Comparison of viral Env proteins from acute and chronic infections with subtype C human immunodeficiency virus type 1 identifies differences in glycosylation and CCR5 utilization and suggests a new strategy for immunogen design

Abstract

Understanding human immunodeficiency virus type 1 (HIV-1) transmission is central to developing effective prevention strategies, including a vaccine. We compared phenotypic and genetic variation in HIV-1 env genes from subjects in acute/early infection and subjects with chronic infections in the context of subtype C heterosexual transmission. We found that the transmitted viruses all used CCR5 and required high levels of CD4 to infect target cells, suggesting selection for replication in T cells and not macrophages after transmission. In addition, the transmitted viruses were more likely to use a maraviroc-sensitive conformation of CCR5, perhaps identifying a feature of the target T cell. We confirmed an earlier observation that the transmitted viruses were, on average, modestly underglycosylated relative to the viruses from chronically infected subjects. This difference was most pronounced in comparing the viruses in acutely infected men to those in chronically infected women. These features of the transmitted virus point to selective pressures during the transmission event. We did not observe a consistent difference either in heterologous neutralization sensitivity or in sensitivity to soluble CD4 between the two groups, suggesting similar conformations between viruses from acute and chronic infection. However, the presence or absence of glycosylation sites had differential effects on neutralization sensitivity for different antibodies. We suggest that the occasional absence of glycosylation sites encoded in the conserved regions of env, further reduced in transmitted viruses, could expose specific surface structures on the protein as antibody targets.

Fig 1

High CD4 dependence and differential use of CCR5 conformers of Env proteins. Subtype C env clones derived from both acute (red) and chronic (blue) infections were used to generate pseudotyped viruses carrying a luciferase reporter gene. (A) The resulting viruses were used to infect Affinofile cells expressing 10 levels of surface CD4 (1,780, 7,360, 2,7983, 36,320, 43,758, 45,508, 45,931, 57,165, 58,152, and 63,498 ABS/cell) and a single high level of CCR5 (31,990 ABS/cell). After 48 h of infection, the cells were lysed and luciferase levels measured as relative light units (RLU). The percentage of RLUs measured at each CD4 level relative to the RLUs for high-surface-CD4 infection for each of the viruses is plotted. Included are controls consisting of Ba-L, to demonstrate the phenotype of a macrophage-tropic virus, and JR-CSF, to demonstrate the phenotype of a virus that requires high levels of CD4 to infect cells (R5 T cell-tropic). (B) Reporter pseudoviruses were also used to infect Affinofile cells expressing high levels of CD4 (88,104 ABS/cell) and CCR5 (35,981 ABS/cell) in the presence of 10 levels of the CCR5 antagonist maraviroc (0.0001, 0.002, 0.003, 0.006, 0.013, 0.025, 0.05, 0.1, 1, and 10 μM). Five of the acute clones were also analyzed in reference . The pseudotyped viruses interpreted to have a plateau of at least 10% infectivity at the highest maraviroc concentration have an asterisk by the subject code. The viruses used in the analysis whose results are shown in Figure 2B have a double cross included by the subject code.

Fig 2

Transmission selects for viruses that use a maraviroc-sensitive conformation of CCR5. (A) We infected Affinofile cells expressing high levels of CCR5 with a panel of viruses (data taken from the experiments whose results are shown in Fig. 1) and observed that, in the presence of high levels of maraviroc, a greater fraction of the viruses from chronic infections had a plateau of infectivity of greater than 10% (Fisher's exact test; odds ratio = 0.23, P = 0.01). (B) In order to determine whether resistance to maraviroc is mediated by CCR5 level, we infected Affinofile cells expressing low levels of CCR5 in the presence of maraviroc (10 μM) using a subset of the viruses described in the legend to Figure 1 (13 from acute infections and 21 from chronic infections, representing a range of sensitivities to maraviroc). All of these viruses were completely sensitive to inhibition by maraviroc when CCR5 levels were reduced. An analysis of three of these acute viruses was included in reference .

Fig 3

env genes from acutely infected individuals encode fewer N-linked glycosylation sites. Amplicons of env genes were generated from acutely infected subjects and chronically infected subjects and sequenced. Each transmitted virus was represented by a single sequence, while all sequences from the chronically infected subjects were included in the analysis. The number of encoded N-linked glycosylation sites was determined for each sequence based on the appearance of the motif encoding NXS/T. (A) env sequences derived from acutely infected subjects were compared to env sequences from chronically infected subjects and were found to encode significantly fewer N-linked glycosylation sites than the env sequences from chronically infected patients (Wilcoxon rank sum; W = 33,681, P = 2 × 10⁻⁶). (B) env sequences derived from acutely infected males encode fewer N-linked glycosylation sites than env sequences derived from chronically infected females (Wilcoxon rank sum; W = 8,547, P = 5 × 10⁻⁶). (C) env sequences derived from acutely infected females do not encode a significantly different number of N-linked glycosylation sites compared to env sequences derived from chronically infected males (Wilcoxon rank sum; W = 7,734, P = 0.13). (D) The frequency of appearance of N-linked glycosylation sites encoded in the conserved domains of the env sequences (and present in at least 50% of sequences) was determined separately for the two groups of env sequences and plotted for each position (HXB numbering). Adjacent positions that encoded mutually exclusive sites were pooled and are indicated by two position numbers. At those positions where the transmitted virus sequences encode N-linked glycosylation sites at a greater frequency, the histogram is oriented up. At those positions where the sequences from the chronically infected subjects encode N-linked glycosylation sites at a greater frequency, the histogram is oriented down. The positions are placed over a map of the Env protein to show their presence in either gp120 or gp41. Four positions showed a statistically significant difference in glycosylation count between the two groups at a P value of <0.05 (Fisher's exact test), and these are boxed and indicated with a single asterisk. One position, 442, showed a difference at a P value of <0.0001 and is shown boxed and with a double asterisk.

Fig 4

Comparison of sensitivity to antibody neutralization. (A and B) Viruses pseudotyped with Env proteins derived from chronically infected (blue) or acutely infected (red) subjects were exposed to differing amounts of the indicated monoclonal antibodies or soluble CD4 (sCD4) (A) or to differing amounts of the indicated polyclonal sera (purified IgG fraction) (B). For each virus, the IC₅₀ was determined and plotted. The antibody that has the largest difference in median values (shown by horizontal bars) is boxed, and the associated P value indicated (Kruskal-Wallis test). (C) Analysis of variance was used to evaluate the linear model that best describes the relationship between neutralization sensitivity to each antibody/serum and 22 N-linked glycosylation sites in gp120 and gp41. Relationships that are significant after correcting for multiple comparisons are plotted (*, P < 0.05; **, P < 0.01).

Fig 5

Distribution of N-linked glycosylation sites among transmitted viruses. Each of the 87 transmitted viruses in this study is represented by a single horizontal block, separated by the horizontal lines. The presence (shaded) or absence (white) of specific N-linked glycosylation sites is indicated. The glycosylation sites are grouped as being proximal to specific surface structures of the gp120 Env protein. HXB numbering is used for the positions of the glycan attachment sites, which are shown at the top.