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Institute of Medical Microbiology and Hygiene, Molecular Microbiology and Gene Therapy, University of Regensburg, 93053 Regensburg, Germany
Correspondence
Ralf Wagner
ralf.wagner{at}klinik.uni-regensburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-hairpin/helix tertiary structure that is stabilized by a buried salt bridge forming between the positively charged primary imino group of a proline residue and the negatively charged carboxyl group of a conserved aspartate. In order to evaluate the contribution of either side-chain length or charge to the formation of infectious virus capsids, aspartate 183 was substituted for glutamate or asparagine in the viral context. It was found that both modifications abolished infectivity of the corresponding viruses in permissive T lymphocytes, although none of particle assembly and release, RNA encapsidation, incorporation of Env glycoproteins and packaging of cyclophilin A were impaired. However, whereas biophysical analyses of mutant virions yielded wild-type-like particle sizes and densities, electron microscopy revealed aberrant core morphologies that could be attributed to either increased (D183N) or reduced (D183E) capsid stability. Although the two amino acid substitutions had opposing effects upon core stability, both mutants were shown to exhibit a severe block in early reverse transcription, underscoring the importance of correct salt-bridge formation for early steps of virus replication. A table showing primers used to amplify intermediates of reverse transcription is available in JGV Online.
These authors contributed equally to this work. 
Present address: Abbott Ireland, Diagnostics Division, Finisklin Business Park, Sligo, Ireland.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Göttlinger et al., 1989; Mergener et al., 1992; Wagner et al., 1992; Wills & Craven, 1991), which finally bud from infected cells as immature, non-infectious particles (Fuller et al., 1997; Nermut et al., 1998). Concurrent with the release of virus particles, the Gag and Gag–Pol precursors are processed proteolytically by the virus-encoded protease to generate the mature structural components p17MA, p24CA, p7NC and p6gag, as well as the viral enzymes protease (PR), reverse transcriptase (RT) and integrase (IN) (reviewed by Vogt, 1996). Immediately following processing, capsid proteins (CA) form a conical shell (Ganser et al., 1999) surrounding the electron-dense ribonucleoprotein complex that harbours two copies of genomic RNA associated with NC, as well as the enzymes RT and IN.
Whereas immature particles of all retroviruses share a widely spherical morphology, the mature, infectious virions are characterized individually by the shape of the CA core structure, which can adopt spherical (most retroviruses), tubular and cylindrical (beta- and type D retroviruses) or conical (lentiviruses, including HIV-1) forms (Mayo et al., 2003). However, three-dimensional CA structures of different genera exhibit a similar 
-helical topology that is highly conserved among retroviruses (Campos-Olivas & Summers, 1999).
Crystallographic and nuclear magnetic resonance (NMR) analyses of mature HIV-1 CA proteins revealed two independently folded domains connected by a flexible linker peptide (Gamble et al., 1997; Gitti et al., 1996; Momany et al., 1996) (Fig. 1). The C-terminal four-helix globular domain contains the major homology region, which is highly conserved among all retroviruses and has been shown to be essential to drive assembly and particle production (Dorfman et al., 1994; Mammano et al., 1994; von Poblotzki et al., 1993). The amino-terminal domain, referred to as NTD, is needed for the morphogenesis of the mature, condensed, cone-shaped core (Dorfman et al., 1994; Tang et al., 2003; von Schwedler et al., 1998), and mutational analyses have suggested implications of the NTD for early steps of the virus life cycle (Reicin et al., 1996; Trono et al., 1989; Wang & Barklis, 1993). High-resolution studies of the NTD have documented a structure composed of seven -helices, two -hairpins and an extended proline-rich loop, which is bound by the cellular peptidyl–prolyl cis–trans isomerase cyclophilin A (CypA) (Gamble et al., 1996). This cellular chaperone is packaged into budding particles, thereby enhancing virus infectivity (Franke et al., 1994; Thali et al., 1994).
