Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.

The hepatitis C virus (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...

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Autores principales:	Ann L Wozniak, Stephen Griffin, David Rowlands, Mark Harris, MinKyung Yi, Stanley M Lemon, Steven A Weinman
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/6ac03d00cdad48649ebc91dc982e3b13
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spelling	oai:doaj.org-article:6ac03d00cdad48649ebc91dc982e3b132021-12-02T19:59:44ZIntracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.1553-73661553-737410.1371/journal.ppat.1001087https://doaj.org/article/6ac03d00cdad48649ebc91dc982e3b132010-09-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20824094/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374The hepatitis C virus (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, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H(+)) 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 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, 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. We conclude that 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 an as yet uncharacterized maturation process.Ann L WozniakStephen GriffinDavid RowlandsMark HarrisMinKyung YiStanley M LemonSteven A WeinmanPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 6, Iss 9, p e1001087 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Ann L Wozniak Stephen Griffin David Rowlands Mark Harris MinKyung Yi Stanley M Lemon Steven A Weinman Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
description	The hepatitis C virus (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, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H(+)) 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 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, 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. We conclude that 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 an as yet uncharacterized maturation process.
format	article
author	Ann L Wozniak Stephen Griffin David Rowlands Mark Harris MinKyung Yi Stanley M Lemon Steven A Weinman
author_facet	Ann L Wozniak Stephen Griffin David Rowlands Mark Harris MinKyung Yi Stanley M Lemon Steven A Weinman
author_sort	Ann L Wozniak
title	Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
title_short	Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
title_full	Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
title_fullStr	Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
title_full_unstemmed	Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
title_sort	intracellular proton conductance of the hepatitis c virus p7 protein and its contribution to infectious virus production.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/6ac03d00cdad48649ebc91dc982e3b13
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Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.

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