1 AIDS Reference Laboratory, National Institute of Health, Av. Padre Cruz, 1649-016 Lisbon, Portugal
2 Division of Retrovirology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
Correspondence
Neil Berry
nberry{at}nibsc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Differences in the natural history between HIV-1 and HIV-2 are well documented (Marlink et al., 1994; Whittle et al., 1994
), although the mechanism(s) that governs the maintenance of an attenuated phenotype for extended time-periods in a large proportion of HIV-2-infected individuals is not fully understood. Differences in virus subtype may provide one explanation, in particular of HIV-2 variants that resemble simian immunodeficiency virus (SIVSM) infections, which appear apathogenic (Chen et al., 1997
; Gao et al., 1994
; Wolf et al., 2001
; Yamaguchi et al., 2000
), but these are few in number and distinct from infections due to the more pathogenic subtypes A and B. Isolation of virus from asymptomatic HIV-2-infected individuals is more difficult than for HIV-1 (Schulz et al., 1990
; Simon et al., 1993
); many strains are characterized by low levels of cytopathogenicity and reduced virulence. HIV-2 strains, however, exhibit the capacity to utilize a broad spectrum of co-receptors and do not appear to be restricted in their cell tropism (reviewed by Reeves & Doms, 2002
).
The failure of HIV-2 to develop into a pandemic and the more attenuated phenotype in vivo is correlated with low levels of plasma HIV-2 RNA (Berry et al., 1998, 2002
; Andersson et al., 2000
; Ariyoshi et al., 2000
; Popper et al., 1999
) and when associated with high CD4 counts, low plasma viraemia is predictive of normal survival (Berry et al., 2002
). Conversely, advanced HIV-2 infections and disease are associated frequently with high virus load (Ariyoshi et al., 2000
; Berry et al., 1998
, 2002
), with HIV-2 plasma RNA levels representing an independent predictor of disease progression. Hence, host or viral genetic factors that increase virus replication/virus load are likely to have a direct impact on the pathogenesis of HIV-2. Unlike HIV-1 and SIV, the role of nef in the differing phenotypes of HIV-2 infections has not been studied as extensively.
Nef, an auxiliary protein that is highly conserved among different primate lentiviruses, regulates virus replication and acts as an immune modulator (Collette, 1997). Genetically and structurally, HIV-2 nef is more similar to SIVMAC/SM nef than to HIV-1 nef and is approximately 260 amino acids in length. In vivo studies of SIVMAC in Asian macaques indicate that an intact nef gene is essential for full disease induction and maintenance of high virus load (Kestler et al., 1991
). Virus populations attenuated due to the disruption of nef have been demonstrated in both SIV-infected macaques (Whatmore et al., 1995
) and long-term survivors with HIV-1 (Deacon et al., 1995
; Michael et al., 1995
; Geffin et al., 2000
; Learmont et al., 1999
). However, no consistent relationship between nef disruption and prolonged survival in HIV-1-infected individuals has been shown (Carl et al., 2000a
, b
; Kirchhoff et al., 1999
; Mourich et al., 1999
), suggesting a complex role for nef in the development of human AIDS. Studies of transgenic mice have also indicated a major role for nef in inducing a pathogenic phenotype (Hanna et al., 1998
, 2001
). One previous report based on a mixture of HIV-2 subtypes indicated a higher proportion of truncated nef alleles in asymptomatic HIV-2 infections than expected for HIV-1, an observation that might explain differences in pathogenesis (Switzer et al., 1998
) and which, therefore, warrants further investigation.
Nef accomplishes different functions for virus infectivity and virulence (reviewed by Piguet & Trono, 1999; Fackler & Baur, 2002
). HIV-1 nef downregulates cell surface CD4 expression and probably MHC class I expression (Greenberg et al., 1997
; Mangasarian et al., 1999
), connecting the viral receptor and cellular components by adaptor protein (AP)-forming complexes with clathrin-coated pits (Foti et al., 1997
), which mediate endocytosis (Piguet et al., 1998
). Di-leucine-based motifs in the C-terminal part of nef are important in this process (Craig et al., 1998
). In SIV nef, both tyrosine- and leucine-based motifs are involved in CD4 downmodulation (Bresnahan et al., 1999
). Although AP interactions vary for different virushost interactions among different primate lentiviruses (Greenberg et al., 1997
; Karn et al., 1998
; Lock et al., 1999
), combined data from functional studies indicate the same net effect of nef-induced CD4 downregulation. Other cellular partners such as V-ATPase and
-COP (Piguet et al., 1999
) may also be involved in these complex processes.
