Experimental Pathology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel1
Department of Organic Chemistry, The Hebrew University, Givat Ram, Jerusalem 91904, Israel2
Author for correspondence: Moshe Kotler. Fax +972 2 6758190. e-mail mkotler{at}cc.huji.ac.il
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Abstract |
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HIV-1, like other lentiviruses, expresses six auxiliary genes, tat, rev, vif, vpr, vpu and nef, in addition to the canonical retroviral genes gag, pol and env. Vif (virion infectivity factor) is synthesized in the late phase of infection (Garrett et al., 1991 ) and is predominantly cell associated (Camaur & Trono, 1996
; Goncalves et al., 1994
). Recent reports demonstrated that Vif is associated with viral core structures (Liu et al., 1995
) and specifically packed into HIV particles either by interaction with viral genomic RNA (Khan et al., 2001
) or by interaction with Gag and GagPol precursors (Bardy et al., 2001
). Sova et al. (2001)
demonstrated that Vif incorporates mainly into particles containing unprocessed Gag molecules but is largely absent from mature virions.
There is a wide consensus that Vif is absolutely essential for production of infectious particles in HIV-infected peripheral blood lymphocytes (PBLs) and macrophages (Aldrovandi & Zack, 1996 ; Chowdhury et al., 1996
; Courcoul et al., 1995
; Gabuzda et al., 1992
; Sakai et al., 1993
; Strebel et al., 1987
; von Schwedler et al., 1993
). While the phenotype of the vif gene is quite clear, the mechanism and location of Vif activity is still controversial. Simm et al. (1995)
showed abnormally processed viral proteins in PBLs infected with
vif HIV-1. However, other reports demonstrated that the protein composition of
vif and wild-type HIV-1 particles released from restrictive cells is indistinguishable (Bouyac et al., 1997b
; Fouchier et al., 1996
; Khan et al., 2001
). A direct effect of Vif on autoprocessing of viral precursors was shown in bacterial cells and cell-free systems (Kotler et al., 1997
). In addition, Vif peptides derived from aa 30 to 65 and 78 to 98 bind PR and inhibit both PR-mediated hydrolysis of synthetic peptides in vitro and production of infectious virus from HIV-1-infected human cells (Baraz et al., 1998
; Friedler et al., 1999
; Potash et al., 1998
). Inhibition of the autoprocessing of viral precursors due to interaction with Vif was also shown by Bardy et al. (2001)
. The experiments described in this report were addressed to determine whether Vif binds PR and to map the interaction sites of Vif with PR.
ELISA microwells were coated for 18 h at 4 °C with 200 µl containing 0·2 µM BSA, thyroglobulin or recombinant HIV-1 Vif, purified as described by Yang et al. (1996) . The wells were aspirated and blocked with low-fat milk for 1 h at room temperature. Following washes with PBS containing 0·05% Tween 20, increasing concentrations of HIV-1 and avian sarcoma leukaemia virus (ASLV) PRs, purified as described previously (Baraz et al., 1998
; Kotler et al., 1988
), in 200 µl of 0·1 M NaCl in 50 mM sodium phosphate buffer (pH 7·4) were added to each well and incubated for 2 h at room temperature. The amount of PRs that specifically bound to Vif was determined by anti-HIV-1 or anti-ASLV PR polyclonal sera. Fig. 1(A)
shows that the PR of HIV-1 but not that of ASLV bound to Vif in a dose-dependent manner. There was no binding of PRs to thyroglobulin or BSA, which were used as negative controls (Fig. 1A
). In a reciprocal experiment (Fig. 1B
), the ELISA microwells were coated with HIV-1 wild-type PR and active site-mutated PRD25I, ASLV PR, thyroglobulin or BSA, and increased amounts of Vif protein were added to the plate. The Vif protein bound to either PRs or control proteins was quantified by an anti-HIV-1 Vif polyclonal serum. Wild-type and mutated HIV-1 PRs bound equal amounts of Vif, indicating that alteration of catalytic residues in the active site of PR does not affect the binding of Vif to the enzyme. ASLV PR bound only small amounts of Vif compared to HIV-1 PR, while thyroglobulin- or BSA-coated microwells did not bind Vif, demonstrating the specificity of Vif binding to HIV-1 PR. The binding of Vif to PR was confirmed further by using CNBr-activated beads and immunoprecipitation with specific anti-PR or anti-Vif sera (data not shown).
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In order to identify the regions in the whole Vif molecule that interact with PR, we have determined which of the Vif-derived synthetic peptides interfere with the binding of PR to Vif (Baraz et al., 1998 ). Purified Vif was coupled to the ELISA microwells and HIV-1 PR was then added, following preincubation with Vif7892 and Vif8898 peptides, which were found previously to be PR inhibitors. As a control, we used a peptide derived from the C terminus of Vif (Vif170191), which is essential for Vif function (Goncalves et al., 1995
) and for binding of Vif to Pr55Gag (Bouyac et al., 1997a
; Huvent et al., 1998
). The results of this experiment clearly show that preincubation of PR with Vif8898 and Vif7892 prevents the binding of PR to Vif in a dose-dependent manner. It was demonstrated that a concentration of 32 µM of the Vif8898 peptide is sufficient to reduce the binding of PR to Vif by more than 50%. Vif7892 also blocks binding but not as efficiently as Vif8898. However, Vif1729 and Vif170191 do not interfere with Vif to PR binding (Fig. 3
). Taken together, these results strongly suggest that the aa 7898 region in Vif interacts with the N terminus of HIV-1 PR (aa 19).
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Consensus sequence: 94T V/I S/T Y D/E/N L/Y/W99
Vif: 85V S I E W89
It is unknown yet whether the interaction site of Vif bound to PR is limited solely to PR19 and Vif7898. Others and we have demonstrated that an additional region in the Vif molecule, namely aa 3065, is involved in PR inhibition and that peptides derived from both regions inhibit PR (Baraz et al., 1998 ; Friedler et al., 1999
; Potash et al., 1998
). Thus, it is plausible that the interaction of Vif with PR involves two discontinuous regions of Vif and that the mechanism of inhibition is more complex.
In accordance with our results, Bardy et al. (2001) demonstrated that Vif inhibits the PR-mediated processing of Gag precursors in vitro. Vif is encapsidated more efficiently into virus-like particles containing GagPol rather than Gag precursors. However, these authors could not find significant interaction between Vif and PR in vivo or in vitro. This discrepancy can be explained by the differences in the applied systems.
The time window and the location of Vif activity are not yet known. Previously, we suggested that Vif is active at the late phase of infection and is responsible for the delay of PR activation when it is part of the GagPol precursor. This lag period may prevent PR from digesting cellular proteins, assures the migration of viral structural components to the site of virion assembly at an appropriate molar ratio and allows the organization of precursors in a pattern required for maturation. However, others and we were unable to show that the presence or absence of Vif caused a measurable effect on the protein composition of viruses produced by restrictive or permissive host cells (Bouyac et al., 1997b ; Fouchier et al., 1996
; Khan et al., 2001
). Now that it is clear that Vif and PR are present in the mature virions (Khan et al., 2001
) and that PR is also present in the preintegration complex (Karageorgos et al., 1993
), it is plausible that Vif regulates PR in the virion and/or at the early stage of infection. The notion that
vif virions released from restrictive cells exhibit less stable cores than wild-type cores (Ohagen & Gabuzda, 2000
) supports this suggestion. Experiments designed to assess the relevance of this model in HIV-1-infected cells are now being carried out in our laboratory.
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Acknowledgments |
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References |
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Received 28 January 2002;
accepted 3 May 2002.
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