Forschungsschwerpunkt Angewandte Tumorvirologie, F02001, and Forschungsschwerpunkt Krebsentstehung und Differenzierung, A01002, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
Pathogénie des Infections à Lentivirus, INSERM U372, BP178, 13276 Marseille, France3
Author for correspondence: Valerie Bosch. Fax +49 6221 424932. e-mail V.Bosch{at}dkfz-heidelberg.de
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Abstract |
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Introduction |
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The vif gene does not display significant sequence homology with any genes of known function so it has been difficult to gain insight into its mode of action. Immunofluorescence analysis of the subcellular distribution of Vif indicates that it is predominantly localized within the cytoplasmic region, with only weak staining of the nucleus (Goncalves et al., 1995 ; Karczewski & Strebel, 1996
). In vitro assays indicate that Vif has an affinity for microsomal membranes (Goncalves et al., 1994
, 1995
). The C terminus of Vif, which contains a cluster of basic amino acids, has been implicated in this in vitro membrane association and, additionally, to be important for overall Vif function, as measured in infectivity assays (Goncalves et al., 1994
, 1995
). Fractionation analyses of transfected cells in various detergents have indicated that the Vif protein can be differentially solubilized. Thus, some cellular Vif is soluble in aqueous buffers, some can be solubilized in weak detergents and some can only be dissolved in strong ionic detergent solutions (Goncalves et al., 1994
, 1995
; Karczewski & Strebel, 1996
; Simon et al., 1999
). It is not clear whether these solubility properties reflect specific subcellular localizations of expressed Vif. Thus, it has been controversially discussed that Vif may be associated with cellular membranes (Goncalves et al., 1994
, 1995
), cytoskeletal components (Karczewski & Strebel, 1996
) or insoluble components distinct from cytoskeletal components (Simon et al., 1999
).
It is likely that Vif exerts its function by interacting with cellular and/or viral components. In this context, it is of note that in a subpopulation of transfected HeLa cells expressing Vif from a subvirus vector, the distribution of the intermediate filament protein, vimentin, was altered such that vimentin was present in perinuclear aggregates to which Vif also localized (Karczewski & Strebel, 1996 ). Concerning putative interactions with viral proteins, it has been reported that Vif colocalizes with Gag in infected cells (Simon et al., 1997
, 1999
), whereby more recent evidence suggests that this may reflect independent targetting to the same subcellular compartment without a specific interaction (Simon et al., 1999
). Furthermore, it has been reported that Vif specifically and strongly interacts with Gag in vitro (Bouyac et al., 1997
). The C terminus of Vif has also been implicated in Gag-binding and, again, this interaction has been proposed to be important for Vif function.
We have previously described the properties of a patient-derived HIV-1 vif gene product, VifA45-2 (referred to here as VifA45; Ochsenbauer et al., 1996 ), which has a C-terminal truncation of 19 amino acids as a result of a premature stop codon. Despite the C-terminal truncation, VifA45 is functional and HIV virions, derived from pNL-VifA45, were infectious in both non-permissive H9 cells and peripheral blood mononuclear cells. This was somewhat surprising due to the proposed importance of the C-terminal region in mediating binding to cellular membranes and Gag, properties that had been previously implied to be essential to Vif function. It has previously not been possible to further analyse the properties of VifA45 since available antibodies, reactive with VifWt, do not recognize the variant VifA45 protein. We have generated specific antibodies to VifA45 and, in this study, we characterize further the properties of Vif with respect to intracellular distribution, putative interactions with Gag proteins and effects on components of the cellular intermediate filament network.
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Methods |
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The subvirus construct pNL-A1(CD4-) (Willey et al., 1992 ; Strebel et al., 1987
) with the intron between SD1 and SA2 missing, i.e. nucleotide 743 has been joined to nucleotide 4913, contains vif as the first ORF and results in high-level expression of Vif (Strebel et al., 1987
; Karczewski & Strebel, 1996
). A BssHIIEcoRI fragment (nucleotides 7115743) from this plasmid was replaced by BssHII/EcoRI-digested PCR fragments derived from the different pNL derivatives. The upstream chimeric primer employed, which contains the BssHII site, was from nucleotides 706716 fused to nucleotides 49114933 and the downstream primer from nucleotides 58905869 within the tat gene. In comparison to pNL-A1(CD4-), this results in the deletion of 27 nucleotides downstream of BssHII and the insertion of two nucleotides upstream of SA2. These changes had no effect on Vif expression. The new Vif expression constructs based on pNL-A1(CD4-) are referred to as pNL-A1-VifWt [the vif sequence has been derived from HIV strain BH10 as in pNL-A1(CD4-)], pNL-A1-VifA45, pNL-A1-VifA45open and pNL-A1-Vif-minus. This latter construct expresses all the gene products from pNL-A1(CD4-), except Vif, i.e. including mutated Env. Expression of Gag and pol sequences in eukaryotic cells was achieved employing pK-R-gpV (Mergener et al., 1992
).
