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The genotype of early-transmitting HIV gp120s promotes α (4) β(7)-reactivity, revealing α (4) β(7) +/CD4+ T cells as key targets in mucosal transmission.

Nawaz F, Cicala C, Van Ryk D, Block KE, Jelicic K, McNally JP, Ogundare O, Pascuccio M, Patel N, Wei D, Fauci AS, Arthos J - PLoS Pathog. (2011)

Bottom Line: Using primary α₄β₇/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin-α₄β₇.High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed.Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4+ T cells. Low-level viral replication occurs initially in mucosal CD4+ T cells, but within days high-level replication occurs in Peyer's patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α₄β₇/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin-α₄β₇. High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α₄β₇ affinity is mediated by sequences encoded in gp120 V1/V2. α₄β₇-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α₄β₇+/CD4+ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.

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A comparison of steady-state CD4-reactivity and α4β7-reactivity of a panel of gp120s.A) Levels of CD4-reactivity (red) and α4β7-reactivity (blue) were assessed for each patient 205F gp120, B) the QA203 month 1 and month 41 chimeric gp120s, C) w.t. and N144Q 92Ug037 gp120s, D) the 1-month and 12 month CAP88 gp120s and a panel of well characterized gp120s including an ancestral B (AN1) and an ancestral C (ANC1) gp120. Inset indicates the staining of the cells employed in this assay with PE conjugated CD4 (red) and β7 (blue) mAbs. Error bars represent the standard deviation of two replicates, and results are representative of three independent experiments using independent donor CD4+ T cells.
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ppat-1001301-g006: A comparison of steady-state CD4-reactivity and α4β7-reactivity of a panel of gp120s.A) Levels of CD4-reactivity (red) and α4β7-reactivity (blue) were assessed for each patient 205F gp120, B) the QA203 month 1 and month 41 chimeric gp120s, C) w.t. and N144Q 92Ug037 gp120s, D) the 1-month and 12 month CAP88 gp120s and a panel of well characterized gp120s including an ancestral B (AN1) and an ancestral C (ANC1) gp120. Inset indicates the staining of the cells employed in this assay with PE conjugated CD4 (red) and β7 (blue) mAbs. Error bars represent the standard deviation of two replicates, and results are representative of three independent experiments using independent donor CD4+ T cells.

Mentions: The affinity of HIV-1 gp120s for CD4 varies over a wide range and these differences can influence the cell-tropism of a viral isolate. For example, high CD4 affinity facilitates replication in macrophages by compensating for the low density of CD4 appearing on the macrophage membrane[45]. Changes in CD4 affinity could theoretically impact the transmissibility of a viral isolate; however, studies of early replicating gp120s have not, to date, found any clear correlation between CD4 affinity and transmission fitness [25]. Numerous studies have, however, shown that both amino acid substitutions and glycan additions/deletions in V1/V2 can influence the conformation of gp120 in a global way [44], [46]. Because V1/V2 plays an important role in α4β7 recognition we determined whether there was any relationship between α4β7-reactivity and CD4-reactivity. We first employed a steady-state binding assay to compare the reactivity of gp120s for α4β7 and CD4. In this assay, retinoic acid-cultured CD4+ T cells were differentially masked with either a CD4 mAb or an α4 mAb, as described in supporting Figure S1. For gp120s derived from early-transmitting viruses we found that α4β7 mediated a greater degree of binding to the cell surface than did CD4. The amount of 205F 0-month founder gp120 bound to α4β7 was 37-fold greater than that bound to CD4, but this differential disappeared in each of the 205F escape gp120s (Figure 6A). Such differences could be mediated entirely by V1/V2, as demonstrated by comparing the CD4- and α4β7-reactivities of the chimeric QA203M1 and QA203M41 gp120s, which are identical in all domains other than V1/V2. 1-month QA203M1 bound 5-fold more to α4β7 than to CD4 while the 41 month QA203M41 was captured primarily by CD4 (Figure 6B). The deletion of a single transmission-linked PNG was sufficient to achieve a binding profile in which more binding to the cell membrane was mediated by α4β7 than by CD4. For example, while the majority of w.t. 92Ug037 binding to the cell surface was mediated by CD4, PNGΔs at the N terminus of V1/V2 altered this pattern such that more binding was now mediated by α4β7 (Figure 6C). Finally, we compared the two CAP88 gp120s to a panel that included several widely studied gp120s and subtypes B and C ancestral/consensus gp120s[35], [47]. Similar to the 205F 0 month founder and the chimeric QA203M1, both CAP88 proteins showed preferential binding to α4β7 over CD4 (Figure 6D). In contrast to the CAP88 gp120s, all other gp120s in the panel, with one exception, exhibited CD4 binding that was equal to or greater than α4β7 binding. The one exception was SF162, a highly neutralization-sensitive gp120 whose gut tropic characteristics have been well-documented[20], [21]. Because levels of α4β7 expression on the cells we employed were equivalent to, or less than CD4 expression levels (Figure 6 inset and Materials and Methods) we can conclude that the steady-state affinity of gp120s, like the two CAP88 proteins, for Mn++ activated α4β7 is greater than their steady-state affinity for CD4. In addition, this comparison underscores the strong α4β7-reactivity of the early-replicating gp120s we analyzed, relative to a number of well-studied gp120s. In this way, these gp120s appear to be better adapted to interact with CD4+ T cells that express a gut-homing receptor.