Nef augments virus infectivity in vivo by activating target cells through T-cell signalling and signal transduction pathways (Bell et al., 1998; Simmons et al., 2001
; Manninen et al., 1998
; Fackler et al., 2001
; Fackler & Baur, 2002
). Canonical proline-rich (PxxP) regions in nef (Saksela et al., 1995
) mediate interactions with Src homology region 3 (SH3)-binding domains for Src family kinases such as Hck, Lck and Fyn (Cheng et al., 1999
; Lee et al., 1995
; Collette et al., 2000
). In HIV-2 and SIV nef, the core SH3-binding domain is usually represented by a minimal binding consensus motif (P104xxPLR), although this is typically a tetra-proline structure (PxxP)3 in HIV-1.
In this study of HIV-2 infections, we describe in detail HIV-2 nef sequence variability in Portugal and its relation to putative nef function and pathogenesis.
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METHODS |
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Amplifications were performed in 100 mM KCl, 20 mM Tris/HCl (pH 9), 0·1 mM EDTA, 1 mM DTT, 0·5 % Tween 20, 50 % (v/v) glycerol, 1·5 mM MgCl2, 200 µM of each dNTPs (dATP, dTTP, dCTP and dGTP), 300 nM of each internal primer pair and either 2·5 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer) or 2·6 units of Taq/Pwo using the Expand High Fidelity PCR system enzyme mixture (Roche). Primary amplifications were performed as follows: 94 °C for 10 min for 1 cycle and 94 °C for 30 s, 53 °C for 45 s and 72 °C for 1 min 30 sec for 35 cycles. Nested reactions with inner primers were 94 °C for 15 s, 50 °C for 45 s, 72 °C for 1 min 30 s for 35 cycles with a final extension step at 72 °C for 7 min. Products were visualized on 1·75 % agarose gel containing ethidium bromide (0·5 µg ml-1). Aliquots (210 µl) containing 0·6 µg extracted genomic DNA were used in primary amplifications and 2 µl of first-round product was used as template for second-round PCR.
Purification of PCR fragments and sequencing.
PCR products were purified for sequencing reactions by precipitation with 20 % PEG8000 in 2·5 M NaCl. Alternatively, oligonucleotide primers were separated from the PCR product by gel filtration using a Sepharose CL4B column (Pharmacia) under gravity (Almond et al., 1992). Direct sequence analysis of both strands was determined using a modified Sequenase (Amersham) protocol and T7 polymerase (Pharmacia). Terminations were carried out at 50 °C. ABI PRISM Big Dye Terminator kits were used with a cycling profile recommended by the manufacturer. Additional sequencing primers to those used to generate the product were at conserved sites within nef in either the sense orientation [5'-GGGCTACGAGAGAGACTCTT-3' (nt 85618580), 5'-GCGAGTGGATCCAAGAAG-3' (nt 85258542), 5'-GATAAGGGGGGACTGGAAGGGATG-3' (nt 88918914), 5'-CAGCAAGGAGACTTTATG-3' (nt 87008717) and 5'-TGGCTATGGAAGCTAGT-3' (nt 90359051)] or the antisense orientation [5'-TGCTTTCAGTTTTGCCTTC-3' (nt 92509268), 5'-GTACTAGCTTCCATAGCC-3' (nt 90369053), 5'-CATCATCTGAATCTACAT-3' (nt 87858801) and 5'-GTTCTCCATGGGGTATTC-3' (nt 87168733)], also numbered according to HIV-2ISY. Sequence contigs were generated using the Gap4 program (Staden software package; Bonfield et al., 1995
) from a consensus generated from both sense and antisense strands and analyses were performed using the GCG software package and the CLUSTAL W program to generate multiple sequence alignments (Thompson et al., 1994
).
Cloning of nef alleles.
PCR products from a selected number of individuals were cloned into the pGEM-T plasmid vector with a TA Cloning kit (Promega). Ligated DNA was transfected into JM109 cells (Promega) and plasmid DNA purified using QIAprep kits (Qiagen). Clones were sequenced as described above.