Plasmids for in vitro transcription/translation were generated by cloning PCR-amplified copies of the respective vif genes into appropriate plasmids downstream from the bacteriophage T3 promoter. The vif gene from plasmid pNDK (Spire et al., 1990 ) was amplified as described (Bouyac et al., 1997
) and cloned into a pCR-blunt cloning vector (Invitrogen) to give plasmid pT3-VifWt (strain NDK). The respective genes from pNL-VifA45 and pNL-VifA45open were amplified and cloned into the pACT vector (Promega) to give plasmids pT3-VifA45 and pT3-VifA45open. Plasmids for bacterial expression of the glutathione S-transferase (GST) protein alone or as a fusion protein with Gag or NC have been described previously (Bouyac et al., 1997
).
Antisera reactive with VifWt and VifA45.
Rabbit antiserum against wild-type Vif, strain BH10 (Los Alamos; referred to as anti-VifWt), was generated by inoculating a rabbit with a mixture of N- and C-terminally histidine-tagged full-length VifWt proteins. These proteins had been expressed in bacteria using commercially available expression plasmids (Qiagen) and had been purified, after denaturation, by nickelagarose affinity purification. Antiserum reactive with VifA45 (referred to as anti-VifA45) was prepared by inoculating a rabbit with bacterially expressed denatured full-length VifA45 fused to GST, expressed from the commercial plasmid pGEX-2T (Pharmacia) and purified by solubility criteria and gel electrophoresis.
Expression and infectivity of provirus constructs with different Vif alleles.
Permissive 293T cells were transfected with 10 µg of the different provirus DNA constructs using standard calcium phosphate procedures. Equal amounts of virions, quantified by ELISA for HIV CA (Innogenetics), released into the media at 48 h post-transfection (p.t.) were employed to infect fresh cultures of permissive MT-4 cells or non-permissive H9 cells. Infected cells were washed 16 h post-infection (p.i.) to remove input virus and fed with fresh medium. Subsequently, samples of medium were collected every 3 days and the newly synthesized virions were quantified for HIV CA by ELISA.
Expression of different Vif alleles from subvirus expression plasmids in permissive HeLa and COS-7 cells.
Transfection with 2 µg subgenomic pNL-A1-Vif expression plasmids in the presence or absence of 2 µg pK-R-gpV was achieved in HeLa cells employing Lipofectamine (Life Technologies) and in COS-7 cells employing FuGENE (Roche Diagnostics), as described by the manufacturers. Transfected cells growing on glass cover slips were fixed and permeabilized with acetonemethanol (1:1) at 40 h p.t. Indirect immunofluorescence was then carried out using combinations of rabbit anti-VifWt, rabbit anti-VifA45, rabbit anti-HIV gp120 (Bosch & Pawlita, 1990 ), human anti-HIV serum, mouse anti-vimentin 3B4 (Progen) (Herrmann et al., 1993
), mouse anti-tubulin B-5-1-2 (Sigma), guinea pig anti-plectin P1 (Schroder et al., 1999
) or culture supernatant from mouse hybridoma 183 cells (Chesebro et al., 1992
) containing antibody specific for HIV p24 Gag. The respective secondary antibodies labelled with fluorescein or rhodamine were purchased from Dianova. The fluorescent labelled cells were analysed by confocal fluorescence microscopy using a Carl Zeiss (Jena) LSM 510 UV laser scanning microscope equipped with a HeNe (543 nm wavelength) and an argon ion (488 nm wavelength) laser as light sources and the corresponding beam splitters and barrier filters. The microscope was used in the multitracking scanning mode to avoid bleed-through of the fluorescent dyes, i.e. each scan-line is alternately illuminated by only one laser.
In vitro VifGag interaction.
