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Copyright
by
Ann L. Wozniak
2010
The Dissertation Committee for Ann L. Wozniak Certifies that this is the approved version of the following dissertation:
The Function of the Hepatitis C Virus p7 Protein
Committee:
Steven A. Weinman, M.D., Ph.D., SupervisorLisa A. Elferink, Ph.DHenry F. Epstein, M.D.Stephen Griffin, Ph.D.Stanley M. Lemon, M.D.Guangxiang Luo, M.D., MPH
__________________
Dean, Graduate School
The Function of the Hepatitis C Virus p7 Protein
by
Ann L. Wozniak, B.S.
Dissertation
Presented to the Faculty of the Graduate School of
The University of Texas Medical Branch
in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
The University of Texas Medical Branch
August, 2010
Dedication
I dedicate this work to my family and many friends. Particularity to my mother, Kay Wozniak, whose love and support has provided me the strength and perseverance I needed to achieve my goals. To my brother, John, for his love and encouragement and to my step-father, Joseph Drana, who has provided me endless support and motivation. I also dedicate this work to my late father, William Wozniak. I could not have completed this journey without them.
Acknowledgements
I want to first acknowledge my mentor, Dr. Steven A. Weinman. I express my most sincere gratitude for his advice, patience, guidance and support. I have learned so much from him and I cannot thank him enough for being a true mentor in every respect. I want to acknowledge my committee: Drs. Lisa Elferink, Henry Epstein, Stephen Griffin, Stanley M. Lemon and Guangxiang Luo for their time, insightful comments and most importantly their guidance. I thank Dr. Mary Moslen for her support and training. I also want to thank the Cell Biology Graduate Program faculty, students and directors, Drs. Golda A. Leonard and Darren F. Boehning for their endless support. I want to acknowledge my lab mates, past and present, for their encouragement throughout the years. Finally, I owe a special thank you to my family and friends, for their endless support and advice throughout my education.
The Function of the Hepatitis C Virus p7 Protein
Publication No._____________
Ann L. Wozniak, Ph.D.
The University of Texas Medical Branch, 2010
Supervisor: Steven A. Weinman
The 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.
Table of Contents
TOC \f HYPERLINK \l "_List_of_Figures" List of Figures xii
HYPERLINK \l "_Chapter_1:_" Chapter 1: Introduction 1
HYPERLINK \l "_Hepatitis_C_Virus" Hepatitis C Virus Course of Disease and Therapy 1
HYPERLINK \l "_Genome_Organization" Genome Organization 2
HYPERLINK \l "_HCV_Lifecycle" HCV Lifecycle 3
HYPERLINK \l "_The_HCV_p7" The HCV p7 Protein and the Role of Viroporins 5
HYPERLINK \l "_HIV-1Vpu" HIV-1Vpu 7
HYPERLINK \l "_Poliovirus_2B" Poliovirus 2B 7
HYPERLINK \l "_Influenza_Virus_M2" Influenza Virus M2 8
HYPERLINK \l "_Evidence_for_p7" Evidence for p7 Channel Activity 9
HYPERLINK \l "_Structural_Analysis_of" Structural Analysis of p7 11
HYPERLINK \l "_Biology_of_p7" Biology of p7 in Virus Production 14
HYPERLINK \l "_HCV_Therapy_and" HCV Therapy and p7 Inhibitors 16
HYPERLINK \l "_Project_Aims" Project Aims 19
HYPERLINK \l "_Chapter_2:_Materials" Chapter 2: Materials and Methods 22
