAACAGTAGAGGAGATA-3', positions 61436163),
N3 (5'-CGGGATCCGCGGTAAACCACCTACCAAG-3', positions 61736192) 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 63706353),
C2 (5'-CGGAATTCTTAACAGCCTTTCTTGCAATGCAT-3', positions 63286307) or
C3 (5'-CGGAATTCTATATTAAACACAAGTATCTGTATTT-3', positions 62926268). 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. GSTVpx fusion proteins were expressed in E. coli XL2 Blue cells following induction by using IPTG (1 mM final concentration) and purified by binding to glutathioneSepharose 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 25 µg GSTVpx fusion proteins or GST alone, bound to 2550 µl glutathioneSepharose beads and incubated for 24 h at 4 °C. The affinity-purified proteins were washed with NP-40 buffer. For expression controls, GSTVpx 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 346892 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 (1·52·4-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 6085 and 6572 (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.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bearer, E. L. & Satpute-Krishnan, P. (2002). The role of the cytoskeleton in the life cycle of viruses and intracellular bacteria: tracks, motors, and polymerization machines. Curr Drug Targets Infect Disord 2, 247264.[Medline]
Beer, B. E., Foley, B. T., Kuiken, C. L. & 7 other authors (2001). Characterization of novel simian immunodeficiency viruses from red-capped mangabeys from Nigeria (SIVrcmNG409 and -NG411). J Virol 75, 1201412027.
Belshan, M. & Ratner, L. (2003). Identification of the nuclear localization signal of human immunodeficiency virus type 2 Vpx. Virology 311, 715.[CrossRef][Medline]
Bukrinsky, M. I., Haggerty, S., Dempsey, M. P. & 7 other authors (1993a). A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365, 666669.[CrossRef][Medline]
Bukrinsky, M. I., Sharova, N., McDonald, T. L., Pushkarskaya, T., Tarpley, W. G. & Stevenson, M. (1993b). Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc Natl Acad Sci U S A 90, 61256129.[Abstract]
Carpén, O., Pallai, P., Staunton, D. E. & Springer, T. A. (1992). Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and -actinin. J Cell Biol 118, 12231234.[Abstract]
Cossart, P. (2000). Actin-based motility of pathogens: the Arp2/3 complex is a central player. Cell Microbiol 2, 195205.[CrossRef][Medline]
Craig, S. W. & Pardo, J. V. (1979). Alpha-actinin localization in the junctional complex of intestinal epithelial cells. J Cell Biol 80, 203210.[Abstract]
Critchley, D. R. & Flood, G. (1999). -Actinins. In Guidebook to the Cytoskeletal and Motor Proteins. Edited by T. Kreis & R. Vale. San Franscisco: Oxford University Press.
Cudmore, S., Cossart, P., Griffiths, G. & Way, M. (1995). Actin-based motility of vaccinia virus. Nature 378, 636638.[CrossRef][Medline]
Cullen, B. R. (1998). HIV-1 auxiliary proteins: making connections in a dying cell. Cell 93, 685692.[Medline]
Depienne, C., Roques, P., Créminon, C., Fritsch, L., Casseron, R., Dormont, D., Dargemont, C. & Benichou, S. (2000). Cellular distribution and karyophilic properties of matrix, integrase, and Vpr proteins from the human and simian immunodeficiency viruses. Exp Cell Res 260, 387395.[CrossRef][Medline]
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387395.[Abstract]
Di Marzio, P., Choe, S., Ebright, M., Knoblauch, R. & Landau, N. R. (1995). Mutational analysis of cell cycle arrest, nuclear localization, and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol 69, 79097916.[Abstract]
Dixson, J. D., Forstner, M. R. J. & Garcia, D. M. (2003). The -actinin gene family: a revised classification. J Mol Evol 56, 110.[CrossRef][Medline]
Döhner, K., Wolfstein, A., Prank, U., Echeverri, C., Dujardin, D., Vallee, R. & Sodeik, B. (2002). Function of dynein and dynactin in herpes simplex virus capsid transport. Mol Biol Cell 13, 27952809.