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-hairpin/helix structure of the NTD results from substantial conformational rearrangements that coincide with proteolytic release of CA, and was shown to be stabilized by a buried salt bridge, thereby generating a new CA–CA interface (Gitti et al., 1996). Formation of this salt bridge is induced by a positively charged N-terminal proline residue (Pro133) that contacts – following cleavage and at a distance of 4.0 Å (0.4 nm) – the negatively charged side chain of an aspartate residue (Asp183) within helix III (Fig. 1
). Several lines of evidence suggest that the Pro residue found at the N terminus of all retrovirus CA proteins is critical for the generation of mature and infectious virus cores (Fitzon et al., 2000; Forshey et al., 2002; Rulli et al., 2006). However, retroviruses seem to choose different counterparts for proline in salt-bridge formation. Whereas HIV-1, human T-cell leukemia virus (HTLV-1) and Murine leukemia virus (MLV) provide an aspartate, glutamate is used in Bovine immunodeficiency virus (BIV) and Equine infectious anemia virus (EIAV) (von Schwedler et al., 1998). Here, we report the effects of two different, although related, amino acid substitutions of the highly conserved Asp183 in HIV-1 p24CA upon diverse stages of the virus life cycle. Whereas mutation of Asp to Glu had no influence on virus release, introduction of Asn clearly diminished particle production. However, both mutations abolished virus infectivity in permissive T-cell lines and caused aberrant core morphologies. Despite wild-type (WT)-like virion sizes and composition, corresponding mutations had opposite effects on capsid stability that, in both cases, abrogated the capability to carry out DNA synthesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), comprising the Gag-encoding region of the infectious provirus clone HX10 (Ratner et al., 1987; GenBank accession no. M15654
[GenBank]
), was used as template to generate HIV-1 CA mutations. Mutagenesis was performed by using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, along with primers 5'-GGAGCCACCCCACAAAATTTAAACACCATGCTAAACACAGTGGGG-3' and 5'-CTCGGTGGGGTGTTTTAAATTTGTGGTACGATTTGTGTCACCCC-3' to substitute Asp for Asn (D183N), and 5'-GGAGCCACCCCACAAGAATTAAACACCATGCTAAACACAGTGGGG-3' and 5''-CCTCGGTGGGGTGTTCTTAATTTGTGGTACGATTTGTGTCACCCC-3' to introduce Glu (D183E). The plin8Pr55gag constructs carrying the mutated CA regions were digested with ClaI and SpeI to replace the corresponding fragment of HX10. The ClaI–SpeI region of the resulting proviruses was verified by DNA sequencing.
Cell culture, transfections and infections.
The adherent human lung carcinoma cell line H1299 (Mitsudomi et al., 1992) was grown in Dulbecco's modified Eagle's medium containing 10 % fetal bovine serum (FBS), 100 U penicillin ml–1 and 100 µg streptomycin ml–1. For production of recombinant viruses, 106 H1299 cells were plated on 10 cm dishes and transfected 24 h later with 30 µg provirus plasmid DNA by using the Ca3(PO4)2 precipitation technique. Cells and culture supernatants were harvested at 48 or 72 h post-transfection.
HIV-permissive CEM4 and MT-4 T lymphocytes were obtained from the ATCC and were maintained in RPMI 1640 medium supplemented with 10 % FBS, 100 U penicillin ml–1 and 100 µg streptomycin ml–1. To analyse virus replication, 5x106 exponentially growing CEM4 cells were transfected with 10 µg provirus DNA by using DEAE-dextran (200 µg ml–1, 30 min) and DMSO (10 %, 2.5 min). Samples were collected and cultures were diluted with 1 vol. fresh medium every 48 h over a period of 26 days. Productive infections were monitored by quantification of RT activity from culture supernatants.
Cell harvest and particle preparation.
Transfected H1299 cells were washed twice with PBS and pelleted for 10 min at 300 g. Cells were lysed in RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1 % SDS, 1 % (w/v) Nonidet P-40, 0.5 % sodium deoxycholate] containing protease inhibitors for 30 min on ice, and insoluble components were pelleted (30 min, 10 000 g). Total amounts of protein within soluble fractions were determined by using the Bio-Rad protein assay according to the manufacturer's protocol.
