The addition of asparagine (N)-linked polysaccharide chains (i.e., glycans) to the gp120 and gp41 glycoproteins of human immunodeficiency virus type 1 (HIV-1) envelope is not only required for correct protein folding, but also may provide protection against neutralizing antibodies as a “glycan shield.” As a result, strong host-specific selection is frequently associated with codon positions where nonsynonymous substitutions can create or disrupt potential N-linked glycosylation sites (PNGSs). Moreover, empirical data suggest that the individual contribution of PNGSs to the neutralization sensitivity or infectivity of HIV-1 may be critically dependent on the presence or absence of other PNGSs in the envelope sequence. Here we evaluate how glycan–glycan interactions have shaped the evolution of HIV-1 envelope sequences by analyzing the distribution of PNGSs in a large-sequence alignment. Using a “covarion”-type phylogenetic model, we find that the rates at which individual PNGSs are gained or lost vary significantly over time, suggesting that the selective advantage of having a PNGS may depend on the presence or absence of other PNGSs in the sequence. Consequently, we identify specific interactions between PNGSs in the alignment using a new paired-character phylogenetic model of evolution, and a Bayesian graphical model. Despite the fundamental differences between these two methods, several interactions are jointly identified by both. Mapping these interactions onto a structural model of HIV-1 gp120 reveals that negative (exclusive) interactions occur significantly more often between colocalized glycans, while positive (inclusive) interactions are restricted to more distant glycans. Our results imply that the adaptive repertoire of alternative configurations in the HIV-1 glycan shield is limited by functional interactions between the N-linked glycans. This represents a potential vulnerability of rapidly evolving HIV-1 populations that may provide useful glycan-based targets for neutralizing antibodies.
Many viruses exploit the complex machinery of the host cell to modify their own proteins, by the enzymatic addition of sugar molecules to specific amino acids. These sugars, or “glycans,” play several important roles in the infective cycle of the virus. The envelope of the human immunodeficiency virus type 1 (HIV-1), for example, becomes coated with so many glycans that the virus can become invisible to the protein-specific immune response of the host. Although some glycans are evolutionarily conserved, many others may be present within some hosts but absent in others, and may even appear or disappear over the course of an infection in a single host. To understand this variability, we have analyzed HIV-1 envelope sequences to identify cases where the presence of one glycan was dependent on the presence or absence of another (called glycan–glycan interactions). We used two newly developed computational methods to detect these interactions, thereby providing conclusive evidence of a new fundamental pattern: the glycans that exclude each other tend to occur near the same spot on the envelope, whereas glycans that occur together tend to be far apart.