Egerton, M., Moritz, R. L., Druker, B., Kelso, A. & Simpson, R. J. (1996). Identification of the 70kD heat shock cognate protein (Hsc70) and -actinin-1 as novel phosphotyrosine-containing proteins in T lymphocytes. Biochem Biophys Res Commun 224, 666674.[CrossRef][Medline]
Emerman, M. (1996). HIV-1, Vpr and the cell cycle. Curr Biol 6, 10961103.[Medline]
Farnet, C. M. & Haseltine, W. A. (1991). Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol 65, 19101915.[Medline]
Fletcher, T. M., III, Brichacek, B., Sharova, N., Newman, M. A., Stivahtis, G., Sharp, P. M., Emerman, M., Hahn, B. H. & Stevenson, M. (1996). Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV(SM). EMBO J 15, 61556165.[Abstract]
Fouchier, R. A. & Malim, M. H. (1999). Nuclear import of human immunodeficiency virus type-1 preintegration complexes. Adv Virus Res 52, 275299.[Medline]
Gallay, P., Hope, T., Chin, D. & Trono, D. (1997). HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. Proc Natl Acad Sci U S A 94, 98259830.
Gibbs, J. S., Regier, D. A. & Desrosiers, R. C. (1994). Construction and in vitro properties of HIV-1 mutants with deletions in "nonessential" genes. AIDS Res Hum Retrovir 10, 343350.[Medline]
Gibbs, J. S., Lackner, A. A., Lang, S. M., Simon, M. A., Sehgal, P. K., Daniel, M. D. & Desrosiers, R. C. (1995). Progression to AIDS in the absence of a gene for vpr or vpx. J Virol 69, 23782383.[Abstract]
Hahn, B. H., Shaw, G. M., De Cock, K. M. & Sharp, P. M. (2000). AIDS as a zoonosis: scientific and public health implications. Science 287, 607614.
Hansen, M. S. T. & Bushman, F. D. (1997). Human immunodeficiency virus type 2 preintegration complexes: activities in vitro and response to inhibitors. J Virol 71, 33513356.[Abstract]
Heinzinger, N. K., Bukrinsky, M. I., Haggerty, S. A. & 7 other authors (1994). The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci U S A 91, 73117315.[Abstract]
Heiska, L., Kantor, C., Parr, T., Critchley, D. R., Vilja, P., Gahmberg, C. G. & Carpén, O. (1996). Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to -actinin. J Biol Chem 271, 2621426219.
Henderson, L. E., Sowder, R. C., Copeland, T. D., Benveniste, R. E. & Oroszlan, S. (1988). Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2. Science 241, 199201.[Medline]
Henklein, P., Bruns, K., Sherman, M. P. & 7 other authors (2000). Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G2 cell cycle arrest. J Biol Chem 275, 3201632026.
Hirsch, V. M., Sharkey, M. E., Brown, C. R. & 8 other authors (1998). Vpx is required for dissemination and pathogenesis of SIVSM PBj: evidence of macrophage-dependent viral amplification. Nat Med 4, 14011408.[CrossRef][Medline]
Kappes, J. C., Morrow, C. D., Lee, S. W., Jameson, B. A., Kent, S. B. H., Hood, L. E., Shaw, G. M. & Hahn, B. H. (1988). Identification of a novel retroviral gene unique to human immunodeficiency virus type 2 and simian immunodeficiency virus SIVMAC. J Virol 62, 35013505.[Medline]
Kappes, J. C., Parkin, J. S., Conway, J. A., Kim, J., Brouillette, C. G., Shaw, G. M. & Hahn, B. H. (1993). Intracellular transport and virion incorporation of vpx requires interaction with other virus type-specific components. Virology 193, 222233.[CrossRef][Medline]
Kuhlman, P. A., Ellis, J., Critchley, D. R. & Bagshaw, C. R. (1994). The kinetics of the interaction between the actin-binding domain of -actinin and F-actin. FEBS Lett 339, 297301.[CrossRef][Medline]
Kumar, P. R., Singhal, P. K., Vinod, S. S. & Mahalingam, S. (2003). A non-canonical transferable signal mediates nuclear import of simian immunodeficiency virus Vpx protein. J Mol Biol 331, 11411156.[CrossRef][Medline]
La Boissière, S., Hughes, T. & O'Hare, P. (1999). HCF-dependent nuclear import of VP16. EMBO J 18, 480489.