Virions in clarified culture medium (10 min, 300 g) were filtered through a 0.45 µm pore-size filter, pelleted through a 2 ml cushion of 20 % sucrose in STE [100 mM NaCl, 50 mM Tris/HCl (pH 7.5), 1 mM EDTA] at 120 000 g for 2 h at 4 °C and resuspended in PBS overnight.
Quantification of virus production.
HIV-1 CA antigen in cell lysates, culture supernatants and particle preparations was quantified by using a HIV-1 p24 core profile ELISA (DuPont) according to the manufacturer's instructions after appropriate dilution of samples. Virus-associated RT activity was determined by using a non-radioactive RT assay (Retrotech) according to the manufacturer's instructions.
Analysis of virus proteins and composition.
To analyse HIV-1-specific antigens, transfected H1299 cells were harvested as described above and 50 µg total protein was separated by SDS-PAGE and transferred to nitrocellulose. Gag proteins were analysed by using p24-specific (Wolf et al., 1990) and p17-specific (Niedrig et al., 1989) monoclonal antibodies (mAbs), and Env glycoproteins were detected with the antibody NEA9305 (Dupont). Packaging of CypA was analysed upon cotransfection of provirus and an NH2-terminally Asp–Tyr–Lys–Asp-flagged pcDNA3–CypA expression construct and was detected by enhanced chemiluminescence (Supersignal Pierce) using a monoclonal Flag-specific antibody (Kodak). Incorporated RNA was quantified by subjecting purified particle preparations normalized for RT activity to a branched-DNA assay (Chiron Diagnostics).
Analysis of particle density and size.
Following quantitative analysis, particle preparations (in 0.5 ml 5 % sucrose in STE) were layered on top of a continuous gradient of 2 ml sucrose layers [10–50 % (w/v) in STE] and subjected to isopycnography at 120 000 g for 20 h (4 °C). Subsequently, 500 µl fractions were collected, refraction indices were calculated and banded particles were analysed for RT activity. To determine particle size, purified particle preparations were layered on top of a step gradient consisting of 5–20 % sucrose and subjected to velocity centrifugation (2 h, 20 000 g), and fractions were analysed as described above.
Analysis of capsid stability.
Detergent treatment of virus particles was performed essentially as described previously (Wiegers et al., 1998). Briefly, clarified supernatants of transfected H1299 cells were bisected and one half was treated with 0.5 % Triton X-100 for 10 min at 37 °C. Both fractions were then centrifuged through a 2 ml cushion of 20 % sucrose in STE (2 h, 120 000 g). Alternatively, clarified supernatants of transfected H1299 cells were centrifuged (2 h at 120 000 g) through step gradients containing a cushion of 20 % sucrose in STE and a 1 ml layer of 10 % sucrose in STE with or without 0.5 % Triton X-100. Pelleted virus was resuspended overnight in PBS and subjected to Western blot analysis. Band intensities were quantified by phosphorimager analysis and background values were subtracted from each band.
Transmission electron microscopy.
After 48 h, H1299 cells transfected with proviruses were fixed for 2 h with 2.5 % glutaraldehyde and 2 % paraformaldehyde in cacodylate buffer [0.13 M sodium cacodylate (pH 7.3), 35 mM sucrose, 4 mM CaCl2] and 20 % FBS. Following intensive washing with cacodylate buffer, cells were fixed with 1 % OsO4 in cacodylate buffer for 2 h. Following six further washings, cells were dehydrated with a graded series of propylenoxide dilutions ranging from 25 to 100 % prior to embedding in Epon resin. Thin sections of 70–80 nm were counterstained with 2 % uranyl acetate and 1 % lead citrate as described by Reynolds (1963). Morphology of virus cores was analysed with a Zeiss-902 electron microscope operating at 80 kV.
Virus production and infection for analysis of early replication.
