1 Division of Infectious Diseases, University of Lausanne, Lausanne, Switzerland
2 Institute of Microbiology, University of Lausanne, Lausanne, Switzerland
3 University Hospital, and Center of Electron Microscopy, University of Lausanne, Lausanne, Switzerland
Correspondence
Amalio Telenti
amalio.telenti{at}hospvd.ch
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ABSTRACT |
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INTRODUCTION |
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The identification of extensive deletion and insertion polymorphisms in the central protein region, including deletions of up to six amino acids and duplication of the conserved motif K27Q28E29 (KQE) (Peters et al., 2001), led us to analyse the role of this region in the virus life cycle. For this purpose, we constructed HIV-1 molecular clones carrying: (i) p6 deletions observed in circulating virions in HIV-1-infected individuals; (ii) experimental deletions that spanned up to 20 amino acids; and (iii) the KQE duplication.
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METHODS |
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Virus culture conditions.
Peripheral blood lymphocytes (PBLs) were cultured in RPMI 1640 supplemented with Glutamax (2 mM) (Gibco-BRL Life Technologies), gentamicin (50 µg ml-1), foetal calf serum (FCS; 20 %, v/v) and IL-2 (10 U ml-1). 293 T cells were maintained in RPMI 1640, Glutamax and gentamicin, with the addition of 10 % FCS. HeLa, COS-7 and GHOST cells (stably transduced with chemokine receptor CXCR4 and with the green fluorescence protein (GFP) linked to the HIV-1 long terminal repeat; obtained through the NIH AIDS Research and Reference Reagent Program from Drs D. Littman and V. K. Ramani) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with Glutamax (2 mM), gentamicin (50 µg ml-1) and 10 % FCS. For GHOST cells, selective medium also contained puromycin (1 µg ml-1; Sigma), hygromycin (100 µg ml-1; Gibco-BRL Life Technologies) and G-418 (500 µg ml-1; Gibco-BRL Life Technologies).
Recombinant clones.
Deletions of two to six amino acids observed in vivo, the duplication K27Q28E29 and a number of experimental deletions of up to 20 residues in p6 were introduced into pNL4-3 by site-directed mutagenesis (Stratagene). The small deletions S14E19,
G18T21,
S14R16,
F17G18,
T21T23,
P30I31,
E12E13 and
K27E29 were introduced by a one-step single mutagenesis reaction. The larger deletion
S14T23 used
S14E19 as template,
S14I31 used
S14T23 and
E12I31 was built on
S14I31. All constructs were confirmed by sequencing. Viruses were obtained by HeLa cell transfection (Geneporter transfection reagent; Axon Laboratories).
Virion infectivity.
GHOST/CXCR4 cells in 48-well plates (3x104 cells per well) were infected in triplicate with recombinant virus (3000 pg p24 antigen) as described earlier (Bleiber et al., 2001). The infectious titre was determined by FACS analysis as the proportion of GFP-positive cells.
Replication kinetics.
PBLs (3x106 cells) were infected with virus (1500 pg p24 antigen) in 1 ml of supplemented RPMI 1640 for 2 h. Residual inoculum was removed by washing. Aliquots of culture supernatant were collected to monitor virus replication using an HIV-1 p24 antigen ELISA (HIV AG-1 Monoclonal; Abbott).
Protease autoprocessing.
Autocleavage efficiency of viral protease was assessed by expression of a nucleocapsid-transframe-p6Pol-protease (NC-TF-p6Pol-PR) polyprotein in a transcription and translation TNT T7 rabbit reticulocyte lysate (Promega), following a published protocol (Peters et al., 2001). Deletions and duplication in p6 were introduced into pET3/NC-TF-p6Pol-PR by site-directed mutagenesis. Constructs were confirmed by sequencing. Samples of the TNT reaction were resolved by 15 % SDS-PAGE and processing efficiency evaluated by Instant Imager (Packard Instruments). Results were expressed as the ratio of unprocessed precursor (NC-TF-p6Pol-PRx100) to total protein synthesis (precursor+NC-TF-p6Pol+PR) after a 30 min reaction.
Protein analysis.
Particle- and cell-associated viral proteins were analysed as previously described (Bleiber et al., 2001). Briefly, transfected COS-7 cells in a 100 mm dish were metabolically labelled with 5 ml DMEM (methionine/cysteine-free, 10 % dialysed FCS) containing 40 µCi [35S]methionine/cysteine ml-1 ([35S]EasyTag EXPRESS; NEN Life Science Products) for 1217 h. Virions in the culture supernatant were concentrated by ultracentrifugation (90 min, 10 0000 g, 4 °C) and lysed in radioimmunoprecipitation buffer (1 % NP-40, 0·5 % deoxycholic acid, 0·9 % SDS, 2 mM EDTA, 150 mM NaCl, 50 mM Tris/HCl, pH 8·0), supplemented with complete protease inhibitor cocktail (Roche Diagnostics). Cells were washed, lysed in radioimmunoprecipitation assay buffer and cellular debris was removed by centrifugation. Particle- and cell-associated viral proteins were immunoprecipitated from lysates using anti-HIV human immunoglobulin (NIH AIDS Research and Reference Reagent Program) and protein ASepharose CL-4B beads (Amersham Pharmacia Biotech) and separated by SDS-PAGE (515 % gradient gel). Radioactivity content was quantified using Instant Imager.