Lazarides, E. & Burridge, K. (1975). Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell 6, 289298.[Medline]
Liu, B., Dai, R., Tian, C.-J., Dawson, L., Gorelick, R. & Yu, X.-F. (1999). Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin. J Virol 73, 29012908.
Louis, H. A., Pino, J. D., Schmeichel, K. L., Pomiès, P. & Beckerle, M. C. (1997). Comparison of three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression. J Biol Chem 272, 2748427491.
Mabit, H., Nakano, M. Y., Prank, U., Saam, B., Döhner, K., Sodeik, B. & Greber, U. F. (2002). Intact microtubules support adenovirus and herpes simplex virus infections. J Virol 76, 99629971.
Mahalingam, S., Van Tine, B., Santiago, M. L., Gao, F., Shaw, G. M. & Hahn, B. H. (2001). Functional analysis of the simian immunodeficiency virus Vpx protein: identification of packaging determinants and a novel nuclear targeting domain. J Virol 75, 362374.
McDonald, D., Vodicka, M. A., Lucero, G., Svitkina, T. M., Borisy, G. G., Emerman, M. & Hope, T. J. (2002). Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159, 441452.
Millake, D. B., Blanchard, A. D., Patel, B. & Critchley, D. R. (1989). The cDNA sequence of a human placental -actinin. Nucleic Acids Res 17, 6725.[Medline]
Miller, M. D., Farnet, C. M. & Bushman, F. D. (1997). Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J Virol 71, 53825390.[Abstract]
Mueller, S. M. & Lang, S. M. (2002). The first HxRxG motif in simian immunodeficiency virus mac239 Vpr is crucial for G2/M cell cycle arrest. J Virol 76, 1170411709.
Ott, D. E., Coren, L. V., Kane, B. P., Busch, L. K., Johnson, D. G., Sowder, R. C., II, Chertova, E. N., Arthur, L. O. & Henderson, L. E. (1996). Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 70, 77347743.[Abstract]
Pancio, H. A. & Ratner, L. (1998). Human immunodeficiency virus type 2 Vpx-Gag interaction. J Virol 72, 52715275.
Pancio, H. A., Vander Heyden, N. & Ratner, L. (2000). The C-terminal proline-rich tail of human immunodeficiency virus type 2 Vpx is necessary for nuclear localization of the viral preintegration complex in nondividing cells. J Virol 74, 61626167.
Pavalko, F. M. & LaRoche, S. M. (1993). Activation of human neutrophils induces an interaction between the integrin 2-subunit (CD18) and the actin binding protein
-actinin. J Immunol 151, 37953807.
Pavalko, F. M., Walker, D. M., Graham, L., Goheen, M., Doerschuk, C. M. & Kansas, G. S. (1995). The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via -actinin: receptor positioning in microvilli does not require interaction with
-actinin. J Cell Biol 129, 11551164.[Abstract]
Podlubnaya, Z. A., Tskhovrebova, L. A., Zaalishtsbvili, M. M. & Stefanenko, G. A. (1975). Electron microscopic study of -actinin. J Mol Biol 92, 357359.[Medline]
Rey, O., Canon, J. & Krogstad, P. (1996). HIV-1 Gag protein associates with F-actin present in microfilaments. Virology 220, 530534.[CrossRef][Medline]
Schmeichel, K. L. & Beckerle, M. C. (1994). The LIM domain is a modular protein-binding interface. Cell 79, 211219.[Medline]
Selig, L., Pages, J.-C., Tanchou, V. & 7 other authors (1999). Interaction with the p6 domain of the Gag precursor mediates incorporation into virions of Vpr and Vpx proteins from primate lentiviruses. J Virol 73, 592600.