Phylogenetic analysis.
Nucleotide sequences were aligned and evaluated with CLUSTAL W and phylogenetic trees were constructed from the matrix built by the neighbour-joining method (NEIGHBOR, version 3.5c; Felsenstein, 1989). Nucleotide substitution rates per site were estimated for each pairwise sequence comparison on the basis of the Kimura two-parameter model using DNADIST. Bootstrap analysis was performed with SEQBOOT and CONSENSE with 100 re-samplings. Trees were rooted with SIVMM239 and drawn with NJPLOT; reference sequences were obtained from the Los Alamos database. Nucleotide sequences reported in this study have been submitted to the EMBL nucleotide sequence database (accession nos AJ344369AJ344415). Epidemiological and clinical aspects were evaluated statistically with SPSS (statistical package for social science).
Quantification of HIV-2 RNA levels in plasma.
EDTA-treated plasma samples were stored at -80 °C until levels of HIV-2 viral RNA were assessed using a quantitative RT-PCR assay (Berry et al., 1998). In brief, RNA was extracted according to the method of Boom et al. (1990)
and RT-PCR performed, in duplicate, with HIV-2-specific LTR oligonucleotide sequences in single-tube assays using Titan reagents (Roche). Amplification products were quantified by chemiluminescence using an internal HIV-2 probe linked to alkaline phosphatase (Oswel). The reliable level of sensitivity of the assay was 200 copies ml-1 using an input plasma volume of 100 µl. For statistical analyses, plasmas with <200 copies ml-1 were assigned an arbitrary value of 20 copies ml-1.
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RESULTS |
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HIV-2 nef sequences in Portugal are subtype A and full-length
A total of 44 nef/LTR proviral sequences spanning the entire nef gene were amplified from 37 HIV-2-infected individuals (792 bp, Fig. 1). Consensus sequences derived from each of the 37 individuals from bulk product were substantially full-length with little evidence of deletions, truncations or other disruptions to nef. Predicted amino acid sequences (see below) suggested a functional HIV-2 Nef protein to be present in all individuals studied, irrespective of any other factor. Subsequent bleeds from six individuals yielded a nef sequence with few nucleotide or amino acid differences from the first sequence; hence, comparisons of nef variation were made on the first, bulk amplification of each patient. This observation was confirmed further by dilution experiments of template DNA. Phylogenetic relationships between Portuguese nef sequences were compared with HIV-2 subtype A (HIV-2ROD, HIV-2BEN, HIV-2ALI, HIV-2ISY and HIV-2ST), subtype B (HIV-2D205/ALT, HIV-2UC1 and HIV-2EHO) and SIVMM239 (Fig. 1
). All Portuguese sequences were HIV-2 subtype A, with no other HIV-2/SIVSM subtypes (BG) identified. No phylogenetic relationship was established between nef sequence and disease status and no statistical link between HIV-2 nef sequence and route of transmission, either sexual or blood transfusion, was apparent (data not shown).
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HIV-2 nef amino acid variation
Alignment of predicted amino acid sequences for the entire nef ORF for all 37 Portuguese HIV-2-infected individuals is shown in Fig. 2; the first sequence obtained for each individual is presented. The Portuguese HIV-2 nef consensus sequence was also compared with other lentivirus nef sequences, including a prototypic HIV-1 group M virus (HIV-1LAI), SIVCPZ (Gab), from chimpanzees, and SIVMM239, an experimental infection of Asian macaques derived originally from SIVSM. Predicted coding sequences were compared for 10 putative regions considered important for HIV-1, HIV-2 or SIV nef function (Fig. 2
). Throughout nef, a number of key regions were conserved among the Portuguese sequences, irrespective of clinical status, virus load or CD4 count, and deletions that might disrupt such sites were not evident. Moreover, only one patient showed evidence of a deleted nef sequence: patient 379 (symptomatic/AIDS) exhibited a nef deletion of 8 aa downstream of a suggested N-terminal RNA-binding motif (Echarri et al., 1997
).
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(i) Analysis of HIV-2 nef for putative tyrosine-based sorting motifs.