Virus-containing culture supernatants were harvested 60 h post-transfection as described above. Virus particles were purified by filtration using successively Jumbosep 300-kD (25 min, 2000 g) and Macrosep 1,000-kD (25 min, 3500 g) concentrators (Pall Filtron). To eliminate residual plasmid DNA, virus preparations were treated with 100 U RNase-free DNase I (Roche) in 10 mM MgCl2 for 30 min at 37 °C and analysed for RT activity. Virus preparations were used to infect 7x106 MT-4 cells (m.o.i. 0.1) in 2 ml in the presence of 20 µg polybrene ml–1 in STBS [25 mM Tris/HCl (pH 7.4), 5 mM KCl, 0.7 mM CaCl2, 137 mM NaCl, 0.6 mM Na2HPO4, 0.5 mM MgCl2]. After a 30 min incubation at 4 °C allowing cell attachment, followed by 4 h infection at 37 °C, cells were harvested, washed in PBS and resuspended in fresh medium.
PCR and Southern blot.
Aliquots of 5x105 MT-4 cells were taken at different intervals post-infection, washed to remove extracellular virus and resuspended in 100 µl PCR lysis buffer [50 mM KCl, 10 mM Tris/HCl (pH 8.3), 1.5 mM MgCl2, 0.01 % gelatin, 0.45 % Nonidet P-40, 0.45 % Tween 20] containing fresh proteinase K (60 µg ml–1). After incubation at 55 °C for 5–12 h, samples were heated (5 min at 95 °C) and 3 µl (approx. 1.5x103 cells) was subjected to PCR as described by Bukrinsky et al. (1993). Intermediates of reverse transcription were amplified by using the primers depicted in Supplementary Table S1 (available in JGV Online).
To determine yield and size of the amplified PCR fragments, one-tenth of the PCR sample was separated by gel electrophoresis and transferred to a nylon membrane. Radioactive probes used for hybridization were amplified by nested PCR using specific primers along with [-32P]dATP (50 µCi/1.85 MBq). PCR products were visualized by autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gitti et al., 1996; Momany et al., 1996; von Schwedler et al., 1998). These conformational changes are stabilized by a buried salt bridge between the amino-terminal Pro133 and Asp183 (Fig. 1). Whereas BIV and EIAV contain Glu instead of Asp (von Schwedler et al., 1998), only one exception among HIV-1 strains has been reported, for the isolate VI354 (GenBank accession no. AF076474
[GenBank]
), comprising an Asn at Gag position 183 (Louwagie et al., 1993). However, this variation has not yet been functionally characterized. Thus, in order to evaluate the capacity of these related amino acid residues to drive virus core maturation, Asp183 was replaced either by Glu or Asn within the infectious provirus HX10 (WT). By substituting the negatively charged Asp for its amide derivative Asn (D183N), we intended to evaluate the importance of amino acid charge for the formation of infectious capsids, whereas Glu was introduced (D183E) to determine the influence of side-chain size.
Influence of Asp183 substitutions on virus release
To analyse whether the mutated proviruses were capable of producing virus, H1299 cells were transfected with WT virus or the mutants D183E and D183N. Seventy-two hours post-transfection, virus particles released into culture supernatants were sucrose-purified and analysed for p24 content by ELISA. For determination of intracellular CA amounts, cells were harvested 48 h post-transfection followed by p24 quantification. As demonstrated in Fig. 2, transfections of H1299 cells with the mutant D183E resulted in WT-like cellular CA amounts. However, significantly reduced amounts of CA were detected in cells transfected with the mutant D183N. This effect was even more pronounced when extracellular levels of p24 were compared. Whereas equal amounts of virus-associated p24 were found in supernatants containing WT and D183E particles, no pelletable CA was detected in supernatants from D183N transfections. To clarify further whether these observations were due to differential antibody recognition of modified CA or whether the D183N mutation interfered with expression or stability of the Gag proteins, virus-associated RT activity was determined. As expected, D183E virions yielded WT levels of RT activity, confirming the data obtained in the p24 ELISA. Interestingly, we found substantial RT activity within D183N preparations, suggesting production and release of virus particles, as well as correct packaging and processing of Pol components. Furthermore, this result implies that the D183N mutation induced conformational changes upon extracellular processing of CA, which abrogated recognition by the antibody used in the sandwich ELISA completely. Nevertheless, the D183N mutation appears to affect particle release, as extracellular RT activity was found to represent only 58±20 % of WT levels. These results indicate that the uncharged Asn183 interferes with efficient virus production and correct folding of processed Gag proteins, whereas particle release and conformation-dependent antibody recognition were not influenced by the slightly larger side chain of Glu.
