RESUMO
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.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , HIV-2/genética , HIV-2/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Motivos de Aminoácidos , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , HIV-2/imunologia , Humanos , Mutação , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais Reguladoras e Acessórias/metabolismo , Vírion , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Dietary potassium loading results in rapid kaliuresis, natriuresis, and diuresis associated with reduced phosphorylation (p) of the distal tubule Na(+)-Cl(-) cotransporter (NCC). Decreased NCC-p inhibits NCC-mediated Na(+) reabsorption and shifts Na(+) downstream for reabsorption by epithelial Na(+) channels (ENaC), which can drive K(+) secretion. Whether the signal is initiated by ingesting potassium or a rise in plasma K(+) concentration ([K(+)]) is not understood. We tested the hypothesis, in male rats, that an increase in plasma [K(+)] is sufficient to reduce NCC-p and drive kaliuresis. After an overnight fast, a single 3-h 2% potassium (2%K) containing meal increased plasma [K(+)] from 4.0 ± 0.1 to 5.2 ± 0.2 mM; increased urinary K(+), Na(+), and volume excretion; decreased NCC-p by 60%; and marginally reduced cortical Na(+)-K(+)-2Cl(-) cotransporter (NKCC) phosphorylation 25% (P = 0.055). When plasma [K(+)] was increased by tail vein infusion of KCl to 5.5 ± 0.1 mM over 3 h, significant kaliuresis and natriuresis ensued, NCC-p decreased by 60%, and STE20/SPS1-related proline alanine-rich kinase (SPAK) phosphorylation was marginally reduced 35% (P = 0.052). The following were unchanged at 3 h by either the potassium-rich meal or KCl infusion: Na(+)/H(+) exchanger 3 (NHE3), NHE3-p, NKCC, ENaC subunits, and renal outer medullary K(+) channel. In summary, raising plasma [K(+)] by intravenous infusion to a level equivalent to that observed after a single potassium-rich meal triggers renal kaliuretic and natriuretic responses, independent of K(+) ingestion, likely driven by decreased NCC-p and activity sufficient to shift sodium reabsorption downstream to where Na(+) reabsorption and flow drive K(+) secretion.
Assuntos
Hiperpotassemia/sangue , Rim/metabolismo , Natriurese , Potássio/sangue , Sódio/urina , Animais , Modelos Animais de Doenças , Canais Epiteliais de Sódio/metabolismo , Hiperpotassemia/induzido quimicamente , Hiperpotassemia/fisiopatologia , Hiperpotassemia/urina , Infusões Intravenosas , Rim/fisiopatologia , Masculino , Fosforilação , Potássio/administração & dosagem , Potássio/urina , Canais de Potássio/metabolismo , Potássio na Dieta/sangue , Potássio na Dieta/urina , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Sódio/sangue , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Fatores de TempoRESUMO
BST-2/tetherin is an interferon-inducible host restriction factor that blocks the release of newly formed enveloped viruses. It is enriched in lipid raft membrane microdomains, which are also the sites of assembly of several enveloped viruses. Viral anti-tetherin factors, such as the HIV-1 Vpu protein, typically act by removing tetherin from the cell surface. In contrast, the Ebola virus glycoprotein (GP) is unusual in that it blocks tetherin restriction without apparently altering its cell surface localization. We explored the possibility that GP acts to exclude tetherin from the specific sites of virus assembly without overtly removing it from the cell surface and that lipid raft exclusion is the mechanism involved. However, we found that neither GP nor Vpu had any effect on tetherin's distribution within lipid raft domains. Furthermore, GP did not prevent the colocalization of tetherin and budding viral particles. Contrary to previous reports, we also found no evidence that GP is itself a raft protein. Together, our data indicate that the exclusion of tetherin from lipid rafts is not the mechanism used by either HIV-1 Vpu or Ebola virus GP to counteract tetherin restriction.
Assuntos
Antígenos CD/metabolismo , Ebolavirus/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Doença pelo Vírus Ebola/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos CD/genética , Linhagem Celular , Ebolavirus/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Doença pelo Vírus Ebola/virologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/virologia , Ligação Proteica , Transporte Proteico , Proteínas do Envelope Viral/genética , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
We have developed a modified blue native polyacrylamide gel electrophoresis (PAGE) protocol that can overcome aggregation of lipases seen in native PAGE. We have shown that two lipases, Pseudomonas aeruginosa lipase and Candida rugosa lipase, which aggregate in the native gel, can be resolved using our protocol. Activity staining was done to test for the functionality of the two lipases.