The Experts below are selected from a list of 12162 Experts worldwide ranked by ideXlab platform
Michael L Goldberg - One of the best experts on this subject based on the ideXlab platform.
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human zwint 1 specifies localization of zeste white 10 to kinetochores and is essential for mitotic checkpoint signaling
Journal of Biological Chemistry, 2004Co-Authors: Hongmei Wang, Don W Cleveland, Michael L Goldberg, Xia Ding, Zhen Dou, Zhihong Yang, Andrew W Shaw, Maikun Teng, Liwen Niu, Xuebiao YaoAbstract:Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10) in HeLa cells. Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain. Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZW10 kinetochore localization. In addition, depletion of Zwint-1 affects no mitotic arrest but causes aberrant premature chromosome segregation. These Zwint-1-suppressed cells display chromosome bridge phenotype with sister chromatids inter-connected. Moreover, Zwint-1 is required for stable association of CENP-F and dynamitin but not BUB1 with the kinetochore. Finally, our studies show that Zwint-1 is a new component of the mitotic check-point, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding interphase cells with multinuclei. As ZW10 and Zwint-1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.
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zwilch a new component of the ZW10 rod complex required for kinetochore functions
Molecular Biology of the Cell, 2003Co-Authors: Byron C Williams, Tim J Yen, Songtao Liu, Erika V Williams, Garmay Leung, Michael L GoldbergAbstract:The Zeste-White 10 (ZW10) and Rough Deal (ROD) proteins are part of a complex necessary for accurate chromosome segregation. This complex recruits cytoplasmic dynein to the kinetochore and participates in the spindle checkpoint. We used immunoaffinity chromatography and mass spectroscopy to identify the Drosophila proteins in this complex. We found that the complex contains an additional protein we name Zwilch. Zwilch localizes to kinetochores and kinetochore microtubules in a manner identical to ZW10 and ROD. We have also isolated a zwilch mutant, which exhibits the same mitotic phenotypes associated with ZW10 and rod mutations: lagging chromosomes at anaphase and precocious sister chromatid separation upon activation of the spindle checkpoint. Zwilch's role within the context of this complex is evolutionarily conserved. The human Zwilch protein (hZwilch) coimmunoprecipitates with hZW10 and hROD from HeLa cell extracts and localizes to the kinetochores at prometaphase. Finally, we discuss immunoaffinity chromatography results that suggest the existence of a weak interaction between the ZW10/ROD/Zwilch complex and the kinesin-like kinetochore component CENP-meta.
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the ZW10 and rough deal checkpoint proteins function together in a large evolutionarily conserved complex targeted to the kinetochore
Journal of Cell Science, 2001Co-Authors: Frederic Scaerou, Roger E Karess, Daniel A Starr, Fabio Piano, Ophelia Papoulas, Michael L GoldbergAbstract:The zeste-white 10 (ZW10) and rough deal (rod) genes of Drosophila both encode kinetochore components, and mutations in either gene greatly increase the missegregation of sister chromatids during mitosis. Here, we present genetic, cytological and biochemical evidence for a close, evolutionarily conserved relationship between the ROD and ZW10 proteins. We show that the phenotypes caused by disruption of either gene's function are similar in Drosophila and in C. elegans. No additive effects are observed in ZW10; rod double null mutants. In flies, the two proteins always colocalize and, moreover, require each other for their recruitment to the mitotic apparatus. The human ROD and ZW10 homologs also colocalize on HeLa cell kinetochores or kinetochore microtubules throughout most but not all of mitosis. Finally, we show that in both Drosophila and human cells, ROD and ZW10 are in fact physically associated, and in Drosophila these proteins are together constituents of a large (700-900 kDa), soluble macromolecular complex.
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human ZW10 and rod are mitotic checkpoint proteins that bind to kinetochores
Nature Cell Biology, 2000Co-Authors: Gordon K Chan, Michael L Goldberg, Sandra A Jablonski, Daniel A Starr, Tim J YenAbstract:Here we show that human Zeste White 10 (ZW10) and Rough deal (Rod) are new components of the mitotic checkpoint, as cells lacking these proteins at kinetochores fail to arrest in mitosis when exposed to microtubule inhibitors. Checkpoint failure and premature mitotic exit may explain why cells defective for hZW10 and hRod divide with lagging chromosomes. As ZW10 and Rod are not conserved in yeast, our data, combined with an accompanying study of Drosophila ZW10 and Rod, indicate that metazoans may require an elaborate spindle checkpoint to monitor complex kinetochore functions.
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the rate of poleward chromosome motion is attenuated in drosophila ZW10 and rod mutants
Nature Cell Biology, 2000Co-Authors: Matthew S Savoian, Michael L Goldberg, Conly L RiederAbstract:Here we show that the rate of poleward chromosome motion in ZW10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in production and/or regulation of the force reponsible for poleward chromosome motion.