HYPERLINK \l "_Materials" Materials 22
HYPERLINK \l "_Western_Blotting" Western Blotting 22
HYPERLINK \l "_Plasmids" Plasmids 23
HYPERLINK \l "_Cell_Culture_and" Cell Culture and Transfection 23
HYPERLINK \l "_Human_Embryonic_Kidney" Human Embryonic Kidney (HEK) 293FT Cells: 23
HYPERLINK \l "_HCV_Replicon_Cell" HCV Replicon Cell Lines: 24
HYPERLINK \l "_Huh-7.5_Cells:" Huh-7.5 Cells: 26
HYPERLINK \l "_Transfections:" Transfections: 26
HYPERLINK \l "_RNA_Transcription,_Electroporation" RNA Transcription, Electroporation and HCV Infectivity 26
HYPERLINK \l "_RNA_Electroporation:" RNA Electroporation: 26
HYPERLINK \l "_Assessment_of_Viral" Assessment of Viral Titers: 27
HYPERLINK \l "_Subcellular_Fractionation_and" Subcellular Fractionation and Isolation of Membrane Vesicles 28
HYPERLINK \l "_Cell_Surface_Biotinylation" Cell Surface Biotinylation 29
HYPERLINK \l "_Measurement_of_Proton" Measurement of Proton Permeability 29
HYPERLINK \l "_Live_Cell_Imaging" Live Cell Imaging of Vesicular pH 31
HYPERLINK \l "_Measurement_of_Acidic" Measurement of Acidic Compartments in Fixed Cells 32
HYPERLINK \l "_Bafilomycin_A1_Rescue" Bafilomycin A1 Rescue of Defective p7KR 33
HYPERLINK \l "_Cytotoxicity_of_Bafilomycin." Cytotoxicity of Bafilomycin. 33
HYPERLINK \l "_Effect_of_Bafilomycin" Effect of Bafilomycin A1 on Viral Entry. 33
HYPERLINK \l "_Concentration_Dependence_of" Concentration Dependence of Bafilomycin A1 on Vesicular pH. 34
HYPERLINK \l "_Bafilomycin_A1_Treatment" Bafilomycin A1 Treatment of p7-defective HCV RNA Electroporated Cells. 34
HYPERLINK \l "_M2_Trans-Complementation" M2 Trans-Complementation 35
HYPERLINK \l "_Low_pH_Treatment" Low pH Treatment of Extracellular and Intracellular Virus 35
HYPERLINK \l "_Statistics" Statistics 36
HYPERLINK \l "_Chapter_3:_The" Chapter 3: The Effect of p7 on the Proton Conductance of Intracellular Membrane Vesicles 37
HYPERLINK \l "_Background" Background 37
HYPERLINK \l "_Results" Results 40
HYPERLINK \l "_Isolation_of_p7-Containing" Isolation of p7-Containing Membrane Vesicles 40
HYPERLINK \l "_Preparation_and_pH" Preparation and pH Indicator Loading of p7-Containing Membrane Vesicles 43
HYPERLINK \l "_Measurement_of_Proton_1" Measurement of Proton Permeability in Isolated Membrane Vesicles 46
HYPERLINK \l "_Conductive_Properties_of" Conductive Properties of p7 Proteins from Different HCV Strains Expressed in Native Intracellular Membranes 48
HYPERLINK \l "_HCV_p7-Induced_H+" HCV p7-Induced H+ Conductance is Sensitive to Known Viroporin Inhibitors 51
HYPERLINK \l "_Vesicles_Isolated_from" Vesicles Isolated from p7-Containing Replicon Bearing Cells Have Increased H+ Permeability 53
HYPERLINK \l "_Discussion" Discussion 55
HYPERLINK \l "_Chapter_4:_Effects" Chapter 4: Effects of p7 on the pH of Intracellular Organelles 59
HYPERLINK \l "_Background_1" Background 59
HYPERLINK \l "_Results_1" Results 61
HYPERLINK \l "_The_Effect_of" The Effect of p7 on Intracellular pH in HCV Replicon Bearing Cells 61
HYPERLINK \l "_Effects_of_Viroporin" Effects of Viroporin Inhibitors on Vesicular pH 66
HYPERLINK \l "_HCV_p7_Induces" HCV p7 Induces Channel Activity Within Infected Cells 68
HYPERLINK \l "_Discussion_1" Discussion 70
HYPERLINK \l "_Chapter_5:_The" Chapter 5: The Role of pH in the HCV Viral Lifecycle 74
