Institute of Medical Technology and Tampere University Hospital, FIN-33014 University of Tampere, Finland1
Author for correspondence: Kalle Saksela. Fax +358 3 215 8597. e-mail kalle.saksela{at}uta.fi
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Abstract |
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Introduction |
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The ability of Nef to modulate signal transduction pathways, as well as many other of its cellular functions, such as enhancement of HIV-1 replication and down-modulation of cell-surface expression of human leukocyte antigen class I (HLA-I), have been shown to depend on its SH3-binding (PxxP) motif (Piguet et al., 1999 ; Renkema & Saksela, 2000
; Geyer et al., 2001
). Notably, AIDS-like pathology observed in CD4HIV-1 Nef transgenic mice (Hanna et al., 1998
) was found to be absent in mice similarly expressing a Nef transgene with a disrupted PxxP motif (Hanna et al., 2001
). The relevant SH3 proteins involved in mediating these different functions of Nef remain incompletely characterized. SH3 domain-containing cellular binding partners of HIV-1 Nef reported to date include the Src family tyrosine kinases Hck, Lyn, Fyn and Lck and the adapter protein/guanine exchange factor Vav (Renkema & Saksela, 2000
). Breeding of CD4HIV-1 Nef transgenic mice into an Hck -/- background has been reported and was found to delay, although not abolish, HIV-1 Nef-induced pathogenesis in this animal model (Hanna et al., 2001
).
Binding of HIV-1 Nef to the Hck SH3 domain is relatively strong (KD 0·2 µM), representing one of the tightest known naturally occurring SH3ligand complexes (Lee et al., 1995 ). Detailed studies on this interaction have shown that most of the binding affinity in this case, and presumably in many other tight SH3ligand complexes, is due to molecular contacts involving discontinuously positioned amino acid residues outside the primary SH3 domain docking site formed by the PxxP motif region (Lee et al., 1995
; Manninen et al., 1998
). On the SH3 surface, the matching contacts are provided by a structure known as the RT loop, a region that represents maximal sequence diversity among different SH3 domains (Lee et al., 1995
, 1996
).
Despite the overall similarity of HIV-1 and SIV/HIV-2 Nef proteins, the role of SH3 binding in mediating the cellular functions of SIV/HIV-2 Nef remains unclear. Although SIV Nef and HIV-1 Nef share the ability to interact with Src family tyrosine kinases, in the case of SIV Nef these interactions appear to be predominantly SH3-independent (Greenway et al., 1999 ). Moreover, while both HIV-1 and SIV Nef can down-modulate HLA-I cell-surface expression, only HIV-1 Nef is dependent on its PxxP motif in this function (Swigut et al., 2000
). Experimental infection of macaques with isogenic SIV strains carrying wild-type or PxxP-mutated viruses has yielded conflicting data. P. A. Luciw and colleagues concluded that this motif is critical for pathogenesis of simian AIDS (Khan et al., 1998
). However, studies by F. Kirchhoff and co-workers have indicated that although mutations introduced to disrupt the SIV Nef PxxP motif are under pressure to revert, such reversions typically occur late during disease progression (Lang et al., 1997
; Carl et al., 2000
), suggesting that SH3 binding may play only a limited role in the in vivo functions of SIV Nef.
While many SH3 domains, such Hck and Fyn, show some affinity for SIV Nef (Greenway et al., 1999 ; Collette et al., 2000
), so far no SH3 domain has been identified that will bind SIV Nef with high affinity. This is unlikely to be caused by a failure to test binding of SIV Nef with the relevant simian orthologues of human SH3-binding proteins that interact with HIV-1 Nef. For example, Picard et al. (2002)
have recently cloned simian Hck, which was found to be 97·8% identical overall and 100% identical in its SH3 region with human Hck; nevertheless, it was not any better than its human counterpart in binding to SIV Nef. Moreover, protein engineering studies by Collette et al. (2000)
have shown that if the SH3 RT loop-accommodating amino acid residues from HIV-1 Nef are introduced into the corresponding positions in SIV Nef, binding to Src family SH3 domains can be significantly improved. Thus, the observed low SH3-binding capacity of SIV Nef might be due to poorly matching RT loop regions in the Src family and other SH3 domains tested so far. Nevertheless, in light of the studies introduced above, the question arises as to whether SIV Nef is at all competent for high-affinity SH3 binding.
In this study we have addressed this problem by taking advantage of a strategy that we have previously developed to generate artificial SH3 domains showing strong binding to HIV-1 Nef (Hiipakka et al., 1999 ) for use as intracellular inhibitors of HIV-1 Nef (Hiipakka et al., 2001
). This strategy is based on phage-display libraries presenting Hck-derived modified SH3 domains that carry random sequences in place of six amino acid residues forming the non-conserved region of the RT loop of Hck-SH3. Here we report successful development of novel SH3 domains that bind tightly to SIVmac Nef. In addition to providing further support for the applicability of our strategy for generating SH3 domains with tailored binding properties, these results clearly show that SIVmac Nef has a capacity for high-affinity SH3 binding.
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Methods |
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Phage library construction and affinity selection.
The multivalent library of artificial Hck-derived SH3 domains (RRT-SH3s) was created in pG8H6 using a degenerate PCR primer, as previously described for pCANTAB-5E (Hiipakka et al., 1999 ). In addition, a TAG stop codon was inserted in front of the RRT-SH3 sequence to reduce expression of the fusion protein using a supE host (TG1) in which amber termination is incompletely suppressed. A total of 4·2x108 individual recombinant colonies were obtained by electroporation into TG1 cells and used to produce recombinant phage preparations via infection with the M13KO7 helper phage (Hiipakka et al., 1999
). Sublibraries of approximately 500000 recombinant clones consisting of PCR-amplified RRT-SH3 inserts initially selected using the multivalent system were inserted into pCANTAB-5EP, as previously described (Hiipakka et al., 1999
). Affinity selection of clones from the multi- and monovalent libraries was also carried out as described previously (Hiipakka et al., 1999
).