Mitsuo Tagaya - One of the best experts on this subject based on the ideXlab platform.
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identification of the neuroblastoma amplified gene product as a component of the syntaxin 18 complex implicated in golgi to endoplasmic reticulum retrograde transport
Molecular Biology of the Cell, 2009Co-Authors: Takehiro Aoki, Sarah Ichimura, Ayano Itoh, Mami Kuramoto, Takashi Shinkawa, Toshiaki Isobe, Mitsuo TagayaAbstract:Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.
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n terminal region of ZW10 serves not only as a determinant for localization but also as a link with dynein function
Genes to Cells, 2008Co-Authors: Mamiko Inoue, Kohei Arasaki, Akihiro Ueda, Takehiro Aoki, Mitsuo TagayaAbstract:ZW10 interacts with dynamitin, a subunit of the dynein accessory complex dynactin, and functions in termination of the spindle checkpoint during mitosis and in membrane transport between the endoplasmic reticulum (ER) and Golgi apparatus during interphase. Its associations with kinetochores and ER membranes are mediated by Zwint-1 and RINT-1, respectively. A previous yeast two-hybrid study showed that the C-terminal region of ZW10 interacts with dynamitin, and part of this region has been used as an inhibitor of ZW10 function. In the present study, we reinvestigated the interaction between ZW10 and dynamitin, and showed that the N-terminal region of ZW10 is the major binding site for dynamitin and, like full-length ZW10, could potentially move along microtubules to the centrosomal area in a dynein-dynactin-dependent manner. Competitive binding experiments demonstrated that dynamitin and RINT-1 occupy the same N-terminal region of ZW10 in a mutually exclusive fashion. Consistent with this, over-expression of RINT-1 interfered with the dynein-dynactin-mediated movement of ZW10 to the centrosomal area. Given that the N-terminal region of ZW10 also interacts with Zwint-1, this region may be important for switching partners; one partner is a determinant for localization (kinetochore and ER) and the other links ZW10 to dynein function.
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Correlation of Golgi localization of ZW10 and centrosomal accumulation of dynactin.
Biochemical and biophysical research communications, 2007Co-Authors: Kohei Arasaki, Katsuko Tani, Takefumi Uemura, Mitsuo TagayaAbstract:ZW10 participates in the termination of the spindle checkpoint during mitosis by interacting with dynamitin, a subunit of the dynein accessory complex dynactin. We previously showed that ZW10 is attached to the endoplasmic reticulum through RINT-1 in interphase HeLa cells and involved in membrane transport between the endoplasmic reticulum and Golgi. Although a recent study demonstrated that ZW10 is localized in the Golgi in COS7 cells, the mechanism that regulates ZW10 localization remains unknown. In this study we showed a correlation between the Golgi localization of ZW10 and the centrosomal accumulation of dynactin. The amounts of ZW10 associated with dynactin were larger in cells where ZW10 was present in the Golgi than those where ZW10 was not in the Golgi. The targeting of ZW10 to the perinuclear Golgi region was found to depend on the perinuclear accumulation of dynactin, suggesting that dynactin regulates ZW10 localization.
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rint 1 regulates the localization and entry of ZW10 to the syntaxin 18 complex
Molecular Biology of the Cell, 2006Co-Authors: Kohei Arasaki, Katsuko Tani, May Taniguchi, Mitsuo TagayaAbstract:RINT-1 was first identified as a Rad50-interacting protein that participates in radiation-induced G2/M checkpoint control. We have recently reported that RINT-1, together with the dynamitin-interacting protein ZW10 and others, is associated with syntaxin 18, an endoplasmic reticulum (ER)-localized SNARE involved in membrane trafficking between the ER and Golgi. To address the role of RINT-1 in membrane trafficking, we examined the effects of overexpression and knockdown of RINT-1 on Golgi morphology and protein transport from the ER. Overexpression of the N-terminal region of RINT-1, which is responsible for the interaction with ZW10, caused redistribution of ZW10. Concomitantly, ER-to-Golgi transport was blocked and the Golgi was dispersed. Knockdown of RINT-1 also disrupted membrane trafficking between the ER and Golgi. Notably, silencing of RINT-1 resulted in a reduction in the amount of ZW10 associated with syntaxin 18, concomitant with ZW10 redistribution. In contrast, no redistribution or release of RINT-1 from the syntaxin 18 complex was observed when ZW10 expression was reduced. These results taken together suggest that RINT-1 coordinates the localization and function of ZW10 by serving as a link between ZW10 and the SNARE complex comprising syntaxin 18.