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, Gag expression patterns were nearly identical in all transfectants, with similar levels of unprocessed Pr55gag and p41 MA–CA precursors, mature p24CA and trace amounts of the p25CA–SP1 intermediate. Interestingly, Western blot analysis revealed WT-like Gag amounts in cells expressing the D183N mutant, contrasting with previous ELISA results where Gag expression of the respective mutant appeared to be clearly diminished. These findings suggest that cell-associated Gag proteins were not recognized quantitatively by the mAb used in ELISA, whereas another p24-specific antibody (Wolf et al., 1990) detected Gag proteins readily in Western blot. Using a V3 loop-specific antibody for detection of the virus envelope protein (Env), we found that cell-associated Env expression was not affected by the introduced mutations (Fig. 3a).
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, mutant virions contained WT-like amounts of mature Gag cleavage products CA and MA, whereas no residual Pr55gag precursor or related intermediates were detected. This implied that neither amino acid substitution affected cell-associated or intraviral processing of Gag polyproteins. Moreover, both mutants were capable of packaging properly processed Env glycoproteins (Fig. 3b).
As binding of CypA to CA depends strictly on an exposed CypA-binding loop (Fig. 1), we analysed further the impact of the introduced mutations on CypA binding to exclude lack of CypA incorporation. To detect packaged CypA specifically, H1299 cells were cotransfected with provirus plasmids and a Flag-tagged CypA expression construct. Released virus particles were normalized for RT activity and analysed for CypA incorporation by Western blot (Fig. 3c). By using an anti-Flag antibody for detection, we found similar amounts of CypA packaged into WT and mutant virions. This demonstrates that the corresponding mutations in CA did not affect the conformation of the CypA-binding loop.
To determine further whether NC-mediated packaging of virus RNA was influenced by the precursor-associated CA variants, purified WT and mutant particles were normalized for RT activity and subjected to branched-DNA-based quantification of virus RNA. As specified in Fig. 3(d), none of the mutants showed a reduced capacity to package virus genomic RNA. Together, these data indicate that major structure-related functions of the Gag precursor during late steps of virus assembly and morphogenesis were not affected significantly by mutation of Asp183.
Effect of salt-bridge mutations on virus infectivity
To evaluate the capability of the mutated viruses to replicate in permissive lymphocytes, CEM4 cells were transfected with WT and mutant provirus DNAs, and virus growth was monitored by quantifying extracellular RT activity over a period of 4 weeks (Fig. 4). As expected, WT viruses replicated to high titres, with a peak in virus production at day 8 post-transfection. In contrast, none of the viruses carrying a CA mutation replicated to detectable levels within the observed period. As this assay system is based on exponential virus amplification, single infectious units might be outcompeted over time. Therefore, virus infectivity was additionally determined in a highly sensitive, single-round infection assay. Confirming and extending the results obtained with virus growth curves, both CA mutants turned out to be non-infectious in this assay system (data not shown).
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, virions carrying CA mutations sedimented in the same fractions as WT particles, corresponding to the expected density of 1.16–1.17 g cm–3. Particle preparations were then analysed further by velocity gradients to determine size distributions. Fractions analysed for RT activity did not reveal any differences between WT and mutant viruses, implying that particle size was not affected by the corresponding CA mutations (Fig. 5b).