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RESULTS |
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Late virus cycle phenotype of p6 clones
p6Pol has been implicated in the regulation of viral protease autoactivation (Louis et al., 1999; Paulus et al., 1999
). As changes introduced in p6Gag may also modify p6Pol, we assessed the rate of protease autocleavage for the various constructs using an in vitro transcription and translation assay (Fig. 2
). A range of autocleavage activity was observed with respect to the wild-type (100 %), with
F17G18 exhibiting the highest (mean±SEM, 150±1 %, P=0·02) and
E12E13 the lowest (73±5 %, P=0·02) activity. Particularly relevant was the observation that the clone with the most profound replication impairment and the largest deletion (
R20D39, 42 % of p6Pol) did not present a deficit in autoprocessing (Fig. 1C
). The inter-assay coefficient of variation was 7·5 %. Overall, only clone
E12E13 presented deficits in infectivity, replication and protease autocleavage (Fig. 2
).
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DISCUSSION |
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In vivo, viruses with deletions of up to six amino acids are not associated with unusual patterns of disease progression in patients infected with those strains. As T helper and CTL recognition epitopes have been described in p6 (HIV molecular immunology database, http://hiv-web-lanl.gov), one possibility is that deletion in this region contributes to escape from immune surveillance. In vitro, no deleterious phenotype of the corresponding molecular clones was observed. It is conceivable that there is a fitness cost associated with such deletions and that it could have been better estimated by competitive replication kinetics. However, testing of sequence variation outside its original genetic context makes interpretation of small differences in replication problematic. Surprisingly, more extensive deletions in the region between S14 and I31 or of the conserved KQE motif polymorphisms that are never observed in vivo had little effect on virus propagation in vitro. The central region of p6 could be relevant in determining the level of phosphorylation of p6Gag (Muller et al., 2002). However, analysis of the phosphorylation status of various clones did not yield reliable quantitative data to assess the relevance of this phenomenon in the retention of this region in nature. The significance of the infectivity deficit of natural clone
P30I31 in the absence of a replication deficit is unclear. We did not test for a potential role of central p6Gag region on Vpr incorporation, as no such impairment has been identified in previous analyses (Lu et al., 1995
; Kondo & Gottlinger, 1996
).
We assessed the effect of KQE domain deletion or duplication on virus replication. The lysine residue is the substrate for ubiquitin modification. Ubiquitin is attached by an isopeptide linkage between the C-terminal carboxyl group and the -amino acid group of lysine. An estimated 2 % of the mature p6Gag of HIV-1 is monoubiquitinated (Ott et al., 1998
) and there is a relationship between the level of free ubiquitin in the infected cell and the efficiency of virus release (Schubert et al., 2000
). We have observed duplication of this motif in 4·7 % of clinical strains, a variation not associated with a specific in vitro or in vivo phenotype. KQE duplication could lead to changes in the interaction with the ubiquitination machinery and therefore the ubiquitination pattern of Gag. In contrast, KQE is never deleted in nature, although its absence in molecular clones was not deleterious in vitro. This is consistent with previous work indicating that substitution of the two lysine residues in p6Gag that are substrates for ubiquitin modification (K27 and K33) had no apparent effect on virus assembly or release (Ott et al., 2000
). It has been proposed that when the preferred sites of ubiquitination are not available, other lysine residues may be used (Hou et al., 1994
; Vogt, 2000
; VerPlank et al., 2001
).
Only extension of the deletion to include amino acid residues E12 and E13 led to markedly diminished replication. The role of the acidic motif (E)E is not well established. This motif is present as AE, ME, VE or LE following the L domain PTAP among various lentiviruses: simian immunodeficiency virus, feline immunodeficiency virus and caprine arthritis encephalitis virus. Rarely, EE, ME or AD can follow a PPPY or a double PPPY/PTAP late domain characteristic of various non-lentiviral retroviruses or some enveloped RNA viruses: vesicular stomatitis virus, Ebola and Marburg virus. The human T cell leukaemia virus 1 has a VE motif within a complex PPPYVEPTAP L domain.
Work by Garrus et al. (2001) indicates that substitution of E13 for alanine resulted in a modest reduction in binding affinity of Tsg101. Work by Martin-Serrano et al. (2001)
described a GG for EE substitution leading to a minor decrease in Tsg101Gag interaction and virion production and a moderately diminished viral infectivity. In our work, deletion of EE diminished viral infectivity and replication without evidence of major changes in protein maturation. Electron microscopy did not reveal changes in budding reminiscent of those reported for mutants lacking L-domain function a failure of particles to pinch off. Demirov et al. (2002)
indicated that premature termination at E12 markedly disrupts particle production, whereas truncations immediately downstream have no effect on virus production yet impaired virus replication, a phenotype reminiscent of
E12E13. Similarly, mutations in p9 of equine infectious anaemia virus have been reported that have no apparent effect on virus production yet impair virus replication (Chen et al., 2001
).
Thus, this paper highlights the fact that regions of the p6 gag open reading frame and the overlapping region in pol can be deleted without major consequences for virus replication in vitro or in vivo. The tolerance for polymorphism in this region may reflect a role of the central region of p6 as a linker structure that accommodates the constraints of the overlapping gag/pol open reading frames. Finally, this study underlines the need to investigate the conserved motif KQE using new approaches, given the questions that remain about its precise role in virus physiology.
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ACKNOWLEDGEMENTS |
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Received 15 August 2003;
accepted 23 December 2003.