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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 the α4β7-reactivities mediated by the V1/V2 regions of a month-1 gp120 and month 41 gp120 isolated from a single patient.A) The gp120 V1/V2 sequences from patient QA203 obtained at month 1 and month 41 are listed. PNGs are highlighted in red. Variants, 1, 2 and 3 of QA203M41 are included with the N/Q substitutions highlighted in red. B) α4β7-reactivities of chimeric gp120s encoding the V1/V2 regions of QA203M1, QA203M41 and QA203M41 variant 1 are reported as mean fluorescence intensity. C) A comparison of the α4β7-reactivities of variants 1, 2 and 3 of QA203M41. QA203M41 is included for reference. 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-g002: A comparison of the α4β7-reactivities mediated by the V1/V2 regions of a month-1 gp120 and month 41 gp120 isolated from a single patient.A) The gp120 V1/V2 sequences from patient QA203 obtained at month 1 and month 41 are listed. PNGs are highlighted in red. Variants, 1, 2 and 3 of QA203M41 are included with the N/Q substitutions highlighted in red. B) α4β7-reactivities of chimeric gp120s encoding the V1/V2 regions of QA203M1, QA203M41 and QA203M41 variant 1 are reported as mean fluorescence intensity. C) A comparison of the α4β7-reactivities of variants 1, 2 and 3 of QA203M41. QA203M41 is included for reference. Error bars represent the standard deviation of two replicates, and results are representative of three independent experiments using independent donor CD4+ T cells.

Mentions: Length-shortening in V1/V2 is also associated with early transmitting gp120s. However, because those deletions often result in PNGΔs, it is difficult to determine whether short V1/V2s are favored independently of PNGΔs [34]. We examined the influence of V1/V2 length shortening on α4β7-reactivity by taking advantage of a previously characterized pair of subtype A envelopes isolated from an individual at ∼1-month and ∼41-months post-infection[23]. The month 1 V1/V2 (QA203M1) encodes 63 amino acids and 5 PNGs, while the month 41 V1/V2 (QA203M41) encodes 70 amino acids and 8 PNGs such that the 41 month envelope encodes two additional PNGs near the N-terminus of V1 and one additional PNG near the C-terminus of V2 (Figure 2A). Both V1/V2s were grafted into a subtype A gp120 backbone isolated ∼1year post-infection from a second patient. QA203M1 gp120 displayed ∼20× greater α4β7-reactivity than did w.t. QA203M41 (Figure 2B). To distinguish the influence of V1/V2 length from the influence of the number of PNGs on α4β7 reactivity we constructed a variant of the month-41 V1/V2 (QA203M41variant1) lacking the two V1 PNGs that were missing in QA203M1 without changing its length (Figure 2A). QA203M41 variant 1, exhibited relatively strong binding to α4β7 that was nearly identical to that of QA203M1 gp120. These results indicate that increased α4β7-reactivity mediated by the early-transmitting QA203M1 relative to QA203M41 was due to PNGΔs rather than to a shorter V1/V2. We extended this analysis by removing additional PNGs from QA203M41. QA203M41 variant 2 in which PNGs were removed from the C-terminal region of V2 mediated a small increase in α4β7-reactivity, while QA203M41 variant 3, which combines variant 1 and variant 2 PNGΔs mediated an increase in α4β7-reactivity that was intermediate between variant 1 and variant 2 (Figure 2A, C). These results demonstrate that PNGΔs do not necessarily enhance α4β7-reactivity in an additive manner.


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 the α4β7-reactivities mediated by the V1/V2 regions of a month-1 gp120 and month 41 gp120 isolated from a single patient.A) The gp120 V1/V2 sequences from patient QA203 obtained at month 1 and month 41 are listed. PNGs are highlighted in red. Variants, 1, 2 and 3 of QA203M41 are included with the N/Q substitutions highlighted in red. B) α4β7-reactivities of chimeric gp120s encoding the V1/V2 regions of QA203M1, QA203M41 and QA203M41 variant 1 are reported as mean fluorescence intensity. C) A comparison of the α4β7-reactivities of variants 1, 2 and 3 of QA203M41. QA203M41 is included for reference. 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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044691&req=5

ppat-1001301-g002: A comparison of the α4β7-reactivities mediated by the V1/V2 regions of a month-1 gp120 and month 41 gp120 isolated from a single patient.A) The gp120 V1/V2 sequences from patient QA203 obtained at month 1 and month 41 are listed. PNGs are highlighted in red. Variants, 1, 2 and 3 of QA203M41 are included with the N/Q substitutions highlighted in red. B) α4β7-reactivities of chimeric gp120s encoding the V1/V2 regions of QA203M1, QA203M41 and QA203M41 variant 1 are reported as mean fluorescence intensity. C) A comparison of the α4β7-reactivities of variants 1, 2 and 3 of QA203M41. QA203M41 is included for reference. Error bars represent the standard deviation of two replicates, and results are representative of three independent experiments using independent donor CD4+ T cells.
Mentions: Length-shortening in V1/V2 is also associated with early transmitting gp120s. However, because those deletions often result in PNGΔs, it is difficult to determine whether short V1/V2s are favored independently of PNGΔs [34]. We examined the influence of V1/V2 length shortening on α4β7-reactivity by taking advantage of a previously characterized pair of subtype A envelopes isolated from an individual at ∼1-month and ∼41-months post-infection[23]. The month 1 V1/V2 (QA203M1) encodes 63 amino acids and 5 PNGs, while the month 41 V1/V2 (QA203M41) encodes 70 amino acids and 8 PNGs such that the 41 month envelope encodes two additional PNGs near the N-terminus of V1 and one additional PNG near the C-terminus of V2 (Figure 2A). Both V1/V2s were grafted into a subtype A gp120 backbone isolated ∼1year post-infection from a second patient. QA203M1 gp120 displayed ∼20× greater α4β7-reactivity than did w.t. QA203M41 (Figure 2B). To distinguish the influence of V1/V2 length from the influence of the number of PNGs on α4β7 reactivity we constructed a variant of the month-41 V1/V2 (QA203M41variant1) lacking the two V1 PNGs that were missing in QA203M1 without changing its length (Figure 2A). QA203M41 variant 1, exhibited relatively strong binding to α4β7 that was nearly identical to that of QA203M1 gp120. These results indicate that increased α4β7-reactivity mediated by the early-transmitting QA203M1 relative to QA203M41 was due to PNGΔs rather than to a shorter V1/V2. We extended this analysis by removing additional PNGs from QA203M41. QA203M41 variant 2 in which PNGs were removed from the C-terminal region of V2 mediated a small increase in α4β7-reactivity, while QA203M41 variant 3, which combines variant 1 and variant 2 PNGΔs mediated an increase in α4β7-reactivity that was intermediate between variant 1 and variant 2 (Figure 2A, C). These results demonstrate that PNGΔs do not necessarily enhance α4β7-reactivity in an additive manner.

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