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; Wiegers et al., 1998). In order to analyse mutation-associated alterations in core stability, WT and mutant particles were pelleted through a sucrose cushion either containing or lacking 0.5 % Triton X-100. This short-term treatment was shown to be moderately destabilizing and thus sufficient to reveal even subtle anomalies in capsid stability. Western blot analysis of the pelleted particles with a p24-specific antibody demonstrated clearly that the amount of CA protein in WT preparations was diminished significantly upon detergent treatment (Fig. 6a, top). Compared with untreated controls, approximately 20–30 % of WT CA remained as a stable complex after centrifugation through a detergent layer, as determined by densitometric calculation (Fig. 6a, bottom). Remarkably, scarcely any residual CA could be detected upon treatment of D183E viruses, suggesting that corresponding cores were particularly sensitive to detergent. In contrast, the amount of pelletable p24 obtained upon treatment of D183N particles did not deviate significantly from that of untreated controls, suggesting a substantial increase in core stability. These results were further confirmed when WT and mutant particles were subjected to detergent treatment prior to centrifugation. The increased stringency of this procedure resulted in a complete disaggregation of D183E, as well as WT, capsids (Fig. 6b), whilst pelletable p24 from D183N virions was only reduced to about 90 % of untreated-control levels. Hence, it follows that the two mutations introduced at Gag position 183 appear to have contrasting effects on capsid stability. Whereas a substitution of Asp for the larger Glu resulted in a considerable loss of core stability, capsids carrying the uncharged Asn exhibit significantly increased stability compared with WT species.
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). D183E particles were similar in size and possessed tubular or partly cone-shaped, occasionally electrolucent capsids resembling WT morphology (Fig. 7d). These data are concordant with previous core-stability analyses. The reduced stability determined for D183E capsids might be explained by the heterogeneous shape of partly aberrant core structures revealed by electron microscopy. In contrast, D183N-derived virions contained larger spherical capsids exhibiting rather amorphous and completely uncondensed core-like structures, which resemble early maturation stages of WT particles (Fig. 7c). As immature capsids have been reported to tolerate higher amounts of detergent than do fully matured viruses, this apparent block in maturation of D183N cores correlates nicely with the striking increase in capsid stability.
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; Wiegers et al., 1998). To provide evidence for this hypothesis, both mutants were analysed for virus cDNA synthesis. For this purpose, particle preparations derived from H1299 transfections were quantified for RT activity and used subsequently to infect MT-4 lymphocytes. Total DNA was isolated from infected cells at different times post-infection and intermediates of virus replication were detected by Southern blot following PCR amplification with HIV-1-specific primers (see Supplementary Table S1, available in JGV Online). Consistent with the loss of infectivity on CEM4 cells, no 2-LTR products could be amplified from MT-4 lymphocytes infected with either D183E or with D183N viruses. This clearly indicates a defect in synthesis or transport of full-length virus DNA to the nucleus (Bukrinsky et al., 1992; Fig. 8, third row). Compared with WT infections, amplification of vif DNA yielded very faint signals for both mutants, suggesting incorrect synthesis of first-strand DNA (Fig. 8, upper row). Together, these data illustrate clearly that any modification of the conserved Asp183 within CA results in a severe defect in reverse transcription, affecting either initiation or synthesis of minus-strand strong-stop DNA, thereby abolishing virus DNA synthesis completely.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and is evocative of zymogen activation in serine proteases, where precursor processing causes the free N terminus to remodel a salt bridge via a conserved aspartate (Sigler et al., 1968).
Within the scope of a previous proline-scanning study, we have reported that an N-terminal Pro133Leu substitution results in virion-associated processing defects and modestly enhanced capsid stability, which abolished full-length virus cDNA synthesis (Fitzon et al., 2000). Likewise, a Pro-to-Gly substitution was shown to have similar effects in MLV (Rulli et al., 2006). Apart from Pro, the functional importance of the conserved Asp residue has also been demonstrated for MLV and HIV-1, where Ala mutations caused aberrant core assembly (von Schwedler et al., 1998). A comparable substitution in HTLV-1, destabilizing the -hairpin completely (Bouamr et al., 2005), was reported to have less dramatic effects in the context of spherical-core formation, where the salt-bridge structure appears to serve a different purpose (Cornilescu et al., 2001).
Although various effects of an Asp-to-Ala mutation within HIV-1 CA on structure–function relations have already been reported, these former approaches did not, however, take into account charge or size of the amino acid side chain (Tang et al., 2001, 2003; von Schwedler et al., 1998). On this account, substitution of Asp183 for Glu or Asn, two residues of similar chemical nature that retain either the negative charge (Glu) or the amino acid backbone (Asn), appeared especially interesting in light of the Dayhoff matrix, according to which, Ala exhibits a very low similarity index (Dayhoff et al., 1978).
