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The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors.

Rossi F, Querido B, Nimmagadda M, Cocklin S, Navas-Martín S, Martín-García J - Retrovirology (2008)

Bottom Line: We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249.Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis.Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology and Center for Molecular Virology and Neuroimmunology, Drexel University College of Medicine, Philadelphia, PA 19102, USA. fpr23@drexel.edu

ABSTRACT

Background: HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env.

Results: Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected.

Conclusion: We have identified that the V1-V3 region of a brain-derived envelope glycoprotein seems to play a crucial role in determining not only the low CD4 dependence and increased macrophage tropism, but also the augmented fusogenicity and reduced sensitivity to T-1249 and BMS-378806. By contrast, increased sensitivity to HNG-105 mostly correlated with low CD4 dependence and macrophage tropism but was not determined by the presence of the brain's V1-V3 region, confirming that viral determinants of phenotypic changes in brain-derived envelope glycoproteins are likely complex and context-dependent.

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Pseudotype infection of HOS-CD4-CCR5 cells (top) and monocyte-derived macrophages (bottom). Pseudotypes containing wild-type, chimeric and mutant Env that had shown low CD4 dependence and high avidity for CD4, as well as reduced sensitivity to fusion inhibitors and BMS-378806, showed statistically significant greater macrophage tropism than those without low CD4 dependence and less avidity for CD4. Results shown are the mean relative light units (RLU) per second ± standard error of 3 independent experiments (each performed at least in triplicate).
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Figure 7: Pseudotype infection of HOS-CD4-CCR5 cells (top) and monocyte-derived macrophages (bottom). Pseudotypes containing wild-type, chimeric and mutant Env that had shown low CD4 dependence and high avidity for CD4, as well as reduced sensitivity to fusion inhibitors and BMS-378806, showed statistically significant greater macrophage tropism than those without low CD4 dependence and less avidity for CD4. Results shown are the mean relative light units (RLU) per second ± standard error of 3 independent experiments (each performed at least in triplicate).

Mentions: In order to evaluate whether a relationship between the degree of low CD4 dependence and/or increased fusogenicity of wild-type, chimeric and mutant Env, and their ability to mediate infection of primary macrophages (or macrophage tropism) could be established, MDMs were infected with equivalent amounts of pseudotype stocks. HOS-CD4-CCR5 cells were infected in parallel. After 2–3 days, the extent of infection was measured by luciferase activity in cell lysates. As shown in Figure 7, HOS-CD4-CCR5 cells were infected to a similar extent by all pseudotypes, with luciferase activity at least 1000-fold above background levels (mock infection refers to supernatants containing viral particles lacking Env produced by cells co-transfected with the Env-deficient luciferase backbone and an empty vector). By contrast, only those pseudotypes with low CD4-dependent Env showed the ability to infect MDMs, although with various efficiencies. Infection with wild-type BR and BS chimera resulted in luciferase activities 1000-fold above mock infection, and greater than a 100-fold increase was observed with the Bv1v3 chimera, the BR(N283T) mutant and the DS17 and DS17(N283T). The Bv1v2 chimera containing the V1/V2 region of the brain Env in the context of the spleen Env mediated infection of macrophages to a lower extent than BR or Bv1v3, although luciferase levels were still 20-fold above background, indicating that brain Env's V3 region plays a role, together with the V1/V2, in increasing macrophage tropism. However, the Sv1v2 chimera containing the V1/V2 region from SPL and the remaining of the Env from BR (including the V3 loop) failed to mediate any infection of macrophages suggesting that, by itself, the brain Env's V3 region does not confer macrophage tropism.


The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors.

Rossi F, Querido B, Nimmagadda M, Cocklin S, Navas-Martín S, Martín-García J - Retrovirology (2008)

Pseudotype infection of HOS-CD4-CCR5 cells (top) and monocyte-derived macrophages (bottom). Pseudotypes containing wild-type, chimeric and mutant Env that had shown low CD4 dependence and high avidity for CD4, as well as reduced sensitivity to fusion inhibitors and BMS-378806, showed statistically significant greater macrophage tropism than those without low CD4 dependence and less avidity for CD4. Results shown are the mean relative light units (RLU) per second ± standard error of 3 independent experiments (each performed at least in triplicate).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2576352&req=5

Figure 7: Pseudotype infection of HOS-CD4-CCR5 cells (top) and monocyte-derived macrophages (bottom). Pseudotypes containing wild-type, chimeric and mutant Env that had shown low CD4 dependence and high avidity for CD4, as well as reduced sensitivity to fusion inhibitors and BMS-378806, showed statistically significant greater macrophage tropism than those without low CD4 dependence and less avidity for CD4. Results shown are the mean relative light units (RLU) per second ± standard error of 3 independent experiments (each performed at least in triplicate).
Mentions: In order to evaluate whether a relationship between the degree of low CD4 dependence and/or increased fusogenicity of wild-type, chimeric and mutant Env, and their ability to mediate infection of primary macrophages (or macrophage tropism) could be established, MDMs were infected with equivalent amounts of pseudotype stocks. HOS-CD4-CCR5 cells were infected in parallel. After 2–3 days, the extent of infection was measured by luciferase activity in cell lysates. As shown in Figure 7, HOS-CD4-CCR5 cells were infected to a similar extent by all pseudotypes, with luciferase activity at least 1000-fold above background levels (mock infection refers to supernatants containing viral particles lacking Env produced by cells co-transfected with the Env-deficient luciferase backbone and an empty vector). By contrast, only those pseudotypes with low CD4-dependent Env showed the ability to infect MDMs, although with various efficiencies. Infection with wild-type BR and BS chimera resulted in luciferase activities 1000-fold above mock infection, and greater than a 100-fold increase was observed with the Bv1v3 chimera, the BR(N283T) mutant and the DS17 and DS17(N283T). The Bv1v2 chimera containing the V1/V2 region of the brain Env in the context of the spleen Env mediated infection of macrophages to a lower extent than BR or Bv1v3, although luciferase levels were still 20-fold above background, indicating that brain Env's V3 region plays a role, together with the V1/V2, in increasing macrophage tropism. However, the Sv1v2 chimera containing the V1/V2 region from SPL and the remaining of the Env from BR (including the V3 loop) failed to mediate any infection of macrophages suggesting that, by itself, the brain Env's V3 region does not confer macrophage tropism.

Bottom Line: We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249.Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis.Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology and Center for Molecular Virology and Neuroimmunology, Drexel University College of Medicine, Philadelphia, PA 19102, USA. fpr23@drexel.edu

ABSTRACT

Background: HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env.

Results: Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected.

Conclusion: We have identified that the V1-V3 region of a brain-derived envelope glycoprotein seems to play a crucial role in determining not only the low CD4 dependence and increased macrophage tropism, but also the augmented fusogenicity and reduced sensitivity to T-1249 and BMS-378806. By contrast, increased sensitivity to HNG-105 mostly correlated with low CD4 dependence and macrophage tropism but was not determined by the presence of the brain's V1-V3 region, confirming that viral determinants of phenotypic changes in brain-derived envelope glycoproteins are likely complex and context-dependent.

Show MeSH
Related in: MedlinePlus