The different pT3-Vif plasmids or T3-luciferase control DNA (Promega) were used for in vitro transcription/translation in the presence of [35S]methionine (>1000 Ci/mmol; Amersham) using the TnT T3 Wheat Germ Extract system (Promega), as recommended by the manufacturer. Radio-labelled products were resolved by SDSPAGE on a 12% gel and revealed by autoradiography using Kodak X-OMat films. Bacteria expressing the GST, GSTGag and GSTNC proteins were kindly provided by B. Spire (Bouyac et al., 1997 ). The different proteins were prepared essentially as follows. Briefly, transformed E. coli Top 10 cells were grown at 37 °C to an OD600 of about 1·0. Expression of the fusion proteins was then induced with 1 mM IPTG for 3 h at 30 °C. Bacterial cultures were pelleted by centrifugation at 5000 g for 15 min at 4 °C and then resuspended in 1/10 vol. of MTPBS (150 mM NaCl, 12·5 mM Na2HPO4, 2·5 mM KH2PO4, 100 mM EDTA, pH 7·5). Bacteria were lysed on ice by mild sonication, the lysates were adjusted to 1% Triton X-100 and incubated on ice for 30 min before clearing by centrifugation at 15000 g for 30 min at 4 °C. Glutathioneagarose beads (Sigma), previously resuspended in MTPBS, were added to the cleared supernatant for 1 h at 4 °C. Beads were then extensively washed in 1 M NaCl and then in PBS0·1% Triton X-100 supplemented with a protease inhibitor cocktail (1 µg/ml each of aprotinin, leupeptin, pepstatin A and antipain, 100 µg/ml PMSF, 200 µg/ml pefabloc). Beads were then resuspended in SDSPAGE sample buffer and the bound GST fusion proteins were resolved by SDSPAGE on a 12% gel. Proteins were visualized by Coomassie-blue staining. Equal amounts of GST or GST fusion proteins bound to glutathioneagarose beads were incubated in vitro with [35S]methionine-labelled Vif proteins in TnT binding buffer containing 50 mM TrisHCl, pH 7·6, 0·2% Tween 20 and 150 mM NaCl, in the presence of BSA (100 µg/ml) for 1 h at 4 °C. Beads were washed in TnT buffer and resuspended in SDSPAGE sample buffer. The bound proteins were resolved by SDSPAGE on a 12% gel, revealed by autoradiography, quantified with a Fuji phosphorimager and the data analysed by MacBas version 2.5 software (Fuji Photo Film).
Differential solubility of VifWt, VifA45 and VifA45open.
HeLa or COS-7 cells transfected with pNL-A1-VifWt, pNL-A1-VifA45 or pNL-A1-VifA45open were metabolically labelled for 5 h at different time-points p.t. with 100 µCi/ml [35S]methionine and [35S]cysteine (Pro-mix, Amersham). Subsequently, labelled cells were suspended in PBS and detergent extracted, essentially as described by Karczewski & Strebel (1996) . Briefly, cells were lysed by four cycles of freezing and thawing (3 min each at -70 °C and 37 °C). The supernatants containing soluble cytoplasmic proteins (referred to here as the soluble fraction, S) were separated by centrifugation at 15000 g for 5 min. Insoluble material was extracted with CHAPS/DOC buffer (50 mM TrisHCl, pH 8·8, 5 mM EDTA, 100 mM NaCl, 0·5% CHAPS, 0·2% deoxycholate) by incubation for 5 min at room temperature and centrifugation for 5 min at 15000 g. The resulting supernatant is referred to as the membrane fraction (M), since it contains membrane proteins. However, this should not imply that all proteins in this fraction necessarily originate from cellular membranes. Detergent-resistant material was solubilized by boiling the samples in 1% SDS, 2·5% mercaptoethanol, 2·5% glycerol and 0·031 M TrisHCl, pH 6·8 for 15 min at 80 °C. This sample, referred to as the insoluble fraction (I) was clarified by centrifugation at 15000 g for 10 min. The buffers in all three fractions were adjusted to contain 1% Triton, 0·5% deoxycholate and 0·1% SDS in PBS (RIPA buffer) and immunoprecipitation performed using anti-VifWt, anti-VifA45 or anti-gp120 sera plus protein ASepharose (Pharmacia). Immunoprecipitates were analysed by PAGE on a 15% gel and autoradiographed as described previously (Pfeiffer et al., 1997
).