The canonical tyrosine-based sorting motif Yxx, present as Y28GRL and Y39SQS in SIVMAC239 and Y39SRF in HIV-2ROD, is Y39LQS in these Portuguese sequences, with changes to this basic configuration infrequent. Patient 984, an AIDS patient with a CD4 count of 195 and viral RNA levels of 8830 copies ml-1, exhibited a R
Y mutation at position 24; patients LF and PL exhibited C
Y mutations at position 28. However, additional tyrosine-based motifs in HIV-2, such as YE nef described for SIVMAC239, were not present in any HIV-2 sequences, including those in the symptomatic group. Interestingly, the most conserved tyrosine residue at position 39 was mutated in two AIDS patients (117-1 and 1378) to phenylalanine, serine or cysteine. There was no apparent relationship between disease status and tyrosine-based motifs.
(ii) Di-leucine-based motifs for CD4 downregulation.
Cellular di-leucine-based sorting pathways required for CD4 downregulation in HIV-1 are characterized by a core hexameric motif ENTSLL (ExxxLL), which conforms to the consensus E/DXXXL found in cellular transmembrane proteins (Craig et al., 1998
). HIV-2 nef sequences analysed for a comparable motif (L198L199) indicated variation at only L199, present as leucine, valine or methionine, with L198 conserved throughout. Interestingly, patients 280, 120, 281, MP and 956 grouped into an EANCLL configuration, three of which (281, MP and 956) had tetra-proline (PxxP)3 motifs. Overall, 15/37 (40·5 %) individuals had the L198L199 motif, with seven (19 %) substituting the leucine at position 199 for valine and 15 (40·5 %) for methionine. The number of LL motifs was distributed evenly between asymptomatic and symptomatic groups at eight and seven, respectively, forming an asymptomatic consensus of LM and symptomatic consensus of LL. Interestingly, the region immediately preceding this was relatively heterogeneous across all individuals (aa 185195, Fig. 2
), covering a putative site for
-COP recruitment. However, there was no apparent relationship between this variability and disease status.
(iii) Putative SH3-binding domains characterized by proline-rich (PxxP) motifs.
The proline-rich motif (P104xxPLR) was assessed for variation in more detail. Within this core region, R105K was observed frequently among these Portuguese sequences to form the sequence P104KVPLR, with the arginine residue at position 109. This adds to the observation that this region, which mediates interaction with SH3 ligands, is evolutionarily conserved among primate lentiviruses and is present as a minus-orientation binding consensus. Moreover, HIV-2 nef sequences generated in this study exhibited a broad range of PxxP configurations (Fig. 2
, Table 2
). An HIV-1-like (PxxP)3 configuration was observed for six individuals (16·2 %), three asymptomatic (MP, 511 and 293) and three symptomatic (281, 956 and 379). Conversely, disrupted minimal PxxP motifs were associated exclusively with an asymptomatic profile (11/26, 42·3 %), where the P107 residue was mutated to glutamine in nine individuals (PL, 1567, 1215, 1570, 1147, 794, 1268, 741 and 1139) and the P104 residue to serine in two (120 and 292), the only individuals not preserving the proline at position 104. Disruption of the core P104xxPLR region, P107
Q (n=9) and P104
S (n=2) was associated strongly with an asymptomatic phenotype, although patient PL who had been infected for 24 years had a relatively low CD4 count in 1997 (127 cells mm-3). The tyrosine (Y145) forming part of a putative hydrophobic pocket (Y145LEKE149E150) distal to the PxxP region implicated in SH3 binding in HIV-1 nef (Collette et al., 2000
) was absolutely conserved, with minimal changes to other residues associated with this motif, with E149 mutated to either a glutamine or a lysine in patients 1543 and 1567 and E150 mutated to lysine in patient 223; all three individuals were asymptomatic. In addition, there was an I144
L substitution in six individuals (281, MP, 120, 984, 1428 and 1096) immediately prior to Y145 and similar mutations in three other individuals: I144
T (n=2) and I144
M (n=1), although the significance of these changes is not known. Other sequences associated with the (PxxP)3 region in HIV-1 is a phenylalanine residue (F122). In HIV-2, this appeared in four individuals (281, 120, MP and 741), although the consensus of the comparable residue is a valine. No additional PxxP motifs in other regions of nef were identified in any of these HIV-2 sequences. Variability in the central PxxP region was investigated further for associations with disease status and virus load.
