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1.
J Biol Chem ; 290(6): 3740-51, 2015 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-25525265

RESUMEN

BST-2/tetherin is a cellular host factor capable of restricting the release of a variety of enveloped viruses, including HIV-1. Structurally, BST-2 consists of an N-terminal cytoplasmic domain, a transmembrane domain, an ectodomain, and a C-terminal membrane anchor. The BST-2 ectodomain encodes three cysteine residues in its N-terminal half, each of which can contribute to the formation of cysteine-linked dimers. We previously reported that any one of the three cysteine residues is sufficient to produce functional BST-2 dimers. Here we investigated the importance of cysteine positioning on the ectodomain for functional dimerization of BST-2. Starting with a cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced at various locations throughout the ectodomain. The resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. We found significant flexibility in the positioning of cysteine residues, although the propensity to form cysteine-linked dimers generally decreased with increasing distance from the N terminus. Interestingly, all BST-2 variants, including the one lacking all three ectodomain cysteines, retained the ability to form non-covalent dimers, and all of the BST-2 variants were efficiently expressed at the cell surface. Importantly, not all BST-2 variants capable of forming cysteine-linked dimers were functional, suggesting that cysteine-linked dimerization of BST-2 is necessary but not sufficient for inhibiting virus release. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell.


Asunto(s)
Antígenos CD/química , Cisteína/química , Multimerización de Proteína , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antígenos CD/genética , Antígenos CD/metabolismo , Membrana Celular/metabolismo , Cisteína/genética , Cisteína/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Internalización del Virus
2.
J Biol Chem ; 287(53): 44278-88, 2012 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-23152502

RESUMEN

BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.


Asunto(s)
Antígenos CD/química , Antígenos CD/metabolismo , Regulación hacia Abajo , Infecciones por VIH/metabolismo , VIH-1/fisiología , Virión/fisiología , Liberación del Virus , Secuencia de Aminoácidos , Antígenos CD/genética , Dimerización , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Virión/genética
3.
J Biol Chem ; 286(46): 39967-81, 2011 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-21937450

RESUMEN

BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.


Asunto(s)
Antígenos CD/metabolismo , Secuencias de Aminoácidos , Antígenos CD/genética , Antígenos CD40/genética , Antígenos CD40/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/metabolismo , Células HeLa , Humanos , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
4.
J Virol ; 85(6): 2611-9, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21191020

RESUMEN

BST-2/CD317/tetherin is a host factor that inhibits the release of HIV-1 and other unrelated viruses. A current model proposes that BST-2 physically tethers virions to the surface of virus-producing cells. The HIV-1-encoded Vpu protein effectively antagonizes the activity of BST-2. How Vpu accomplishes this task remains unclear; however, it is known that Vpu has the ability to down-modulate BST-2 from the cell surface. Here we analyzed the effects of Vpu on BST-2 by performing a series of kinetic studies with HeLa, 293T, and CEMx174 cells. Our results indicate that the surface downregulation of BST-2 is not due to an accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell. While our data confirm previous reports that the high-level expression of Vpu can cause the endoplasmic reticulum (ER)-associated degradation of BST-2, we found no evidence that Vpu targets endogenous BST-2 in the ER in the course of a viral infection. Instead, we found that Vpu acts in a post-ER compartment and increases the turnover of newly synthesized mature BST-2. Our observation that Vpu does not affect the recycling of BST-2 suggests that Vpu does not act directly at the cell surface but may interfere with the trafficking of newly synthesized BST-2 to the cell surface, resulting in the accelerated targeting of BST-2 to the lysosomal compartment for degradation.


Asunto(s)
Antígenos CD/metabolismo , VIH-1/inmunología , VIH-1/patogenicidad , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Antígenos CD/inmunología , Línea Celular , Regulación hacia Abajo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Humanos , Transporte de Proteínas , Proteínas Reguladoras y Accesorias Virales/inmunología
5.
Proc Natl Acad Sci U S A ; 106(8): 2868-73, 2009 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-19196977

RESUMEN

HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.


Asunto(s)
Antígenos CD/fisiología , Regulación hacia Abajo , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas Reguladoras y Accesorias Virales/fisiología , Antígenos CD/metabolismo , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Citometría de Flujo , Proteínas Ligadas a GPI , Humanos , Sueros Inmunes , Glicoproteínas de Membrana/metabolismo , Virión , Ensamble de Virus
6.
Retrovirology ; 6: 80, 2009 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-19737401