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Results |
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Confocal microscopy analyses of VifWt, VifA45, VifA45open and Gag in transfected HeLa cells
It has been implied that the basic C-terminal region of VifWt plays a role in the subcellular localization and putative interactions of Vif with cellular and viral components. Thus, making use of the newly generated antisera, we initially employed indirect immunofluorescence and confocal microscopy to analyse the subcellular distributions of the different Vif proteins in HeLa cells, and compared these to the subcellular distribution of coexpressed Gag protein. Fig. 2 illustrates the different types of Vif protein localizations that were observed and the percentages of cells, transfected with pNL-A1-VifWt, pNL-A1-VifA45 and pNL-A1-VifA45open, respectively, which exhibit these distributions. The bulk of the cells (approximately 8090%) transfected with pNL-A1-VifWt exhibited a somewhat punctate distribution of VifWt throughout the cytoplasmic region and a weaker fluorescence signal in the nucleus (Fig. 2
, left). This intracellular distribution was not affected by the fixation procedure, as has been reported to be the case with the vif gene product from feline immunodeficiency virus (Chatterji et al., 2000
). Thus, our usual fixation procedure in either acetonemethanol or paraformaldehyde yielded similar results. However, in agreement with previously published results (Karczewski & Strebel, 1996
), about 1020% of the cells expressing VifWt showed a different distribution of the Vif protein; in these cases, VifWt exhibited either stringy aggregation on a background of diffuse cytoplasmic staining (Fig. 2
, middle) or almost complete aggregation to a perinuclear structure (Fig. 2
, right). In strong contrast to VifWt, most of the cells (8090%) expressing VifA45 exhibit stringy or perinuclear Vif staining. The localization to stringy or perinuclear aggregates is even more obvious in the case of VifA45open and virtually all of the transfected cells (99%) exhibit this Vif localization. This localization was not a property of the anti-VifA45 serum but was also observed with anti-VifWt serum, which also reacts well with VifA45open. This distinct localization in comparison to VifWt is thus independent of the C-terminal 19 amino acids and must be due to one or a combination of further sequence differences that exist between VifWt and VifA45open (Wieland et al., 1994
).
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Discussion |
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By employing an in vitro membrane-binding assay, it was previously reported that Vif has an affinity for membranes in vitro and that this association is mediated by the basic C-terminal region (Goncalves et al., 1994 , 1995
). In cell fractionation analyses, a fraction of the Vif protein is soluble in aqueous buffers, another fraction is soluble in weak detergent buffers and a last fraction is insoluble in weak detergent. The fact that the detergent buffer fraction contains integral and peripheral membrane proteins like Env, which localizes exclusively to this fraction (data not shown), further suggests that a fraction of Vif could be associated with cellular membranes. However, our observation that approximately the same amounts of Vif, which represents about 1020% of the total VifWt, VifA45 and VifA45open, are present in the detergent fraction of transfected HeLa and COS-7 cells, indicates that regions of the protein other than the C terminus mediate solubility in weak detergent. In confocal microscopy analyses, both VifA45 and VifA45open, expressed in HeLa cells, exhibit distinctly different immunofluorescence patterns as compared to VifWt. In most transfected cells, VifA45 and VifA45open are localized in stringy or perinuclear aggregates, whereas VifWt is localized and more evenly distributed within the cytoplasmic region. This distinct localization is thus not related to the presence or absence of the 19 C-terminal Vif amino acids but is presumably mediated by one or several other amino acids which differ between VifA45 and VifWt. It is perhaps surprising that this distinct localization, observed by confocal microscopy, does not result in a more significant difference in the solubility of VifA45 and VifA45open in HeLa cells in comparison to VifWt. In fact, the only difference observable is a slight decrease in the solubility of VifA45 and VifA45open in comparison to VifWt, so that slightly less of these components are present in the S fraction and slightly more in the I fraction.
In contrast to the localizations in HeLa cells, confocal microscopy analyses demonstrate that VifWt, VifA45 and VifA45open all exhibit a relatively even distribution within the cytoplasmic region of monkey COS-7 cells. This cell-specific difference in the distributions of VifA45 and VifA45open in HeLa and COS-7 cells, as observed by confocal microscopy, is intriguing but its significance, if any, is unclear.