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ZW10 links mitotic checkpoint signaling to the structural kinetochore
Journal of Cell Biology, 2005Co-Authors: Geert J P L Kops, Mitsuo Tagaya, Yumi Kim, Beth A Weaver, Yinghui Mao, Ian Mcleod, John R Yates, Don W ClevelandAbstract:The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80–Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1–Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived “stop anaphase” inhibitor.
Judith Klumperman - One of the best experts on this subject based on the ideXlab platform.
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dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis
Nature Cell Biology, 2018Co-Authors: Carlos Sacristan, Eelco Tromer, Misbha Ud Din Ahmad, Jenny Keller, Job Fermie, Vincent Groenewold, Roberto Melero, Alexander Fish, José María Carazo, Judith KlumpermanAbstract:Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. The outer layer of kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD–Zwilch–ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.
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dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis
Nature Cell Biology, 2018Co-Authors: Carlos Sacristan, Eelco Tromer, Misbha Ud Din Ahmad, Jenny Keller, Job Fermie, Vincent Groenewold, Roberto Melero, Alexander Fish, José María Carazo, Judith KlumpermanAbstract:Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. The outer layer of kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD–Zwilch–ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments. Sacristan et al. show that the dynein adaptor Spindly facilitates oligomerisation of the RZZ complex to expand the kinetochore, after which Spindly-associated dynein compacts the kinetochore to allow for faithful chromosome segregation.
Wei Zheng - One of the best experts on this subject based on the ideXlab platform.
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Original Research Mechanism of copper transport at the blood–cerebrospinal fluid barrier: influence of iron deficiency in an in vitro model
2016Co-Authors: Andrew D Monnot, Gang Zheng, Wei ZhengAbstract:Copper (Cu) is an essential trace element that requires tight homeostatic regulation to ensure appropriate supply while not causing cytotoxicity due to its strong redox potential. Our previous in vivo study has shown that iron deficiency (FeD) increases Cu levels in brain tissues, particularly in the choroid plexus, where the blood–cerebrospinal fluid (CSF) barrier resides. This study was designed to elucidate the mechanism by which FeD results in excess Cu accumulation at the blood–CSF barrier. The effect of FeD on cellular Cu retention and transporters Cu transporter-1 (Ctr1), divalent metal transporter 1 (DMT1), antioxidant protein-1 (ATOX1) and ATP7A was examined in choroidal epithelial Z310 cells. The results revealed that deferoximine treatment (FeD) resulted in 70 % increase in cellular Cu retention (P, 0.05). A significant increase in the mRNA levels of DMT1, but not Ctr1, was also observed after FeD treatment, suggesting a critical role of DMT1 in cellular Cu regulation during FeD. Knocking down Ctr1 or DMT1 resulted in significantly lower Cu uptake by Z310 cells, whereas the knocking down of ATOX1 or ATP7A led to substantial increases of cellular retention of Cu. Taken together, these results suggest that Ctr1, DMT1, ATOX1 and ATP7A contribute to Cu transport at the blood–CSF barrier, and that the accumulation of intracellular Cu found in the Z310 cells during FeD appears to be mediated, at least in part, via the upregulation of DMT1 after FeD treatment
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use of z310 cells as an in vitro blood cerebrospinal fluid barrier model tight junction proteins and transport properties
Toxicology in Vitro, 2008Co-Authors: Lewis Zhichang Shi, Shunzhen Wang, Wei ZhengAbstract:Immortalized rat choroidal epithelial Z310 cells have the potential to become an in vitro model for studying transport of materials at blood-cerebrospinal fluid barrier (BCB) (Shi and Zheng, 2005) [Shi, L.Z., Zheng, W., 2005. Establishment of an in vitro brain barrier epithelial transport system for pharmacological and toxicological study. Brain Research 1057, 37-48]. This study was designed to demonstrate the presence of tight junction properties in Z310 cells and the functionality of Z310 monolayer in transport of selected model compounds. Western blot analyses revealed the presence of claudin-1, ZO-1, and occludin in Z310 cells. Transmission electron microscopy showed a "tight junction" type of structure in the sub-apical lateral membranes between adjacent Z310 cells. Real-time RT-PCR revealed that Z310 cells expressed representative transporters such as DMT1, MTP1, TfR, p-glycoprotein, ATP7A, ZnT1, ABCC1, Oat3, OCT1 and OB-Ra. Moreover, Z310 cells cultured in a two-chamber Transwell device possessed the ability to transport zidovudine (anionic drug), thyroxine (hormone), thymidine (nucleoside), and leptin (large polypeptide) with kinetic properties similar to those obtained from the in vitro model based on primary culture of choroidal epithelial cells. Taken together, these data indicate that the Z310 BCB model expresses major tight junction proteins and forms a tight barrier in vitro. The model also exhibits the ability to transport substances of various categories across the barrier.