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Of the 20 individuals with an intact P104xxPLR motif but not a tetra-proline configuration, eight (117-1, 423, 280, 268, 984, 138, 1069 and 546) had evidence of symptomatic infection/AIDS. The second sample taken from patient 117 at 5 years later indicated an A107P change, forming a (PxxP)3 motif, and an A106
V change. The Nef protein of all 20 would seem to be capable of interacting with SH3 in this region. Further subdivisions of this group indicate that 11 individuals have only the minimal P104xxPLR sequence (n=3 symptomatic: 268, 984 and 138; n=8 asymptomatic: 1378, 223, EP, 1096, 1544, 1320, BL and LF); eight (n=4 symptomatic: 280, 1069, 423 and 117; n=4 asymptomatic: 1543, 1428, 483 and 1227) had an additional P101 (P101xxPxxPLR) and one symptomatic patient (546) had a PxxPLRP110 configuration.
Finally, 11 individuals had a disruption in the central region of the PxxP motif, mostly as a P107Q mutation (n=9), all of which were asymptomatic; this was statistically significant (Fisher's exact test, P=0·026). There was also a non-significant trend for lower virus loads with fewer prolines present (Table 2
). Levels of viral RNA correlated more closely to clinical status, with a significantly higher plasma virus load in symptomatic than asymptomatic individuals (mean HIV-2 RNA levels were 17 051 and 817 copies ml-1, respectively; unpaired Student t-test, P=0·0026), whereby all symptomatic individuals possessed an intact core P104xxPLR motif. Conversely, disruptions to the P104xxPLR core were associated with lower virus loads, which only exceeded 5000 copies ml-1 in one asymptomatic individual (PL) who had been infected for 24 years and who had a low (127 cells mm-3) CD4 count.
Longitudinal changes in nef and cloned nef sequences
A second sample obtained from each of six individuals, two symptomatic (281-2 and 117-2) and four asymptomatic (LF-2, MP-2, 1147-2 and 1139-2), allowed comparison with nef sequences from earlier time-points. The close genetic relationship between the two sequences (Fig. 1) indicated no difference in nef sequence measured at two distinct time-points. However, some evidence for increased nucleotide distances was observed, relating to sampling times that ranged from 0·6 to 1·5 % for 6 months to 1 year (sequences 1147-1/1147-2, 1139-1/1139-2 and 281-1/281-2), 1·62·4 % at 3 years (LF-1/LF-2 and MP-1/MP-2) and 3·4 % for a 5 year break between sampling (117-1/117-2). These findings (approximately 0·51 % per year) are similar to values estimated previously (Shankarappa et al., 1999
; Pieniazek et al., 1999
).
Clones isolated from three patients with disparate nef sequences were also sequenced: one AIDS patient (sample 117-2), one asymptomatic individual (sample MP-2) and one asymptomatic individual (1268) with disruptions in both proline-rich and di-leucine regions. No major variation in nef sequence, either synonymous or non-synonymous, was identified in individual clones compared to bulk sequence derived from a swarm of proviruses and overall, the cloned data confirmed the observed changes in nef sequence to be present as majority sequence (Fig. 3).
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DISCUSSION |
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Studies of SIVMAC have indicated that subtle differences in nef can alter dramatically the in vivo characteristics of virus replication (Whatmore et al., 1995) and a broad body of data exists relating the structure of HIV-1 nef with functional characteristics (Geyer et al., 2001
; Piguet & Trono, 1999
). Sequence-specific motifs involved in key functions of nef, such as enhancement of virus infectivity, downregulation of CD4 and interaction with T-cell signalling pathways, have the potential to invoke a variety of cellular pathways that can have an impact on pathogenesis. Hence, we embarked on a more systematic study of the HIV-2 nef sequence based on known properties of HIV-1 or SIV nef.