Cell culture, transfections and reporter gene assays.
293T and Jurkat (JE-6) cells were obtained from ATCC and cultured using standard procedures. 293T cells were transfected using the Lipofectamine reagent (Invitrogen) and the JE-6 cells using DMRIE-C transfection reagent (Invitrogen), as previously described (Manninen et al., 2000 ). The reagents and protocols used for the luciferase assays have also been described previously (Hiipakka et al., 2001
).
In vitro binding experiments.
Expression and purification of bacterial GSTSH3 fusion proteins was carried out as previously described (Hiipakka et al., 1999 ). SIVmac and HIV-1 Nef protein-containing lysates were produced by transient transfection of 293T cells. Approximately 2x107 transfected cells were collected 48 h after transfection and lysed in lysis buffer (1% NP-40, 150 mM NaCl, 20 mM TrisHCl, pH 7·4, 0·5% sodium deoxycholate, 50 mM NaF, 1 mM PMSF and 10 µg/ml aprotinin). The lysates were serially diluted (fourfold dilutions) into a similarly prepared lysate of untransfected 293T cells. After removal of small aliquots to monitor their Nef content, 200 µl of these dilutions were incubated for 2 h at 4 °C with glutathioneSepharose 4B beads (Amersham Biosciences) coated with 8 µg plain GST or GSTSH3 proteins. After the incubation, the beads were washed three times with PBS and boiled in SDSPAGE sample buffer and analysed by SDSPAGE to examine the amount of GSTSH3 and associated Nef proteins bound to the beads following immunoblotting with anti-HA and anti-Myc antibodies, respectively, and subsequent detection by the enhanced chemilumiscence (ECL) system, as suggested by the manufacturer (Amersham Biosciences).
In vivo binding experiments.
Forty-eight hours after transfection with expression vectors for the GSTSH3 and Nef proteins, 293T cells were harvested, washed with PBS and lysed into 50 mM HEPES, pH 7·4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1·5 mM MgCl2, 10 mM NaF, 1 mM Na-orthovanadate, 1 mM PMSF and 10 µg/ml aprotinin. Lysates (1 mg total cellular protein) were incubated with glutathioneSepharose 4B beads for 4 h at 4 °C. After three washes with PBS, the proteins associated with the beads were examined by SDSPAGE followed by immunoblotting with anti-Myc and anti-HA antibodies and detection by ECL. Thirty µg from each lysate was removed before addition of the beads to verify uniform expression of the transfected GSTSH3 and Nef proteins.
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Results and Discussion |
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Selection of the sequences EGWWG and DGWWG from the library of Hck-derived SH3 domains designed to carry six randomized RT loop residues suggested a strong selective advantage for a shorter RT loop in binding to SIVmac Nef. Possibly the original library contained a sufficiently large collection of clones with five-residue substitutions from which these sequences were selected, or, alternatively, they may have evolved via a deletion during the phage selection/re-amplification process. In either case, this appears to be specific for SIVmac Nef, since the length of six modified RT loop residues has been strictly conserved among the large number of RRT-SH3 domains binding with high affinity to HIV-1 Nef that we have selected previously (Hiipakka et al., 1999 ). It should be noted that the RT loop region in various natural SH3 domains is not fixed to six residues but, in addition to sequence diversity, also shows considerable variation in length.
To characterize further the ability of the phage display-selected SH3 clones E and D to bind to SIVmac Nef, they were expressed as GST fusion proteins in E. coli and used to precipitate lysates of human 293T cells transfected with an SIVmac Nef expression vector. Native Hck-SH3 as well as Fyn-SH3, which has been implicated as the natural SH3 domain with most affinity for SIV Nef (Collette et al., 2000 ), were similarly expressed as GST fusion proteins and tested in parallel with E and D. For comparison, HIV-1 Nef-transfected 293T cell lysates were also used in order to include the HIV-1 Nef/Hck-SH3 interaction with the known affinity of KD 250 nM (Lee et al., 1995
) as an internal control.
Equal amounts of the GSTSH3 fusion proteins of Hck, Fyn, D and E were coated on glutathioneSepharose beads (Fig. 1a) and incubated with Nef-expressing lysates serially diluted with lysates of untransfected 293T cells (Fig. 1b
). The amount of Nef proteins bound to the different GSTSH3 proteins after washing of the beads is shown in Fig. 1(c)
. The SIVmac Nef-selected clones E and D precipitated readily detectable amounts of SIVmac Nef, even from the 64-fold diluted lysate (Fig. 1c
). In contrast, the affinity of Hck-SH3 and Fyn-SH3 was too weak to bring down enough SIVmac Nef to be visible, even in very long exposures of the immunoblots (
Fig. 1c and data not shown). Thus, we concluded that the EAIHHE to E/DGWWG modification in the Hck-SH3 RT loop was associated with a very significant, apparently at least two orders of magnitude, improvement in binding to SIVmac Nef. Unlike SIVmac Nef, HIV-1 Nef could be precipitated under the same experimental conditions by the natural Hck-SH3, as expected based on the known affinity of this interaction (KD 250 nM). Although the assay was not strictly quantitative, the ability of E and D to precipitate from a 64-fold diluted lysate an amount of SIVmac Nef comparable with HIV-1 Nef precipitated by Hck-SH3 from an undiluted lysate suggested that the affinity of E and D binding to SIVmac Nef was probably in the low nanomolar range.
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Acknowledgments |
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References |
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Received 5 July 2002;
accepted 15 August 2002.