In eukaryotic cells, three distinct yet interconnected filament systems are of central importance for the mechanical stability and dynamic behaviour of the cytoarchitecture. These are microfilaments consisting of actin, microtubules made from /
-tubulin subunits and intermediate filaments made from fibrous proteins, including vimentin. Plectin and its isoforms are ubiquitous cytoskeletal cross-bridging proteins of very large size with characterized binding domains for all three types of filament systems (for review see Herrmann & Aebi, 2000
). In both HeLa and COS-7 cells, the cellular localizations of vimentin and plectin but not microtubular protein tubulin are affected by coexpression of Vif protein from the pNL-A1 constructs. The fact that the microtubular network has remained unaffected shows that Vif does not exert a detrimental effect on all of the cellular filament systems. There are again, however, intriguing cell-specific differences. In HeLa cells, when the respective Vif protein species forms aggregates (as is especially prominent for VifA45 and VifA45open), vimentin and plectin also aggregate to similar stringy or perinuclear locations. Thus, there appears to be colocalization between Vif and vimentin or plectin in transfected HeLa cells. In contrast, although the respective Vif proteins all localize throughout the cytoplasmic region in COS-7 cells, vimentin and plectin form perinuclear aggregates, i.e. in these cells, there is very little colocalization, if any, between the respective Vif proteins and aggregated vimentin and plectin. These results in COS-7 cells suggest an indirect mechanism of action of Vif on vimentin and plectin organization whereby the nature of this putative indirect effect can only be speculated upon. It is known that intermediate filaments undergo dynamic changes during the cell-cycle and that this may be regulated by phosphorylation of vimentin, which is a substrate for several kinases involved in cell-cycle regulation (reviewed by Herrmann & Aebi, 2000
). In another virus system, frog virus 3, phosphorylation of vimentin has been shown to be involved in virus-induced intermediate filament reorganization (Chen et al., 1986
). On the other hand, it has recently been shown that in an early step of apoptosis, plectin is proteolytically cleaved at a defined position by caspase-8 and that this leads to a reorganization of the microfilament system (Stegh et al., 2000
). Further investigations will be required to establish if any of these types of mechanism also play a role in the effect of HIV-1 Vif on intermediate filament organization shown here.
The dramatic effects on vimentin and plectin cellular localization, which have been shown here after transfection of cells with pNL-A1 constructs, were not encountered in HeLa or COS-7 cells transfected with provirus (pNL4-3) constructs from which Vif is less strongly expressed. Only occasionally, although significantly, were aggregated vimentin and plectin structures observed in some COS-7 cells expressing pNL-VifA45 (data not shown). It is also of importance to keep in mind that both HeLa and COS-7 cells are permissive with respect to Vif, i.e. infectious virus is still released from these cells in the absence of any Vif protein. Nevertheless, we find it conceivable that the observations on intermediate filament relocalization do still reflect an essential, even if not such a dramatically visible, function of Vif in non-permissive cells.
In this context, it is of interest that several studies report on effects of virus infection on cytoskeletal structure as well as on associations of viral proteins with cytoskeletal components. For example, as mentioned above, frog virus 3 interacts with the cytomatrix (Chen et al., 1986 ), the E1-E4 protein of human papillomavirus type 16 has been reported to bind to, and cause the collapse of, the cytokeratin matrix (Doorbar et al., 1991
) and the E1B 19 kDa protein of adenovirus causes disruption of the intermediate filament network (White & Cipriani, 1990
). More recently, Theilers murine encephalomyelitis virus, a picornavirus, has been shown to bind to desmin and vimentin and cause a structural rearrangement in the intermediate filament network (Nedellec et al., 1998
). Also in these cases, the importance of these interactions and their putative functional involvement in virus replication and/or pathogenesis have not yet been elucidated.
In the case of HIV-1, it is intriguing that cytoskeletal proteins have also been detected inside released virions (Ott et al., 1996 ) and furthermore that, in a normal HIV infection, establishment of a functional reverse transcription complex involves the cytoskeleton (Bukrinskaya et al., 1998
). It is conceivable that the nature of the virus-producing cell (permissive/non-permissive) may influence the ability, in the target cell, of the preintegration complex to attach to cytoskeletal components and be able to complete reverse transcription and nuclear transport. It is theoretically possible that a cytoskeletal protein itself, as a component of the virus particle, may mediate an essential interaction with the cytoskeleton in the infected target cell. Thus, a theory combining the observations made here with published data on the time-point of the infectivity block of vif-defective virions would be that the incorporation of this essential cytoskeletal component occurs only when virus is produced in permissive cells or in Vif-expressing non-permissive cells. Thus the function of Vif could be to influence the cytoskeleton in the producer cell such that the required cytoskeletal component becomes available for incorporation. While such speculations have no experimental support, they do at least provide a framework for future studies.
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Acknowledgments |
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Footnotes |
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Received 2 October 2000;
accepted 24 November 2000.