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dc.contributor.advisorSteven A. Weinman, M.D., Ph.D.en_US
dc.creatorAnn L Wozniaken_US
dc.date.accessioned2011-12-20T16:05:18Z
dc.date.available2010-09-28en_US
dc.date.available2011-12-20T16:05:18Z
dc.date.created2010-09-02en_US
dc.date.issued2010-08-26en_US
dc.identifier.otheretd-09022010-115940en_US
dc.identifier.urihttp://hdl.handle.net/2152.3/223
dc.description.abstractThe Hepatitis C Virus is the most common cause of chronic liver disease. Current therapy is only partially effective and fraught with side effects. A greater understanding of viral replication and new virus particle formation is thus important for developing new therapeutic targets. The HCV p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, a fluorescent pH sensor was incorporated within native, intracellular vesicles in the presence or absence of p7. p7 increased proton conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel-inactive p7, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid-stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. In conclusion, p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during maturation. This understanding will allow targeting of this mechanism with novel therapeutic agents, and offers insights into the mechanisms of liver pathogenesis during infection.en_US
dc.format.mediumelectronicen_US
dc.language.isoengen_US
dc.rightsCopyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works.en_US
dc.subjectHepatitis C Virusen_US
dc.subjectHCV p7 proteinen_US
dc.titleThe function of the hepatitis C virus p7 proteinen_US
dc.type.materialtexten_US
dc.type.genredissertationen_US
thesis.degree.namePhDen_US
thesis.degree.levelDoctoralen_US
thesis.degree.grantorThe University of Texas Medical Branchen_US
thesis.degree.departmentCell Biologyen_US
dc.contributor.committeeMemberStephen Griffin, Ph.D.en_US
dc.contributor.committeeMemberStanley M. Lemon, M.D.en_US
dc.contributor.committeeMemberLisa A. Elferink, Ph.Den_US
dc.contributor.committeeMemberHenry F. Epstein, M.D.en_US
dc.contributor.committeeMemberGuangxiang Luo, M.D., MPHen_US


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