| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Microbiology and Tumor Biology Center, Karolinska Institutet, Box 280, SE-171 77 Stockholm, Sweden
2 Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, USA
3 Department of Laboratory Medicine, Division of Clinical Virology, Karolinska University Hospital Huddinge, Karolinska Institutet, Box 280, SE-171 77 Stockholm, Sweden
Correspondence
Anneka Ehrnst
Anneka.Ehrnst{at}mtc.ki.se
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Tables showing details of sequences retrieved and sequence alignments are available as supplementary material in JGV Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
) or of the non-syncytium-tropic phenotype is suggested to be more transmissible (Zhang et al., 1993), whilst virus using the CXCR4 receptor (X4 virus; Berger et al., 1998) has been associated with disease progression (Connor et al., 1997; Scarlatti et al., 1997).
The third variable (V3) loop of the HIV-1 gp120 envelope glycoprotein was recognized early on to play an important role in governing the choice of target cells (Hwang et al., 1991; De Jong et al., 1992; Fouchier et al., 1992; Shioda et al., 1992). Few amino acid substitutions and an increasing net charge of the V3 loop were sufficient to confer a change in cellular tropism in vitro (De Jong et al., 1992; De Wolf et al., 1994). A decreased number of N-linked glycosylation sites (sequons) in gp120, especially within and around the V3 region, has been demonstrated during evolution from the R5 to the X4 phenotype (Pollakis et al., 2001; Polzer et al., 2001, 2002).
Computer-based models have been developed to predict the biological phenotype of HIV sequences (Briggs et al., 2000; Resch et al., 2001; Jensen et al., 2003; Pillai et al., 2003), based on multiple linear regression (Briggs et al., 2000), specific amino acids within V3 and overall charge among subtype B viruses (Resch et al., 2001) and other subtypes (Pillai et al., 2003). Machine learning has been used to develop phenotype classifiers (Resch et al., 2001) and position-specific scoring matrices have also been used (Jensen et al., 2003).
These methods relate to the assumption or possibility that the R5 and X4 phenotypes may be evaluated or classified, based on the properties of their specific amino acids, from the same scale or sum of scales. If the R5- and X4-specific properties relate to different characteristics, however, such programs may overlook essential differences in the R5 versus X4 phenotypes.
In a previous study (Clevestig et al., 2005), a phylogenetic analysis was performed on a large set of individual HIV-1 sequence clones from integrated DNA or plasma RNA, connected to sequences of isolates with determined coreceptor use. We made a preliminary observation (Clevestig et al., 2005) that R5 isolates and R5-associated sequences seemed to be connected to the presence of the N-linked glycosylation motif within the V3 loop of the env gp120 gene. As we studied only four individuals, linked epidemiologically as two mother–child pairs, we sought to explore this finding in the most epidemiologically and genetically diverse HIV-1 strains available. We retrieved sequences from the Los Alamos Database in a fashion similar to that of Resch et al. (2001), but included all available HIV-1 types, subtypes and recombinant forms.
We retrieved 1015 sequences, covering the V3 loop of all HIV-1 groups, group M subtypes, sub-subtypes and circulating recombinant forms (CRFs), representing all submitted sequences with a reported coreceptor use as of October 2004. Among the sequences were many multiples per patient, from which we performed a random selection to choose one patient sequence so that all sequences had a unique identity code. The selection was randomized through lottery and without consideration of the different phenotypes present.
We included sequences from HIV-1, spanning at least the V3 loop of the gp120 env gene with a specified coreceptor use, such as CCR5, CXCR4 or dual-tropic (CCR5 and CXCR4). We excluded sequences without a specified unique individual code and/or sequences using other coreceptors. Two amino acid sequences were found to be identical, despite unique identity codes. One sequence was therefore omitted randomly.
We assessed the glycosylation sequon motifs in the remaining 176 HIV-1 sequences with known coreceptor use from the database in Los Alamos (http://www.hiv.lanl.gov). All sequences were aligned manually and translated by using the sequence alignment editor Se-Al (Rambaut, 2002), also available at http://evolve.zoo.ox.ac.uk/software.html?id=seal.