As revealed by in vitro virus replication, the D183N mutation resulted in reduced particle release, correlating with lower intracellular Gag levels measured by p24 ELISA. Whereas part of the intracellular CA species was detected by a conformation-dependent antibody in ELISA, the processed extracellular variant failed to be recognized, probably due to structural reorganization upon proteolytic release of the mutant CA. Indeed, a similar effect was observed previously after non-conservative mutation of the corresponding residue in HTLV-1 CA (D54A) (Bouamr et al., 2005). However, virion-associated CA species of D183N and D183E mutants detected in Western blot analysis by using a different p24-specific antibody appeared to be processed properly, notwithstanding former reports about truncated CA species arising from a corresponding Ala mutation (Tang et al., 2001; von Schwedler et al., 1998). This Ala-related phenotype has been ascribed to unfolding of the modified NTD, which becomes accessible to partial proteolytic degradation, illustrating clearly that overall effects of salt-bridge mutations depend critically on the nature of the chosen amino acid.
Further analysis of particle composition revealed WT-like amounts of properly processed Env molecules and normal RNA contents, indicating preservation of Gag-mediated packaging functions for both mutants. Although the formation of the -hairpin within capsid NTD was reported to induce a 2 Å (0.2 nm) displacement of helix VI and a movement of the CypA-binding loop (Tang et al., 2002), neither mutation of Asp183 affected packaging of CypA, implying integrity of the conserved Pro-rich CypA loop. However, both modifications abolished virus infectivity completely in CEM4 lymphocytes. This demonstrates that the Glu residue found at corresponding positions in other lentiviruses is not capable of proper salt-bridge formation in the HIV-1 context, resulting in severe defects in early virus replication.
Further attempts to clarify the loss of infectivity included biophysical examination of mutant particles, which did not deviate in size or density from WT viruses. However, electron-microscopy analysis revealed clear differences between WT and mutant virions in terms of shape and condensation of virus cores. Whereas D183E particle preparations partly contained cone-shaped, electron-dense cores widely resembling WT morphology, D183N virions displayed only spherical structures. A lack of conical core formation was also reported for the former Ala mutation, speculated to derive from the observed Gag-processing defects (Kaplan et al., 1993; Pettit et al., 1994). NMR analysis of this mutant yielded chemical shifts in 51 of all 138 CA backbone amide protons in the -hairpin and its adjacent helices, which impressively demonstrates the broad effects of a single substitution (von Schwedler et al., 1998).
As electron micrographs of the mutated virions revealed aberrant particle morphologies, we analysed stability of the modified capsids following detergent treatment. Indeed, sensitivity towards detergent was altered significantly for both mutants, albeit resulting in contrary effects. Whereas D183N cores exhibited a dramatic increase in stability, we found D183E capsids to be less stable than WT cores. The lack of negative charge in D183N mutants probably prevented stable salt-bridge formation, arresting particles at a stage resembling immature WT cores, which have been shown to be less fragile than mature, infectious cores. In contrast, the charged Glu residue might have less tremendous effects on overall capsid maturation, as indicated by the partly cone-shaped core structures found in D183E preparations. Nevertheless, this functionally related amino acid abolished virus infectivity completely. It is tempting to speculate that the bulky side chain of Glu loosens the tightly folded amino-terminal -hairpin by steric hindrance, finally resulting in less condensed cores.
Finally, both non-infectious phenotypes could be correlated with severe defects in early steps of reverse transcription. Similar effects have also been documented for the Ala substitution (Tang et al., 2001) and a couple of other CA mutations affecting core stability. Forshey et al. (2002) reported that HIV-1 mutants exhibiting either enhanced or diminished core stability share the same block in minus-strand DNA synthesis, which was explained partly by reduced amounts of RT molecules associated with the mutant capsids (Tang et al., 2003). The authors concluded that optimal core stability is critical for the time frame in which uncoating is supposed to occur to guarantee successful reverse transcription.
Together, these data clearly underline the necessity of correct salt-bridge formation for HIV-1 infectivity, where aberrant CA proteins of various morphologies have to pass the final bottleneck of optimal core stability.
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ACKNOWLEDGEMENTS |
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Received 1 February 2006;
accepted 26 September 2006.
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