Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in Trichoplusia ni Indicates High Multiplicity of Cellular Infection

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.     

Restriction Map of Rachiplusia ou and Rachiplusia ou-Autographa californica Baculovirus Recombinants

The restriction sites of Rachiplusia ou nuclear polyhedrosis virus (RoMNPV) DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. Of the 60 DNA restriction sites of RoMNPV, 35 mapped in similar positions as compared to the restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA. Two plaque-purified viruses, obtained from randomly picked plaques of a wild-type isolate of RoMNPV, were recombinants of RoMNPV and AcMNPV. The recombinants were shown to have RoMNPV and AcMNPV restriction fragments as well as structural polypeptides from each parental virus. Both recombinant viruses had a major RoMNPV capsid protein but were occluded in the AcMNPV polyhedrin protein.     

Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus

Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.     

In Vivo and In Vitro Analysis of Baculovirus ie-2 Mutants

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.     

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.     

Restriction Maps of Five Autographa californica MNPV Variants, Trichoplusia ni MNPV, and Galleria mellonella MNPV DNAs with Endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI

The restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) E2 DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. The restriction maps of four other AcMNPV variants, Trichoplusia ni (TnMNPV), and Galleria mellonella (GmMNPV) genomes were determined and compared to the endonuclease cleavage maps of AcMNPV E2 DNA. The viral structural polypeptides of AcMNPV variants S3, E2, S1, M3, and R9 were the same when analyzed by polyacrylamide gel electrophoresis. The major structural polypeptides of GmMNPV and TnMNPV had the same pattern in polyacrylamide gels as did AcMNPV structural polypeptides. GmMNPV and TnMNPV had several minor structural protein differences as compared with AcMNPV. AcMNPV variants, TnMNPV, and GmMNPV were distinct but with very similar genomes and protein structures.     

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3′ sialyllactosamine (3′-SLN) and 6′-SLN sialosides with equilibrium dissociation constant (K_D) values of 30.0 μM and 645 μM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus

In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.     

Identification of Genes Involved in the Biosynthesis of the Third and Fourth Sugars of the Methanococcus maripaludis Archaellin N-Linked Tetrasaccharide

N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes—mmp1084, mmp1085, mmp1086, and mmp1087—were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV.     

Glycosylation Provides Both Stimulatory and Inhibitory Effects on Cell Surface and Soluble CD44 Binding to Hyaluronan

Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44–immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal α2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non–N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (K_d = 5 to >150 μM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.     

Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway.     

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.     

Studies of the Nucleopolyhedrovirus Infection Process in Insects by Using the Green Fluorescence Protein as a Reporter

A recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) expressing the green fluorescence protein (GFP) under the control of the AcMNPV polyhedrin promoter was constructed to study the spatial and temporal regulation of baculovirus infection in a permissive host. Larvae that ingested AcMNPV-GFP showed localized expression of GFP in the midgut epithelial cells, as well as hemocytes, at 24 h postinfection. The presence of fluorescence in these tissues indicated not only that the virus was replicating but also that the very late viral proteins were being synthesized. Secondary infection occurred within the tracheal cells throughout the body cavity, confirming earlier reports, and these foci of infection allowed entry of the virus into other tissues, such as the epidermis and the fat body.     

Display of heterologous proteins on gp64null baculovirus virions and enhanced budding mediated by a vesicular stomatitis virus G-stem construct

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.     

Multiple Nucleocapsid Packaging of Autographa californica Nucleopolyhedrovirus Accelerates the Onset of Systemic Infection in Trichoplusia ni

Among the nucleopolyhedroviruses (Baculoviridae), the occlusion-derived virus (ODV), which initiates infection in host insects, may contain only a single nucleocapsid per virion (the SNPVs) or one to many nucleocapsids per virion (the MNPVs), but the significance of this difference is unclear. To gain insight into the biological relevance of these different packaging strategies, we compared pathogenesis induced by ODV fractions enriched for multiple nucleocapsids (ODV-M) or single nucleocapsids (ODV-S) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) containing a β-galactosidase reporter gene. In time course experiments wherein newly molted fourth-instar Trichoplusia ni were challenged with doses of ODV-S or ODV-M that yielded the same final mortality (~70%), we characterized viral foci as either being restricted to the midgut or involving tracheal cells (the secondary target tissue, indicative of systemic infection). We found that while the timing of primary infection by ODV-S and ODV-M was similar, ODV-S established significantly more primary midgut cell foci than ODV-M, but ODV-M infected tracheal cells at twice the rate of ODV-S. The more efficient establishment of tracheal infections by ODV-M decreased the probability that infections were lost by midgut cell sloughing, explaining why higher numbers of primary infections established by ODV-S within larvae were needed to achieve the same final mortality. These results showed that the multiple nucleocapsid packaging strategy of AcMNPV accelerates the onset of irreversible systemic infections and may indicate why MNPVs have wider individual host ranges than SNPVs.     

Sugar–lectin interactions investigated through affinity capillary electrophoresis

The affinity interactions of Concanavalin A (Con A) with various saccharide oligomers (dextrins, dextrans, and selected N-linked glycans from various glycoproteins) have been investigated through a capillary electrophoresis approach. Con A has shown a notable binding discrimination between the α-1,6-linked dextran and α-1,4-linked dextrin oligomers. Both the binding capacity and binding discrimination appear to decrease with an increase in sugar chainlength. While the core structure of N-linked glycans is deemed to be responsible for the overall binding of various glycans to Con A, the presence of mannose units at the non-reducing ends was found to be very beneficial to the affinity interaction with Con A. Finally, a connection between the glycan–lectin interaction and glycoprotein–lectin interaction has also been suggested.     

Evolutionary interactions between N-linked glycosylation sites in the HIV-1 envelope

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.