The sequons were governed by the amino acid order asparagine–X–threonine/serine–Y (N–X–S/T–Y) (Marshall, 1972), where X can be any amino acid except proline (P) in the threonine (T) context (Gavel & von Heijne, 1990; Kasturi et al., 1997; Mellquist et al., 1998) and also not tryptophan (W), aspartic acid (D) or glutamine (E) in a serine (S) context (Kasturi et al., 1997). For the Y position, only proline would completely hinder oligosaccharide addition through steric hindrance in both contexts (Gavel & von Heijne, 1990; Mellquist et al., 1998). These parameters provided us with a high probability of oligosaccharide addition (Gavel & von Heijne, 1990; Shakin-Eshleman et al., 1996; Kasturi et al., 1997; Mellquist et al., 1998) and the criteria for evaluating each sequon as a possible N-linked glycosylation site. Sequences lacking data or exhibiting ambiguities in a site were excluded from this calculation.
The amino acid net charge of the V3 loop for each sequence at pH 7·0 was determined by using an online peptide-property calculator from Innovagen (http://www.innovagen.se).
A two-tailed Fisher's exact test was used to compare the significance of the presence or lack of glycosylation sequons in conjunction with CCR5, CXCR4 or dual-tropic use, and a one-tailed test in the analysis of net charge between R5 and X4, and R5 and dual-tropic strains, respectively. The level of significance was set to a probability (P) of 
0·05.
Among the 176 retrieved sequences (see Supplementary Table 1, available in JGV Online), 133 used CCR5, 29 used CXCR4 and 14 were dual-tropic (see Supplementary Table 2, available in JGV Online). However, this does not discount the possibility of inclusion of more than one sequence per patient that may have been submitted in another context, as was in fact the case with two sequences.
The dataset included HIV-1 of groups M (major, n=171), O (outlier, n=4) and N (non-M, non-O, n=1). M included subtypes A (n=22), B (n=62), C (n=28), D (n=12), G (n=5) and one unknown (U); circulating recombinant forms CRF01_AE (n=20), CRF02_AG (n=11); and other recombinants AC (n=4), AD (n=1), A1DGJ (n=1), AU (n=1), BC (n=1) and BF1 (n=2).
We investigated the presence of eight possible N-linked glycosylation sequons across 
100 aa spanning the V3 region, where the fifth site resided inside the V3 loop. The frequency of sequons at each site is shown in Fig. 1.
|
; Shakin-Eshleman et al., 1996; Kasturi et al., 1997; Mellquist et al., 1998), this has been greatly influenced by the amino acid in the Y position (Mellquist et al., 1998). Our NLS-Y sequons, most of which occurred in the sixth site (n=20/23), involved Y-position amino acids that had a high probability of glycosylation, altogether 
70 %.
No significant difference between the R5 and X4 sequences was observed in seven out of eight glycosylation sites (P>0·2 for each site), whereas the difference at the fifth site was remarkable (P4·6x10–12). There was also a tendency towards significance when comparing R5 with dual-tropic sequences (P=0·058). A glycosylation sequon (none with an NLS-Y sequon) was present in 128/133 (96 %) of R5 sequences versus 11/29 (38 %) in X4 sequences and 11/14 (79 %) in dual-tropic sequences. Further, the NNT sequon in the fifth glycosylation site was also conserved within the R5 sequences of known M-group subtypes in all but one sequence (120/121; 99·2 %).
The visual difference between R5 and dual-tropic sequons in site 7 was not significant (P=0·12). The same was observed between dual-tropic and X4 sequons (P=0·14). The stable glycosylation sequons at the flanking sites suggested that these were similarly important, but without discriminating between R5 and X4 strains.
To discern the role of the V3 loop net charge in relation to phenotype, the amino acid charge was calculated (Fig. 2). As expected, the net charge of R5 viruses (median 3·0) was lower than that of X4 viruses (median 5·9) (P=8·8x10–12) and of dual-tropic strains (median 5·5) (P=0·002). A breaking point was discerned at a charge of +4·2, below which there were no X4 sequences and above which there were only 10/133 (7·5 %) R5 sequences.
|
). There was also no significant association between charge and the fifth sequon among the dual-tropic strains (P>0·4).
|
). The sensitivity was very high in both cases (0·98). Moreover, the high positive predictive value of the fifth glycan and CCR5 use indicated a strong link between the two. However, the stern selection criteria provided us with relatively few X4 sequences, which may have influenced the lower predictive values.
Our analysis of available HIV-1 sequence data has provided evidence that the fifth N-linked glycosylation site is conserved in the vast majority of the available primary HIV-1 sequences using the CCR5 coreceptor. This pronounced correlation suggested that the V3 loop sequon, and the subsequently added sugar residues, may play an important role in the interaction with the CCR5 coreceptor. In contrast, X4 viruses seemed to rely preferentially on an increased net charge with a breaking point similar to that described previously (Fouchier et al., 1992).
We do not discount the possibility of other regions on the gp120 molecule also playing a role, either in conjunction with or independently of the V3 loop, but through this study, we would like to present a different perspective on the role of glycans in determining receptor tropism. Previous studies have shown a correlation between a loss of N-linked glycans and basic amino acid substitutions with emerging X4 and dual-tropic viruses (De Jong et al., 1992; Briggs et al., 2000; Polzer et al., 2001, 2002; Pontow & Ratner, 2001; Jensen et al., 2003). Our results are in agreement with previous findings, but emphasize the importance of the fifth N-linked glycan in the context of R5 viruses of the major (M) group of HIV-1. Loss of this glycan may not be enough to acquire the X4 phenotype, where additive changes seem necessary, providing the molecule with a higher net charge (Jensen et al., 2003). Similarly, Losman et al. (1999) found that the presence or absence of the V3 loop glycan did not influence the infectivity of X4 viruses.
It cannot be deduced from the information in the Los Alamos Database whether dual-tropic strains represented clones possessing the capacity to infect both CCR5 and CXCR4 cells or a mixture of R5 and X4 virus strains. Our data may be interpreted so that truly dual-tropic viral strains would be expected to contain the fifth sequon and a positive charge of over 4·2. Sequences lacking the fifth sequon and having a low charge would instead be expected to be less infectious or to use alternative coreceptors. Similarly, in a recent review, Hartley et al. (2005) suggested that dual-tropic strains would retain their CCR5-using property along with acquiring a higher net charge.
It might, therefore, be suggested that some of the original samples, from which the dual-tropic sequences studied here were derived, could have contained a mixture of R5 and X4 viral strains, rather than a population where the dual-tropic property was present in the same strain.
The amino acid sequence does not reveal the nature of the glycosylations with regard to actual addition of glycans or their number. The actual glycosylation may further depend on proximity to the C terminus (Gavel & von Heijne, 1990; Nilsson & von Heijne, 2000) and other proximal glycans (Pollakis et al., 2001). It is also important to include analysis of glycans in the other variable regions of gp120 (Pollakis et al., 2001). However, the presented data provide evidence that the V3 loop glycan appears to be important for CCR5 use.
The present interpretation of the relative role of N-linked glycosylation and charge for the use of CCR5 and CXCR4, respectively, points to the necessity to further elucidate the relative differences/similarities of the CCR5 and CXCR4 receptors and their interaction with their natural ligands or HIV-1 glycoproteins.
Interestingly, the coreceptors CCR5 and CXCR4 are structurally similar. There is an implication that the second extracellular loops (ECL2) of CCR5 and CXCR4 interact with their respective HIV-1 viruses (Brelot et al., 1999; Chabot & Broder, 2000; Pontow & Ratner, 2001). It is interesting to note that CXCR4 has two N-linked glycosylation sites, one in ECL2 and one in the N terminus, that are absent on CCR5 (Chabot et al., 2000). Elimination of these glycans in CXCR4 broadens its coreceptor capacity to include several R5 strains, without affecting entrance of X4 strains (Chabot & Broder, 2000). Instead, eliminating CXCR4 charge may decrease its binding of X4 strains (Brelot et al., 1999).
The binding of R5 viruses of the M group to the CCR5 coreceptor may be associated with the presence of V3 loop N-linked glycosylation and a corresponding absence in the CCR5 coreceptor. The glycan may provide R5 strains with a narrow fitness in affinity to the N terminus and/or ECL2 of CCR5, which maybe lacking in X4/CXCR4 interactions. Instead, these seem to be governed by V3 loop charge (De Jong et al., 1992) and also by the CXCR4 ECL2 charge (Brelot et al., 1999; Chabot & Broder, 2000).
The association between the V3 loop glycosylation site and CCR5-using viruses could be incorporated into current and future prediction algorithms, where presence of this glycan would increase the probability of an R5 origin, whilst lack of it would not be considered a marker for an X4 origin. It is of interest that the three R5 M-group sequences, lacking the fifth sequon, had the amino acid motifs TNT (Gao et al., 1996), HNT (Engelbrecht et al., 2001) and NNA (Tebit et al., 2002), differing by only 1 nt each from the NNT motif.
A question that arises is whether this R5 conserved sequence or other conserved glycosylation motifs could be used as new targets for prophylactic interventions, especially as R5 is suggested to be the major transmissible variant of HIV-1.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Brelot, A., Heveker, N., Adema, K., Hosie, M. J., Willett, B. & Alizon, M. (1999). Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses. J Virol 73, 2576–2586.
Briggs, D. R., Tuttle, D. L., Sleasman, J. W. & Goodenow, M. M. (2000). Envelope V3 amino acid sequence predicts HIV-1 phenotype (co-receptor usage and tropism for macrophages). AIDS 14, 2937–2939.[CrossRef][Medline]
Chabot, D. J. & Broder, C. C. (2000). Substitutions in a homologous region of extracellular loop 2 of CXCR4 and CCR5 alter coreceptor activities for HIV-1 membrane fusion and virus entry. J Biol Chem 275, 23774–23782.
Chabot, D. J., Chen, H., Dimitrov, D. S. & Broder, C. C. (2000). N-linked glycosylation of CXCR4 masks coreceptor function for CCR5-dependent human immunodeficiency virus type 1 isolates. J Virol 74, 4404–4413.
Clevestig, P., Maljkovic, I., Casper, C. & 7 other authors (2005). The X4 phenotype of HIV type 1 evolves from R5 in two children of mothers, carrying X4, and is not linked to transmission. AIDS Res Hum Retroviruses 21, 371–378.[CrossRef][Medline]
Connor, R. I., Sheridan, K. E., Ceradini, D., Choe, S. & Landau, N. R. (1997). Change in coreceptor use correlates with disease progression in HIV-1-infected individuals. J Exp Med 185, 621–628.
De Jong, J.-J., De Ronde, A., Keulen, W., Tersmette, M. & Goudsmit, J. (1992). Minimal requirements for the human immunodeficiency virus type 1 V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution. J Virol 66, 6777–6780.
De Wolf, F., Hogervorst, E., Goudsmit, J. & 52 other authors (1994). Syncytium-inducing and non-syncytium-inducing capacity of human immunodeficiency virus type 1 subtypes other than B: phenotypic and genotypic characteristics. WHO Network for HIV Isolation and Characterization. AIDS Res Hum Retroviruses 10, 1387–1400.[Medline]
Engelbrecht, S., de Villiers, T., Sampson, C. C., zur Megede, J., Barnett, S. W. & van Rensburg, E. J. (2001). Genetic analysis of the complete gag and env genes of HIV type 1 subtype C primary isolates from South Africa. AIDS Res Hum Retroviruses 17, 1533–1547.[CrossRef][Medline]
Fouchier, R. A. M., Groenink, M., Kootstra, N. A., Tersmette, M., Huisman, H. G., Miedema, F. & Schuitemaker, H. (1992). Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule. J Virol 66, 3183–3187.
Gao, F., Morrison, S. G., Robertson, D. L. & 12 other authors (1996). Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization. J Virol 70, 1651–1667.[Abstract]
Gavel, Y. & von Heijne, G. (1990). Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng 3, 433–442.
Hartley, O., Klasse, P. J., Sattentau, Q. J. & Moore, J. P. (2005). V3: HIV's switch-hitter. AIDS Res Hum Retroviruses 21, 171–189.[CrossRef][Medline]
Hwang, S. S., Boyle, T. J., Lyerly, H. K. & Cullen, B. R. (1991). Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 253, 71–74.
Jensen, M. A., Li, F.-S., van't Wout, A. B. & 7 other authors (2003). Improved co-receptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J Virol 77, 13376–13388.
Kasturi, L., Chen, H. & Shakin-Eshleman, S. H. (1997). Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors. Biochem J 323, 415–419.
Losman, B., Biller, M., Olofsson, S., Schønning, K., Lund, O. S., Svennerholm, B., Hansen, J.-E. S. & Bolmstedt, A. (1999). The N-linked glycan of the V3 region of HIV-1 gp120 and CXCR4-dependent multiplication of a human immunodeficiency virus type 1 lymphocyte-tropic variant. FEBS Lett 454, 47–52.[CrossRef][Medline]
Marshall, R. D. (1972). Glycoproteins. Annu Rev Biochem 41, 673–702.[CrossRef][Medline]
Mellquist, J. L., Kasturi, L., Spitalnik, S. L. & Shakin-Eshleman, S. H. (1998). The amino acid following an Asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. Biochemistry 37, 6833–6837.[CrossRef][Medline]
Nilsson, I. & von Heijne, G. (2000). Glycosylation efficiency of Asn-Xaa-Thr sequons depends both on the distance from the C terminus and on the presence of a downstream transmembrane segment. J Biol Chem 275, 17338–17343.