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establishment and characterization of an immortalized z310 choroidal epithelial cell line from murine choroid plexus
Brain Research, 2002Co-Authors: Wei Zheng, Qiuqu ZhaoAbstract:Abstract The choroid plexus plays a wide range of roles in brain development, maturation, aging process, endocrine regulation, and pathogenesis of certain neurodegenerative diseases. To facilitate in vitro study, we have used a gene transfection technique to immortalize murine choroidal epithelial cells. A viral plasmid (pSV3neo) was inserted into the host genome of primary choroidal epithelia by calcium phosphate precipitation. The transfected epithelial cells, i.e., Z310 cells, that survived from cytotoxic selection expressed SV40 large-T antigen throughout the life span, suggesting a successful gene transfection. The cells displayed the same polygonal epithelial morphology as the starting cells by light microscopy. Immunocytochemical studies demonstrate the presence of transthyretin (TTR), a thyroxine transport protein known to be exclusively produced by the choroidal epithelia in the CNS, in both transfected and starting cells. Western blot analyses further confirm the production and secretion of TTR by these cells. The mRNAs encoding transferrin receptor (TfR) were identified by Northern blot analyses. The cells grow at a steady rate, currently in the 110th passage with a population doubling time of 20–22 h in the established culture. When Z310 cells were cultured onto a Trans-well apparatus, the cells formed an epithelial monolayer similar to primary choroidal cells, possessing features such as an uneven fluid level between inner and outer chambers and an electrical resistance approximately 150–200 Ω-cm 2 . These results indicate that immortalized Z310 cells possess the characteristics of choroidal epithelia and may have the potential for application in blood-CSF barrier (BCB) research.
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establishment and characterization of an immortalized z310 choroidal epithelial cell line from murine choroid plexus
Brain Research, 2002Co-Authors: Wei Zheng, Qiuqu ZhaoAbstract:The choroid plexus plays a wide range of roles in brain development, maturation, aging process, endocrine regulation, and pathogenesis of certain neurodegenerative diseases. To facilitate in vitro study, we have used a gene transfection technique to immortalize murine choroidal epithelial cells. A viral plasmid (pSV3neo) was inserted into the host genome of primary choroidal epithelia by calcium phosphate precipitation. The transfected epithelial cells, i.e., Z310 cells, that survived from cytotoxic selection expressed SV40 large-T antigen throughout the life span, suggesting a successful gene transfection. The cells displayed the same polygonal epithelial morphology as the starting cells by light microscopy. Immunocytochemical studies demonstrate the presence of transthyretin (TTR), a thyroxine transport protein known to be exclusively produced by the choroidal epithelia in the CNS, in both transfected and starting cells. Western blot analyses further confirm the production and secretion of TTR by these cells. The mRNAs encoding transferrin receptor (TfR) were identified by Northern blot analyses. The cells grow at a steady rate, currently in the 110th passage with a population doubling time of 20-22 h in the established culture. When Z310 cells were cultured onto a Trans-well apparatus, the cells formed an epithelial monolayer similar to primary choroidal cells, possessing features such as an uneven fluid level between inner and outer chambers and an electrical resistance approximately 150-200 omega-cm(2). These results indicate that immortalized Z310 cells possess the characteristics of choroidal epithelia and may have the potential for application in blood-CSF barrier (BCB) research.
Toshiyuki Miura - One of the best experts on this subject based on the ideXlab platform.
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hla b57 b 5801 human immunodeficiency virus type 1 elite controllers select for rare gag variants associated with reduced viral replication capacity and strong cytotoxic t lymphocyte recognition
Journal of Virology, 2009Co-Authors: Florencia Pereyra, Toshiyuki Miura, Brian L Block, Mark A Brockman, Arne Schneidewind, Michael A Lobritz, Almas Rathod, Zabrina L Brumme, Chanson J BrummeAbstract:Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.
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escape and compensation from early hla b57 mediated cytotoxic t lymphocyte pressure on human immunodeficiency virus type 1 gag alter capsid interactions with cyclophilin a
Journal of Virology, 2007Co-Authors: Mark A Brockman, Toshiyuki Miura, Arne Schneidewind, Matthew P Lahaie, Aaron G Schmidt, Ivna Desouza, Faina Ryvkin, Cynthia A DerdeynAbstract:Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8(+) T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T(242)N substitution in the capsid protein is associated with upstream mutations at residues H(219), I(223), and M(228) in the cyclophilin A (CypA)-binding loop in B57(+) individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T(242)N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T(242)N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T(242)N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8(+) T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.