The genotype of early-transmitting HIV gp120s promotes α (4) β(7)-reactivity, revealing α (4) β(7) +/CD4+ T cells as key targets in mucosal transmission.

Nawaz F, Cicala C, Van Ryk D, Block KE, Jelicic K, McNally JP, Ogundare O, Pascuccio M, Patel N, Wei D, Fauci AS, Arthos J - PLoS Pathog. (2011)

A comparison of steady-state CD4-reactivity and α4β7-reactivity of a panel of gp120s.A) Levels of CD4-reactivity (red) and α4β7-reactivity (blue) were assessed for each patient 205F gp120, B) the QA203 month 1 and month 41 chimeric gp120s, C) w.t. and N144Q 92Ug037 gp120s, D) the 1-month and 12 month CAP88 gp120s and a panel of well characterized gp120s including an ancestral B (AN1) and an ancestral C (ANC1) gp120. Inset indicates the staining of the cells employed in this assay with PE conjugated CD4 (red) and β7 (blue) mAbs. Error bars represent the standard deviation of two replicates, and results are representative of three independent experiments using independent donor CD4+ T cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044691&req=5

ppat-1001301-g006: A comparison of steady-state CD4-reactivity and α4β7-reactivity of a panel of gp120s.A) Levels of CD4-reactivity (red) and α4β7-reactivity (blue) were assessed for each patient 205F gp120, B) the QA203 month 1 and month 41 chimeric gp120s, C) w.t. and N144Q 92Ug037 gp120s, D) the 1-month and 12 month CAP88 gp120s and a panel of well characterized gp120s including an ancestral B (AN1) and an ancestral C (ANC1) gp120. Inset indicates the staining of the cells employed in this assay with PE conjugated CD4 (red) and β7 (blue) mAbs. Error bars represent the standard deviation of two replicates, and results are representative of three independent experiments using independent donor CD4+ T cells.
Mentions: The affinity of HIV-1 gp120s for CD4 varies over a wide range and these differences can influence the cell-tropism of a viral isolate. For example, high CD4 affinity facilitates replication in macrophages by compensating for the low density of CD4 appearing on the macrophage membrane[45]. Changes in CD4 affinity could theoretically impact the transmissibility of a viral isolate; however, studies of early replicating gp120s have not, to date, found any clear correlation between CD4 affinity and transmission fitness [25]. Numerous studies have, however, shown that both amino acid substitutions and glycan additions/deletions in V1/V2 can influence the conformation of gp120 in a global way [44], [46]. Because V1/V2 plays an important role in α4β7 recognition we determined whether there was any relationship between α4β7-reactivity and CD4-reactivity. We first employed a steady-state binding assay to compare the reactivity of gp120s for α4β7 and CD4. In this assay, retinoic acid-cultured CD4+ T cells were differentially masked with either a CD4 mAb or an α4 mAb, as described in supporting Figure S1. For gp120s derived from early-transmitting viruses we found that α4β7 mediated a greater degree of binding to the cell surface than did CD4. The amount of 205F 0-month founder gp120 bound to α4β7 was 37-fold greater than that bound to CD4, but this differential disappeared in each of the 205F escape gp120s (Figure 6A). Such differences could be mediated entirely by V1/V2, as demonstrated by comparing the CD4- and α4β7-reactivities of the chimeric QA203M1 and QA203M41 gp120s, which are identical in all domains other than V1/V2. 1-month QA203M1 bound 5-fold more to α4β7 than to CD4 while the 41 month QA203M41 was captured primarily by CD4 (Figure 6B). The deletion of a single transmission-linked PNG was sufficient to achieve a binding profile in which more binding to the cell membrane was mediated by α4β7 than by CD4. For example, while the majority of w.t. 92Ug037 binding to the cell surface was mediated by CD4, PNGΔs at the N terminus of V1/V2 altered this pattern such that more binding was now mediated by α4β7 (Figure 6C). Finally, we compared the two CAP88 gp120s to a panel that included several widely studied gp120s and subtypes B and C ancestral/consensus gp120s[35], [47]. Similar to the 205F 0 month founder and the chimeric QA203M1, both CAP88 proteins showed preferential binding to α4β7 over CD4 (Figure 6D). In contrast to the CAP88 gp120s, all other gp120s in the panel, with one exception, exhibited CD4 binding that was equal to or greater than α4β7 binding. The one exception was SF162, a highly neutralization-sensitive gp120 whose gut tropic characteristics have been well-documented[20], [21]. Because levels of α4β7 expression on the cells we employed were equivalent to, or less than CD4 expression levels (Figure 6 inset and Materials and Methods) we can conclude that the steady-state affinity of gp120s, like the two CAP88 proteins, for Mn++ activated α4β7 is greater than their steady-state affinity for CD4. In addition, this comparison underscores the strong α4β7-reactivity of the early-replicating gp120s we analyzed, relative to a number of well-studied gp120s. In this way, these gp120s appear to be better adapted to interact with CD4+ T cells that express a gut-homing receptor.

Bottom Line: Using primary α₄β₇/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin-α₄β₇.High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed.Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4+ T cells. Low-level viral replication occurs initially in mucosal CD4+ T cells, but within days high-level replication occurs in Peyer's patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α₄β₇/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin-α₄β₇. High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α₄β₇ affinity is mediated by sequences encoded in gp120 V1/V2. α₄β₇-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α₄β₇+/CD4+ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.

Show MeSH
Related in: MedlinePlus