Limits...
Determinants in HIV-2 Env and tetherin required for functional interaction.

Exline CM, Yang SJ, Haworth KG, Rengarajan S, Lopez LA, Droniou ME, Seclen E, Cannon PM - Retrovirology (2015)

Bottom Line: The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein.Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env.These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 502, Los Angeles, CA, 90033, USA. colinexline@gmail.com.

ABSTRACT

Background: The interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity.

Results: By examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin.

Conclusions: These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

No MeSH data available.


Related in: MedlinePlus

HIV-2 Env mutants that disrupt the interaction with tetherin. a HIV-1 Gag-Pol VLPs were created by transfecting 293A cells with pHIV-1-pack, together with a control CMV expression plasmid (−) or expression plasmids for tetherin and the indicated Envs. 24 h later, VLP release was measured as the ratio of p24-reacting bands in supernatants versus cell lysates following Western blot analysis. Results were normalized to the no tetherin control which was set at 100% VLP release. Graphs show mean plus standard deviation for n = 3 independent experiments, p < 0.05 (*). b 293T cells were co-transfected with tetherin and GFP-tagged Env variants. Cells were lysed 24 h later and GFP-tagged proteins were immunoprecipitated (IP) with anti-GFP MicroBeads, followed by Western blotting of the input lysates (1%) and IP products using anti-Env, anti-GFP or anti-tetherin antibodies.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4528709&req=5

Fig1: HIV-2 Env mutants that disrupt the interaction with tetherin. a HIV-1 Gag-Pol VLPs were created by transfecting 293A cells with pHIV-1-pack, together with a control CMV expression plasmid (−) or expression plasmids for tetherin and the indicated Envs. 24 h later, VLP release was measured as the ratio of p24-reacting bands in supernatants versus cell lysates following Western blot analysis. Results were normalized to the no tetherin control which was set at 100% VLP release. Graphs show mean plus standard deviation for n = 3 independent experiments, p < 0.05 (*). b 293T cells were co-transfected with tetherin and GFP-tagged Env variants. Cells were lysed 24 h later and GFP-tagged proteins were immunoprecipitated (IP) with anti-GFP MicroBeads, followed by Western blotting of the input lysates (1%) and IP products using anti-Env, anti-GFP or anti-tetherin antibodies.

Mentions: Previously, it was reported that the Env protein from HIV-2 strain ROD10 can be co-immunoprecipitated with tetherin [37]. However it was not established if this interaction was necessary for the Env protein’s anti-tetherin activity. To analyze this further, we selected two closely related mutants of ROD10 Env that do not counteract tetherin [29, 37], and evaluated their ability to bind to the protein. The ROD14 Env differs from ROD10 Env at 5 specific amino acids, and contains a 30 amino acid deletion in its cytoplasmic tail [40], with substitutions K422R and A598T in the ectodomain of the protein being primarily responsible for its loss of tetherin antagonism [41]. In addition, mutant ROD10 EnvY707A contains a point mutation that disrupts an endocytosis motif in its cytoplasmic tail, and this is sufficient to prevent tetherin antagonism [35, 37]. We confirmed that both of these Env variants lacked the ability to counteract tetherin, since they could not stimulate the release of HIV-1 Gag-Pol virus-like particles (VLPs) from tetherin-expressing cells (Fig. 1a).Fig. 1


Determinants in HIV-2 Env and tetherin required for functional interaction.

Exline CM, Yang SJ, Haworth KG, Rengarajan S, Lopez LA, Droniou ME, Seclen E, Cannon PM - Retrovirology (2015)

HIV-2 Env mutants that disrupt the interaction with tetherin. a HIV-1 Gag-Pol VLPs were created by transfecting 293A cells with pHIV-1-pack, together with a control CMV expression plasmid (−) or expression plasmids for tetherin and the indicated Envs. 24 h later, VLP release was measured as the ratio of p24-reacting bands in supernatants versus cell lysates following Western blot analysis. Results were normalized to the no tetherin control which was set at 100% VLP release. Graphs show mean plus standard deviation for n = 3 independent experiments, p < 0.05 (*). b 293T cells were co-transfected with tetherin and GFP-tagged Env variants. Cells were lysed 24 h later and GFP-tagged proteins were immunoprecipitated (IP) with anti-GFP MicroBeads, followed by Western blotting of the input lysates (1%) and IP products using anti-Env, anti-GFP or anti-tetherin antibodies.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4528709&req=5

Fig1: HIV-2 Env mutants that disrupt the interaction with tetherin. a HIV-1 Gag-Pol VLPs were created by transfecting 293A cells with pHIV-1-pack, together with a control CMV expression plasmid (−) or expression plasmids for tetherin and the indicated Envs. 24 h later, VLP release was measured as the ratio of p24-reacting bands in supernatants versus cell lysates following Western blot analysis. Results were normalized to the no tetherin control which was set at 100% VLP release. Graphs show mean plus standard deviation for n = 3 independent experiments, p < 0.05 (*). b 293T cells were co-transfected with tetherin and GFP-tagged Env variants. Cells were lysed 24 h later and GFP-tagged proteins were immunoprecipitated (IP) with anti-GFP MicroBeads, followed by Western blotting of the input lysates (1%) and IP products using anti-Env, anti-GFP or anti-tetherin antibodies.
Mentions: Previously, it was reported that the Env protein from HIV-2 strain ROD10 can be co-immunoprecipitated with tetherin [37]. However it was not established if this interaction was necessary for the Env protein’s anti-tetherin activity. To analyze this further, we selected two closely related mutants of ROD10 Env that do not counteract tetherin [29, 37], and evaluated their ability to bind to the protein. The ROD14 Env differs from ROD10 Env at 5 specific amino acids, and contains a 30 amino acid deletion in its cytoplasmic tail [40], with substitutions K422R and A598T in the ectodomain of the protein being primarily responsible for its loss of tetherin antagonism [41]. In addition, mutant ROD10 EnvY707A contains a point mutation that disrupts an endocytosis motif in its cytoplasmic tail, and this is sufficient to prevent tetherin antagonism [35, 37]. We confirmed that both of these Env variants lacked the ability to counteract tetherin, since they could not stimulate the release of HIV-1 Gag-Pol virus-like particles (VLPs) from tetherin-expressing cells (Fig. 1a).Fig. 1

Bottom Line: The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein.Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env.These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 502, Los Angeles, CA, 90033, USA. colinexline@gmail.com.

ABSTRACT

Background: The interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity.

Results: By examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin.

Conclusions: These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

No MeSH data available.


Related in: MedlinePlus