Sells, M. A. & Chernoff, J. (1995). Epitope-tag vectors for eukaryotic protein production. Gene 152, 187189.[CrossRef][Medline]
Smith, D. B. & Johnson, K. S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 3140.[CrossRef][Medline]
Sodeik, B. (2000). Mechanisms of viral transport in the cytoplasm. Trends Microbiol 8, 465472.[CrossRef][Medline]
Sodeik, B., Ebersold, M. W. & Helenius, A. (1997). Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol 136, 10071021.
Stivahtis, G. L., Soares, M. A., Vodicka, M. A., Hahn, B. H. & Emerman, M. (1997). Conservation and host specificity of Vpr-mediated cell cycle arrest suggest a fundamental role in primate lentivirus evolution and biology. J Virol 71, 43314338.[Abstract]
Suomalainen, M., Nakano, M. Y., Keller, S., Boucke, K., Stidwill, R. P. & Greber, U. F. (1999). Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. J Cell Biol 144, 657672.
Takemura, T. & Hayami, M. (2004). Phylogenetic analysis of SIV derived from mandrill and drill. Front Biosci 9, 513520.[Medline]
Takubo, T., Hino, M., Suzuki, K. & Tatsumi, N. (1999). Relative distribution of myosin, actin, and alpha-actinin in adherent monocytes. Eur J Histochem 43, 7177.[Medline]
Tristem, M., Marshall, C., Karpas, A., Petrik, J. & Hill, F. (1990). Origin of vpx in lentiviruses. Nature 347, 341342.[Medline]
Tristem, M., Marshall, C., Karpas, A. & Hill, F. (1992). Evolution of the primate lentiviruses: evidence from vpx and vpr. EMBO J 11, 34053412.[Abstract]
Trono, D. (1998). When accessories turn out to be essential. Nat Med 4, 13681369.[CrossRef][Medline]
Ueno, F., Shiota, H., Miyaura, M. & 7 other authors (2003). Vpx and Vpr proteins of HIV-2 up-regulate the viral infectivity by a distinct mechanism in lymphocytic cells. Microbes Infect 5, 387395.[CrossRef][Medline]
Wachsstock, D. H., Wilkins, J. A. & Lin, S. (1987). Specific interaction of vinculin with -actinin. Biochem Biophys Res Commun 146, 554560.[Medline]
Weil, R., Sirma, H., Giannini, C., Kremsdorf, D., Bessia, C., Dargemont, C., Bréchot, C. & Israël, A. (1999). Direct association and nuclear import of the hepatitis B virus X protein with the NF-B inhibitor I
B
. Mol Cell Biol 19, 63456354.
Wu, X., Conway, J. A., Kim, J. & Kappes, J. C. (1994). Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein. J Virol 68, 61616169.[Abstract]
Wyszynski, M., Lin, J., Rao, A., Nigh, E., Beggs, A. H., Craig, A. M. & Sheng, M. (1997). Competitive binding of -actinin and calmodulin to the NMDA receptor. Nature 385, 439442.[CrossRef][Medline]
Yamada, K. M. & Geiger, B. (1997). Molecular interactions in cell adhesion complexes. Curr Opin Cell Biol 9, 7685.[CrossRef][Medline]
Yu, X.-F., Ito, S., Essex, M. & Lee, T.-H. (1988). A naturally immunogenic virion-associated protein specific for HIV-2 and SIV. Nature 335, 262265.[CrossRef][Medline]
Zhao, L.-J., Wang, L., Mukherjee, S. & Narayan, O. (1994). Biochemical mechanism of HIV-1 Vpr function. Oligomerization mediated by the N-terminal domain. J Biol Chem 269, 3213132137.
Zhu, Y., Gelbard, H. A., Roshal, M., Pursell, S., Jamieson, B. D. & Planelles, V. (2001). Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes. J Virol 75, 37913801.
Received 14 April 2004;
accepted 2 July 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |