2 Theoretical Biology Group, Los Alamos National Laboratory, Los Alamos, NM 87544; 3 Seattle Biomedical Research Institute, Seattle, WA 98109; 4 Pathobiology Department, University of Washington, Seattle, WA 98195; and 5 The Santa Fe Institute, Santa Fe, NM 87501
Received on February 13, 2004; revised on May 26, 2004; accepted on May 28, 2004
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Abstract |
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Key words: immune escape / N-linked glycosylation / neutralization antibody / variability / virus
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Introduction |
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Alteration of a glycosylation site can have dramatic consequences for a virus. It can impact protein folding (Hebert et al., 1997; Land and Braakman, 2001
; Slater-Handshy et al., 2004
) and conformation (Meunier et al., 1999) and affect distant parts of a protein through masking or conformational changes. Although gain of a carbohydrate can sterically mask epitopes, the loss of one could result in tighter packing of glycoprotein regions involved in neutralization epitopes, reduce accessibility, and so also facilitate immune escape (Ye et al., 2000
). The loss of sequons can even impact immunogenicity of noncovalently associated proteins, for example a change in sequons in the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope (Env) protein gp41 induces conformational changes in the associated Env gp120 surface protein that dramatically diminish the binding of many gp120-specific antibodies (Si et al., 2001
). Altered patterns of glycosylation in viral proteins can also contribute to escape from T cell responses (Botarelli et al., 1991
; Ferris et al., 1999
; Selby et al., 1999
) and influence receptor binding and phenotypic properties of viruses (Kaverin et al., 2002
; Koito et al., 1995
; Matrosovich et al., 1999
; Ogert et al., 2001
; Pollakis et al., 2001
).
Influenza, glycosylation, and antigenic drift
Some of the earliest studies on the biological and immunological consequences of glycosylation site variation were conducted in influenza proteins (Alexander and Elder, 1984). The number of sequons in the heavily glycosylated influenza A hemagglutinin 1 (HA1) of the pandemic H3 virus has increased from 6 to 10 since it entered the human population in 1968 (Skehel and Wiley, 2000
), and the increase is assumed to make HA1 generally more refractive to antibodies. For example, the amino acids around the N-linked glycosylations site at position N165 of HA stopped participating in antigenic drift (Skehel and Wiley, 2000
; Wiley and Skehel, 1987
).
HIV Env and glycosylation site variation
HIV-1 is highly variable, and variants are grouped through phylogenetic analysis into major clades or subtypes (AK). Recombinant forms of HIV are very common (Robertson et al., 2000), and when a lineage based on recombination between two subtypes becomes an important epidemic lineage, it is called a circulating recombinant form (CRF). HIV-1 varies dramatically within a clade and within infected individuals. HIV Env gp120 is among the most heavily glycosylated proteins in nature (Myers et al., 1992
), far more heavily glycosylated than envelopes of other retroviruses of similar size (e.g., HTLV-1, MuLV) (Polonoff et al., 1982
). The influence of sequon variation on HIV antibody recognition and viral phenotype has been studied in the context of HIV and SIV Env proteins (Chackerian et al., 1997
; Cheng-Mayer et al., 1999
; Losman et al., 2001
; Ly and Stamatatos, 2000
; Malenbaum et al., 2000
; Matthews et al., 1987
; Ratner 1992
; Ye et al., 2000
). Sigvard Olofsson and colleagues first showed that glycosylation of gp120 could change exposure of neutralizing antibody epitopes (Bolmstedt et al., 1996
). In the HIV-1 CRF01, the absence of a sequon near the base of the V3 loop, an important antigenic doman of Env gp120, was correlated with rapid amino acid substitutions and positive selection (Kalish et al., 2002
), suggesting a similar situation to the influenza A HA protein, where a glycosylation site may provide regional protection from antibodies (Skehel and Wiley 2000
). The number of Env sequons in both HIV and SIV infections varies extensively within infected individuals (Overbaugh and Rudensey, 1992
; Wolinsky et al., 1992
), and this variation constitutes a mechanism of immune escape during the course of an HIV or SIV infection (Cheng-Mayer et al., 1999
; Davis et al., 1990
; Simmonds et al., 1991
).
A heavily glycosylated face of the 3D structure of Env gp120 has been called the immunologically silent face (Moore and Sodroski, 1996). Generally carbohydrate moieties appear as self to the immune system, so this face reduces the antigenicity of a large region on the gp120 surface. Glycosylation of variable loops restricts access to conserved receptor binding sites and limits their exposure to the immune system (Wyatt and Sodroski, 1998
), and HIV Env has been described as having a glycan shield (Wei et al., 2003
).
A recent survey of the global collection of HIV-1 Env gp120 surface protein M group sequences in the Los Alamos HIV database showed that gp120 varied in length between 484 and 543 amino acids (Korber et al., 2001). The number of potential sequons in gp120 ranges between 18 and 33 with a median of 25 (Korber et al., 2001
) (Figure 1) (this range is often ignored and HIV is frequently reported to have 25 N-linked glycosylation sites). The dramatic variation in number of sequons partly results from gp120 length variation frequently involving insertions and deletions of potential glycosylation sites inside HIV hypervariable domains, however, evolutionary propensities in base substitution patterns in HIV may also contribute to the rapid flux in numbers of sequons in gp120 (Bosch et al., 1994
; Kuiken et al., 1999
).
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Glycosylation and hepatitis C Env diversity
Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family (Rice, 1996). Like HIV-1, it is highly variable; HCV establishes a chronic infection in most hosts and so is subject to continuous immune pressure and rapid accumulation of mutations. The virus has been classified into six different genotypes, which have each been subdivided into a large number of subtypes. The Env E1 and E2 proteins of HCV form heterodimers on the virion surface, and glycosylation is essential for this dimerization (Meunier et al., 1999). The variability of both proteins is comparable, from 88% nucleotide (90% amino acid) identity between strains from the same subtype to 55% nucleotide (59% amino acid) identity between different genotypes. In both E1 and E2, N-glycosylation sites are limited to the amino terminus of the proteins; the carboxy-terminal region of these proteins is the transmembrane portion. The efficient glycosylation of E1 is dependent on the presence of E2 in a polyprotein (Deleersnyder et al., 1997
), although it does not appear to depend on the specific sequence of E2 (Dubuisson et al., 2000
), and the noncovalent association of E1 and E2 depends on the first and fourth glycosylation sites of E1 (Meunier et al., 1999). It has been shown that the glycans attached to the E1E2 heterodimer prior to budding of the virus are exclusively high-mannose (Deleersnyder et al., 1997
), although E1E2 found circulating on HCV virions also have complex carbohydrates (Sato et al., 1993
). As in HIV, sequon changes have been shown to change antibody exposure in HCV (Fournillier et al., 2001
).
In this study, we characterize patterns in viral glycosylation site variation at the population level for influenza, HIV, and HCV and describe a Web-based program that was used to facilitate tracking sequons in protein alignments for these comparisons.
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Results |
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Global trends in glycosylation patterns in HIV Env
There is no net tendency for the sequon number to increase or decrease over time in the HIV-1 M group, or within subtypes or CRFs (Figure 3). Two kinds of sequons are evident in HIV-1 and HIV-2: those embedded in readily aligned positions and those embedded in hypervariable regions that shift in relative position and regional density by base mutation and by insertions and deletions. Here we refer to these two classes of sequons as fixed and shifting, respectively. Although most of the shifting sequons are found in variable loops, some are also present in the C4 region (conserved, or C, domains in the HIV envelope were called conserved because they are relatively conserved when compared to the variable regions, however, they can also span insertions and deletions that can result in shifting sequon locations). The location and frequency of sequons in gp120 in each major subtype of the HIV-1 M group (Gao et al., 1996; Robertson et al., 2000
) are illustrated in Figure 4. Despite the extreme variability between isolates, there is an essentially conserved pattern of variation in each of the HIV-1 M group subtypes, and even in the genetically very distant HIV-1 group O. (There are insufficient full-length HIV-1 group N sequences for a comparison). Protein regions show the same frequencies for most sequons in each HIV-1 lineage and subtype, suggesting that selective pressures on sequons in these diverse lineages are consistent (Figure 4).
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The 2G12 monoclonal antibody is one of the few broadly cross-reactive human HIV-neutralizing antibodies, and it has an unusual epitope involving several mannose carbohydrates that project from the generally immunorefractive glycosylated face of HIV-1 Env. Not surprisingly, cross-competition studies have shown that B cell responses to 2G12 epitope are very unusual in HIV-1-infected individuals (Moore and Sodroski, 1996). The sites that comprise the epitope, indicated in Figure 4, require mannose at positions of N295, N332, and N392 for 2G12 binding (Sanders et al., 2002
; Scanlan et al., 2002
). (See the HXB2 sequence locator tool in the HIV database [www.hiv.lanl.gov] to determine the specific positions referred to in this text.) These sites are well conserved in most subtypes, and tend to show comparable levels of glycosylation. The exception is subtype C, which only rarely has a glycosylation site immediately next to the Cys at the base of the V3 loop (N295). It has been suggested that the 2G12 epitope may be conserved because its structure enhances gp120mannose interactions with the human protein dendritic cellspecific HIV-1-binding protein (DC-SIGN), an interaction that faciliatates efficient HIV-1 infection (Sanders et al., 2002
).
The global collection of HIV Env gp120 molecules shows small but significant variations in the distributions of sequon frequencies for each subtype (Kruskal-Wallis p-value = 0.0002). Occasionally a site will be completely lost or added in a subtype (Figure 4), like the loss of a shifting sequon in CRF01 (subtype E in Env) in the V4 region or the additional N-terminal fixed site in O group. These differences may simply reflect a founder effect in the lineage, or may confer a critical change to the conformation of Env in the context of a particular lineage. Although the sequon frequencies in different HIV-1 subtypes are significantly different, perhaps more important are the similarities between subtypes: The range of sequon frequencies is broad and basically overlapping within all subtypes (Gao et al., 1996) (Figure 5), and the conserved sites as well as frequencies of variable sites tend to be comparable between subtypes (Figure 4).
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N-linked glycosylation patterns in other primate lentiviruses
Among the primate lentiviruses, Env sequon variation in M group viruses is the most extreme relative to the genetic distances based on base substitution (Wills et al., 1996). However, SIVs do vary extensively and also show some interesting glycosylation patterns (Chackerian et al., 1997
; Nyambi et al., 1997
; Overbaugh and Rudensey, 1992
). Using phylogenetic analysis, we selected HIV-2 and SIV sequences representing the spectrum of natural diversity in each lineage. Shifting sites, analogous to those found in HIV-1, were apparent in HIV-2 and in chimpanzee viral Envs (SIVCPZ). The human HIV-1 epidemic is thought to have resulted from a cross-species transmission from chimpanzee, whereas human HIV-2 and macaque SIVMAC result from cross-species transmission of SIVSMM virus from the sooty mangabey, its natural host (Gao et al., 1996
; Hirsch et al., 1989
). In SIVSMM and African green monkey SIVAGM, shifting sites tended to resolve into fixed sites (Figure 8A), with the exception of the V4 region in SIVAGM. Table I highlights the sequons in the V1 loops from sequences used to generate the frequencies shown in Figure 8. Although there was length variation in SIVSMM and SIVAGM V1 sequences, the relative placement of the sequons and the Cys involved in the disulfide bridge that forms the base of the loop was conserved (Table I). In contrast, shifting sequons are frequently seen in infected macaques (SIVMAC) followed over time through progression to simian AIDS (Chackerian et al., 1994
, 1997
; Overbaugh et al., 1991
) and in HIV-2 sequences in human (Figure 8A, Table I). Because HIV-2 and SIVMAC infections result from cross-species transmission of SIVSMM virus from sooty mangabeys (Gao et al., 1996
; Hirsch et al., 1989
), the degree of variation in shifting sites is host-specific for viruses of this lineage. SIVSMM does not cause disease in sooty mangabeys, but it does in macaques, as does HIV-2 in humans. Like SIVSMM, SIVAGM does not cause disease in its natural host species, African green monkeys, and is readily found in animals in the wild (Norley et al., 1999
; Ohta et al., 1988
).
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There are typically 57 sequons in E1 over a stretch of 194 amino acids. Four of these sites are highly conserved among all genotypes. The site at position N61 in the alignment was present in genotype 6 and 1b but absent in all other genotypes (Figure 9, top). A pair of adjacent sequons at positions N43/N44 also showed subtype-specific variation, the site at position N43 being present in subtype 2b, and the one at N44 in 2a and 2c, as well as in most other sequences. In 35% of the subtype 1b sequences, the N43 and N44 sites were both present; this occurred in 1 sequence of another genotype, a 3b. The sequences with two sequons did not appear to come from a specific geographic region and probably arose independently. The sequons at positions N5 and N136 of E1 have been shown to be essential for formation of E1E2 complexes on the virion surface (Meunier et al., 1999); N136 also is important in reducing the antigenicity of the virus (Fournillier et al., 2001). Little is known about genotype- or subtype-specific differences in viral phenotype.
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Discussion |
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There is a striking recapitulation of sequon patterns in different levels of HIV evolution. The level of sequon variation that can be seen in an individual is mirrored at the level of variation seen within a subtype, which is further reflected at the level of variation seen within the HIV-1 M group global epidemic. Thus the boundaries of the potential to add and eliminate sequons to facilitate immune escape and infect a range of cell types, within what is tolerated in terms of fitness costs, may be explored anew during the course of each HIV-1 infection of a single individual. Although the phylogenetic tree of HIV-1 is clearly expanding over the time period during which we have followed HIV-1 in the human population (Korber et al., 2000; Robbins et al., 2003
; Yusim et al., 2001
), the sequon variations in gp120 and gp41 seem instead to be rapidly fluctuating within inherent boundaries.
There is probably an upper bound to the number of sequons that can be maintained on Env gp120, because carbohydrate structures are quite large and their presence or absence must influence the protein's conformation. For example, there are two sequons next to cysteines that form the base of the V3 loop. A typical glycan is about 2000 Da, and the V3 loop is only about 3000 Da, so the presence or absence of these sequons would logically impact the orientation with which the V3 loop projects from the protein surface. Glycosylation sites may be preserved in vivo to mask neutralizing antibody sites, but replication efficiency and access to receptor binding sites may provide a counterbalancing forcemultiple glycosylation sites can be removed from gp120 without loss of infectivity (Ohgimoto et al., 1998).
The observation that the complex carbohydrates tend to be localized in the shifting positions while high mannose is favored in the fixed positions in HIV-1 Env gp120 could result from multiple contributing factors. The interactions of DC-SIGN and other C-type lectins with gp120 high-mannose oligosaccharides have an important role in HIV infectivity (Lin et al., 2003), suggesting there may be a fitness cost in disrupting their precise orientation. If the high-mannose oligosaccharides are important for successful sexual transmission, there would be selection pressure to preserve the high-mannose forms. It has been suggested that the conservation of the high-mannose epitope for the 2G12 antibody might be related to the preservation of mannose structure that facilitates DC-SIGN interaction (Sanders et al., 2002
). N-linked glycosylation patterns in HIV Env are further complicated by the fact that different cell types, H9 and Chinese hamster ovary (Mizuochi et al., 1988
, 1990
), and primary T cells and macrophages have different patterns of glycan modification (Liedtke et al., 1997
; Lin et al., 2003
; Willey et al., 1996
). DC-SIGN preferentially binds to HIV Env gp120 enriched for high-mannose oligosaccharides, which are typically produced by peripheral blood mononuclear cells and T cells, compared to macrophage-produced gp120, which contains more complex carbohydrates (Lin et al., 2003
). Also, macrophage-derived gp120 carbohydrates are modified by lactosaminoglycans, whereas peripheral blood mononuclear cellsderived gp120 s are not. Interestingly, macrophage-derived virus tends to be more neutralization resistant (Willey et al., 1996
). Bisecting N-glycans have been implicated in the suppression of natural killer (NK) cellmediated responses (Yoshimura et al., 1996
), the suppression of innate immune responses involving NK cells that may be responsible for initiating AIDS pathogenesis (Kottilil et al., 2003
). Thus cell-specific glycosylation profiles may affect immune susceptibility, and AIDS progression may be related to the glycobiology of the virus (Clark et al., 1997
).
Positions that accept complex carbohydrate additions may also be determined simply by being more exposed during passage through the Golgi apparatus, where carbohydrate modifications occur. Such exposure may be related to accessibility in the folded functional protein, and immune evasion and neutralizing antibody escape are likely to be the driving force for the variation in shifting sites. So the complex carbohydrate additions may occur in more exposed sites, and those sites, due to the exposure, may be under greater pressure to shift to escape from antibody recognition.
We did not observe variation in the net number of sequons in the variable loop domains of HIV-1 Env gp120 associated with any particular pattern of coreceptor usage, although a particular sequon in the V3 loop was highly conserved in R5 viruses but not in X4 or R5X4 viruses. Potential involvement of sequon changes in gp41 in cell tropism was also noted, as some sites are rarely found in NSI variants. Glycosylation in gp41 may be more influential than previously thought, given new evidence that additional loops of the transmembrane protein are located extracellularly (Cleveland et al., 2003). The role of alterations of specific sequons in HIV-1 Env associated with changes in coreceptor usage on a population basis can be subtle and may be context-specific. For example, in our analysis of one patient for changes associated with phenotypic variation (Figure 7), significant differences were found at positions that were different from those identified in the cross-sectional population analyses.
Glycosylation in the V1V2 region can be important for coreceptor usage (Ogert et al., 2001), and additional glycosylation sites in V1V2 may in some circumstances potentiate the use of CXCR4 (Pollakis et al., 2001
). V2 elongation and sequon changes have been associated with slow disease progression (Shioda et al., 1997
). The effects may be subtle; for example, removal of three sequons in V1 increased the affinity between gp120 and the CXCR4 receptor but did not alter the infectivity of the virus (Losman et al., 2001
). Limited V1V2 length variation and sequon shifts in rapid progressors (Masciotra et al., 2002
; Shioda et al., 1997
) versus long-term survivors may be a consequence of a poor immune response resulting in weak selection pressure (Delwart et al., 1997
; Wolinsky et al., 1996
) and not related directly to the coreceptor usage. This is particularly plausible because changes in V1 V2 sequons often influence antigenic domains in other regions, for example, they can alter antibody recognition of both the V3 loop (Losman et al., 2001
; Ly and Stamatatos, 2000
; Ye et al., 2000
) and CD4 binding site (Ly and Stamatatos, 2000
). It is intriguing that the capacity for shifting sequons is found not only in HIV but also in two HCV E2 locations in two lineages in genotypes 1 and 6, suggesting they may give a selective advantage in rapidly evolving viruses.
Although the SIVSMM and SIVAGM viruses have some degree of shifting sequons in their natural hosts (particularly in the Env V4 region in SIVAGM), the shifting Env sequon characteristics appear far less pronounced in these lineages than in HIV-1 and CPZ lineages (Figure 8). HIV-2, which stems from cross-species transmission of SIVSMM, also has more extreme levels of shifting sites, suggesting that selective forces in the new host bring out the greater levels of position diversity seen in sequons. Neutralizing antibody responses to SIVSMM and SIVAGM infections in their natural hosts are present but may be relatively reduced (Fultz et al., 1990; Gicheru et al., 1999
; Kaur et al., 1998
; Norley et al., 1990
). However, despite the lack of shifting sequons, both SIVSMM and SIVAGM diversify rapidly in vivo (Broussard et al., 2001
). At least some of this diversification may be due to cytotoxic T lymphocyte escape (Kaur et al., 2001
), which may not select for patterns of shifting sequons. Neutralizing activity of sera from HIV-infected individuals and SIV-infected primates generally lags behind, so serum from one time point can neutralize earlier but not contemporary virus (Albert et al., 1990
; Arendrup et al., 1992
; Bradney et al., 1999
; Montefiori et al., 1991
; Nyambi et al., 1997
). Rapid cycles of response and escape may be related to the gain and loss of sequons (Richman et al., 2003
; Wei et al., 2003
). Thus it is possible that the strength and neutralizing antibody response in the host dictates the extent of the shifting antibody sites in different primate lentiviruses.
N-linked glycosylation sites are a critical component of the external proteins of primate lentiviruses, influenza, and hepatitis C viruses, and their modification can be important for evolution of the immune response. The gain or loss of such sites can play a key role in viral infectivity, antigen conformation, and immune escape. The mechanisms that generate shifting sites and tolerance of such shifting sequons in viral proteins provides a unique evolutionary avenue for immune evasion.
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Materials and methods |
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The protein alignments used for this study were retrieved from the Los Alamos HIV sequence database (www.hiv.lanl.gov), Influenza database (www.flu.lanl.gov), and the Hepatitis C database (www.hcv.lanl.gov). All alignments used in this study are available on request. All hepatitis, SIV, and HIV alignments were restricted so that only sequence from a single individual was included. Thus the sample sets were not biased by including multiple sequences from one person or from closely related infections. The one exception is the sequon change analysis of single patient in Figure 7 (Hu et al., 2000).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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