Pillai, S., Good, B., Richman, D. & Corbeil, J. (2003). A new perspective on V3 phenotype prediction. AIDS Res Hum Retroviruses 19, 145–149.[CrossRef][Medline]
Pollakis, G., Kang, S., Kliphuis, A., Chalaby, M. I. M., Goudsmit, J. & Paxton, W. A. (2001). N-Linked glycosylation of the HIV type-1 gp120 envelope glycoprotein as a major determinant of CCR5 and CXCR4 coreceptor utilization. J Biol Chem 276, 13433–13441.
Polzer, S., Dittmar, M. T., Schmitz, H., Meyer, B., Müller, H., Kräusslich, H.-G. & Schreiber, M. (2001). Loss of N-linked glycans in the V3-loop region of gp120 is correlated to an enhanced infectivity of HIV-1. Glycobiology 11, 11–19.
Polzer, S., Dittmar, M. T., Schmitz, H. & Schreiber, M. (2002). The N-linked glycan g15 within the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage, cellular tropism, and neutralization. Virology 304, 70–80.[CrossRef][Medline]
Pontow, S. & Ratner, L. (2001). Evidence for common structural determinants of human immunodeficiency virus type 1 coreceptor activity provided through functional analysis of CCR5/CXCR4 chimeric coreceptors. J Virol 75, 11503–11514.
Rambaut, A. (2002). Se-Al Sequence Alignment Program, version 2.0a11. Department of Zoology, University of Oxford, Oxford, UK.
Resch, W., Hoffman, N. & Swanstrom, R. (2001). Improved success of phenotype prediction of the human immunodeficiency virus type 1 from envelope variable loop 3 sequence using neural networks. Virology 288, 51–62.[CrossRef][Medline]
Scarlatti, G., Tresoldi, E., Björndal, Å. & 9 other authors (1997). In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat Med 3, 1259–1265.[CrossRef][Medline]
Shakin-Eshleman, S. H., Spitalnik, S. L. & Kasturi, L. (1996). The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency. J Biol Chem 271, 6363–6366.
Shioda, T., Levy, J. A. & Cheng-Mayer, C. (1992). Small amino acid changes in the V3 hypervariable region of gp120 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 89, 9434–9438.
Tebit, D. M., Zekeng, L., Kaptué, L., Salminen, M., Kräusslich, H.-G. & Herchenröder, O. (2002). Genotypic and phenotypic analysis of HIV type 1 primary isolates from western Cameroon. AIDS Res Hum Retroviruses 18, 39–48.[CrossRef][Medline]
Zhang, L. Q., MacKenzie, P., Cleland, A., Holmes, E. C., Leigh Brown, A. J. & Simmonds, P. (1993). Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection. J Virol 67, 3345–3356.
Received 8 September 2005;
accepted 10 November 2005.
This article has been cited by other articles:
|
|
 |
|
  Y. Li, B. Cleveland, I. Klots, B. Travis, B. A. Richardson, D. Anderson, D. Montefiori, P. Polacino, and S.-L. Hu Removal of a Single N-Linked Glycan in Human Immunodeficiency Virus Type 1 gp120 Results in an Enhanced Ability To Induce Neutralizing Antibody Responses J. Virol., January 15, 2008; 82(2): 638 - 651. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Huang, S. H. Eshleman, J. Toma, S. Fransen, E. Stawiski, E. E. Paxinos, J. M. Whitcomb, A. M. Young, D. Donnell, F. Mmiro, et al. Coreceptor Tropism in Human Immunodeficiency Virus Type 1 Subtype D: High Prevalence of CXCR4 Tropism and Heterogeneous Composition of Viral Populations J. Virol., August 1, 2007; 81(15): 7885 - 7893. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Balzarini, Y. Van Herrewege, K. Vermeire, G. Vanham, and D. Schols Carbohydrate-Binding Agents Efficiently Prevent Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin (DC-SIGN)-Directed HIV-1 Transmission to T Lymphocytes Mol. Pharmacol., January 1, 2007; 71(1): 3 - 11. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