Key features of nef function, such as the myristoylation signal, were absolutely conserved among HIV-2 sequences, re-enforcing the notion that N-myristoylation is critical for the biological activity of all nef proteins (Harris, 1995). Analysis of motifs in HIV-2 nef indicated no difference in N-terminal tyrosine-based sorting signals, with no additional tyrosine residues, such as RQ
YE (YE nef), which, under certain circumstances, have been linked to acute lymphocyte activation and pathogenicity of some strains of SIV involving SH2 interactions (Luo & Peterlin, 1997
). Interestingly, interactions of SIV nef with human Lck or Hck do not seem to be mediated via the consensus proline motif, as is the case for HIV-1 nef (Collette et al., 1996
), as SIV nef demonstrates the ability to bind Lck and Hck SH2 domains, suggesting the multiple mechanisms by which nef is able to bind to and regulate Src kinases (Greenway et al., 1999
). The role of Src family kinases and the interaction with either SH2 or SH3 remains to be clarified for HIV-2.
In this respect, the natural variability of proline sequences that could mediate putative SH3 interactions was an important finding in this study. This variability was wider than that recognized previously and appeared to be related, at least in part, to disease status. There was a significant correlation between a P107Q mutation at the centre of the P104xxPLR core and an asymptomatic phenotype, which formed one end of a wide spectrum of proline configurations. Though numbers were relatively small, an increased number of proline residues was accompanied by increased virus load. However, tetra-proline (PxxP)3 sequences in three asymptomatic individuals were also identified and RNA levels in 5/6 patients with (PxxP)2 motifs were below the level of detection. Moreover, the wide spectrum of PxxP configurations encompassed all theoretical combinations ranging from (PxxP)3 to P104xxQ. This situation contrasts markedly with HIV-1 where, even in studies of long-term survivors, the consensus tetra-proline motif remains preserved, with no difference in proline motif configurations between rapid progressors and long-term survivors (Kirchhoff et al., 1999
).
Interestingly, in studies of SIV-infected macaques, there appears to be a strong selection pressure for a functional SH3-binding ligand in vivo, associated with kinase interactions (Khan et al., 1998), although other studies cast doubt on the functional significance of the PxxP motif in leading to disease induction during the acute phase (Lang et al., 1997
). However, the latter study seems unlikely to be representative of the broader picture of disease development in simian AIDS. Moreover, studies in the TgCD4 mouse model for HIV-1 nef have identified the PxxP region to be a crucial determinant of pathogenicity (Hanna et al., 2001
). The natural variability of proline-rich sequences in these HIV-2-infected individuals is compelling, given the potential for this region to interact with a variety of cellular partners and to modulate virus replication at the level of the T-cell receptor signalling environment (Fackler et al., 2001
). The fact that a P107
Q mutation was found only in asymptomatic individuals who also had low levels of peripheral viral RNA suggests a role for PxxPSH3 interactions in influencing pathogenesis. Hence, one could envisage a situation where the secondary structure of the polyproline type II helix is stabilized by the additional proline residues, with the critical P104xxP107 providing the important hydrophobic contacts with binding of the SH3 domain. The trend for increasing plasma virus load with increasing numbers of proline residues may reflect this and disruption of the binding consensus would have a major impact on putative SH3 interactions.
Immunological control also seems more effective for HIV-2 than HIV-1 (Whittle et al., 1998), with lower levels of immune activation and reduced levels of apoptosis (Michel et al., 2000
) reflecting the low virus steady-state of many HIV-2 infections (Andersson et al., 2000
; Ariyoshi et al., 2000
; Berry et al., 1998
, 2002
; Popper et al., 1999
). Recent data have indicated a role for nef in apoptotic regulatory pathways (Geleziunas et al., 2001
; Wolf et al., 2001
; Fackler & Baur, 2002
) and aspects of cell signalling that may have a bearing on virulence (Simmons et al., 2001
). Functional data are required to determine which cellular signalling pathways may be involved and define a more precise role for HIV-2 nef PxxPSH3 interactions. Whether changes in a single region or gene such as nef might be expected to influence pathogenicity directly in the face of multiple virushost interactions, including immunological considerations, is an interesting question. Certainly, the natural variation observed in the PxxP region was unexpected and would suggest that this region may be one of the more relevant regions for HIV-2 nef function in vivo. Molecular clones derived from this study are currently being taken forward to address some of these issues in order to provide a fuller understanding of the role of nef in the pathogenesis of HIV-2 infections.
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ACKNOWLEDGEMENTS |
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Received 17 October 2002;
accepted 27 January 2003.