RESUMEN

BACKGROUND: The Human Immunodeficiency virus type 1 (HIV-1) Vpu protein enhances virus release from infected cells and induces proteasomal degradation of CD4. Recent work identified BST-2/CD317 as a host factor that inhibits HIV-1 virus release in a Vpu sensitive manner. A current working model proposes that BST-2 inhibits virus release by tethering viral particles to the cell surface thereby triggering their subsequent endocytosis. RESULTS: Here we defined structural properties of BST-2 required for inhibition of virus release and for sensitivity to Vpu. We found that BST-2 is modified by N-linked glycosylation at two sites in the extracellular domain. However, N-linked glycosylation was not important for inhibition of HIV-1 virus release nor did it affect surface expression or sensitivity to Vpu. Rodent BST-2 was previously found to form cysteine-linked dimers. Analysis of single, double, or triple cysteine mutants revealed that any one of three cysteine residues present in the BST-2 extracellular domain was sufficient for BST-2 dimerization, for inhibition of virus release, and sensitivity to Vpu. In contrast, BST-2 lacking all three cysteines in its ectodomain was unable to inhibit release of wild type or Vpu-deficient HIV-1 virions. This defect was not caused by a gross defect in BST-2 trafficking as the mutant protein was expressed at the cell surface of transfected 293T cells and was down-modulated by Vpu similar to wild type BST-2. CONCLUSION: While BST-2 glycosylation was functionally irrelevant, formation of cysteine-linked dimers appeared to be important for inhibition of virus release. However lack of dimerization did not prevent surface expression or Vpu sensitivity of BST-2, suggesting Vpu sensitivity and inhibition of virus release are separable properties of BST-2.


Asunto(s)
Antígenos CD/inmunología , VIH-1/inmunología , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/fisiología , Glicoproteínas de Membrana/inmunología , Proteínas Reguladoras y Accesorias Virales/fisiología , Replicación Viral , Sustitución de Aminoácidos/genética , Antígenos CD/metabolismo , Línea Celular , Membrana Celular/química , Cisteína/genética , Dimerización , Disulfuros , Proteínas Ligadas a GPI , Glicosilación , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Mutagénesis Sitio-Dirigida
7.
Mol Biol Cell ; 19(12): 5259-66, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18843040

RESUMEN

Isu, the scaffold protein on which Fe-S clusters are built in the mitochondrial matrix, plays a central role in the biogenesis of Fe-S cluster proteins. We report that the reduction in the activity of several components of the cluster biogenesis system, including the specialized Hsp70 Ssq1, causes a 15-20-fold up-regulation of Isu. This up-regulation results from changes at both the transcriptional and posttranslational level: an increase in ISU mRNA levels and in stability of ISU protein. Its biological importance is demonstrated by the fact that cells lacking Ssq1 grow poorly when Isu levels are prevented from rising above those found in wild-type cells. Of the biogenesis factors tested, Nfs1, the sulfur donor, was unique. Little increase in Isu levels occurred when Nfs1 was depleted. However, its presence was required for the up-regulation caused by reduction in activity of other components. Our results are consistent with the existence of a mechanism to increase the stability of Isu, and thus its level, that is dependent on the presence of the cysteine desulfurase Nfs1.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Chaperonas Moleculares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas HSP70 de Choque Térmico , Mitocondrias/metabolismo , Proteínas Mitocondriales , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genética , Sulfurtransferasas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
8.
Appl Environ Microbiol ; 72(9): 6402-4, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16957271

RESUMEN

The development of lysine 2,3-aminomutase as a robust biocatalyst hinges on the development of an in vivo activation system to trigger catalysis. This is the first report to show that, in the absence of chemical reductants, lysine 2,3-aminomutase activity is dependent upon the presence of flavodoxin, ferredoxin, or flavodoxin-NADP(+) reductase.


Asunto(s)
Transferasas Intramoleculares/metabolismo , Porphyromonas gingivalis/enzimología , Catálisis , Activación Enzimática , Escherichia coli/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Transferasas Intramoleculares/genética , Cinética , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Porphyromonas gingivalis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
J Biol Chem ; 281(21): 14580-7, 2006 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-16551614

RESUMEN

Jac1p is a conserved, specialized J-protein that functions with Hsp70 in Fe-S cluster biogenesis in mitochondria of the yeast Saccharomyces cerevisiae. Although Jac1p as well as its specialized Hsp70 partner, Ssq1p, binds directly to the Fe-S cluster scaffold protein Isu, the Jac1p-Isu1p interaction is not well understood. Here we report that a C-terminal fragment of Jac1p lacking its J-domain is sufficient for interaction with Isu1p, and amino acid alterations in this domain affect interaction with Isu1p but not Ssq1p. In vivo, such JAC1 mutations had no obvious phenotypic effect. However, when present in combination with a mutation in SSQ1 that causes an alteration in the substrate binding cleft, growth was significantly compromised. Wild type Jac1p and Isu1p cooperatively stimulate the ATPase activity of Ssq1p. Jac1p mutant protein is only slightly compromised in this regard. Our in vivo and in vitro results indicate that independent interaction of Jac1p and the Isu client protein with Hsp70 is sufficient for robust growth under standard laboratory conditions. However, our results also support the idea that Isu protein can be "targeted" to Ssq1p after forming a complex with Jac1p. We propose that Isu protein targeting may be particularly important when environmental conditions place high demands on Fe-S cluster biogenesis or in organisms lacking specialized Hsp70s for Fe-S cluster biogenesis.


Asunto(s)
Proteínas Hierro-Azufre/química , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutación , Péptidos/química , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido
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