HYPERLINK \l "_Background_2" Background 74
HYPERLINK \l "_Results_2" Results 76
HYPERLINK \l "_The_Effects_of" The Effects of p7-Induced pH Changes During Infectious Virus Production 76
HYPERLINK \l "_p7_Channel_Activity" p7 Channel Activity is Required for Production of Infectious Virus 78
HYPERLINK \l "_Exposure_to_Acidic" Exposure to Acidic pH Renders Intracellular Infectious Virus Non-Infectious 82
HYPERLINK \l "_Influenza_M2_Protein" Influenza M2 Protein Can Trans-Complement an HCV p7 Channel Mutant 84
HYPERLINK \l "_Discussion_2" Discussion 87
HYPERLINK \l "_Chapter_6:_Conclusions" Chapter 6: Conclusions and Future Directions 91
HYPERLINK \l "_Background_3" Background 91
HYPERLINK \l "_HCV_p7_as" HCV p7 as a Mediator of Viral Entry 91
HYPERLINK \l "_HCV_p7-Induced_Membrane" HCV p7-Induced Membrane Rearrangement and Host Cells pH Manipulation 92
HYPERLINK \l "_HCV_p7_as_1" HCV p7 as an Assembly and Scaffolding Factor 93
HYPERLINK \l "_Role_of_p7" Role of p7 in Secretory Pathway Protection 94
HYPERLINK \l "_Mechanisms_of_pH" Mechanisms of pH Protection by p7 96
HYPERLINK \l "_HCV_Envelope_Proteins" HCV Envelope Proteins as pH-Triggered Fusion Proteins 96
HYPERLINK \l "_Lipoprotein_Particle_Formation" Lipoprotein Particle Formation and HCV pH Sensitivity 97
HYPERLINK \l "_Consequences_of_p7" Consequences of p7 Presence and Future Directions 99
HYPERLINK \l "_Autophagy" Autophagy 99
HYPERLINK \l "_Lysosomal_Integrity" Lysosomal Integrity 100
HYPERLINK \l "_Bibliography" Bibliography 102
HYPERLINK \l "_Vita" Vita 110
List of Figures
TOC \f Figure 1: HYPERLINK \l "_Figure_1.__1" Genetic organization, polyprotein processing and membrane association of hepatitis C virus (HCV). 4
Figure 2: HYPERLINK \l "_HIV-1Vpu" Lifecycle of HCV.. 6
TOC \f Figure 3: HYPERLINK \l "_Figure_3._The" The 3D structure of a hexameric HCV p7 channel. 13
TOC \f Figure 4: HYPERLINK \l "_Figure_4._Schematic" Schematic representation of sub-genomic and full-length HCV replicon bearing cells. 25
Figure 5: HYPERLINK \l "_Figure_5._Isolation" Isolation of p7-containing Membrane Vesicles 42
TOC \f Figure 6: HYPERLINK \l "_Figure_6._Cell" Cell surface biotinylation of p7-expresssing cells.. 43
Figure 7: HYPERLINK \l "_Figure_7._Preparation" Preparation and pH indicator loading of p7-containing membrane vesicles. 45
TOC \f Figure 8: HYPERLINK \l "_Figure_8._Measurement" Measurement of Proton Permeability in Isolated Membrane Vesicles. 47
Figure 9: HYPERLINK \l "_Figure_9._Control_1" Control vesicles have low proton permeability. 49
TOC \f Figure 10: HYPERLINK \l "_Figure_10._Conductive" Conductive Properties of HCV p7 Proteins.. 50
Figure 11: HYPERLINK \l "_Figure_11._Sensitivity" Sensitivity of p7-Associated pH Changes to Known Viroporin Inhibitors. 52
TOC \f Figure 12: HYPERLINK \l "_Figure_12._" Effect of p7 on Intracellular pH in HCV Replicon-bearing Cells. 54
Figure 13: HYPERLINK \l "_Figure_13._" p7 alters the Intracellular pH in HCV Replicon-bearing Cells 62
TOC \f Figure 14: HYPERLINK \l "_Figure_14._Co-localization_1" Co-localization of LysoTracker Red with Cellular Organelles.. 63
Figure 15: HYPERLINK \l "_Figure_15._Live" Live Cell Imaging of LysoSensor Loaded Replicon Cells. 65
TOC \f Figure 16: HYPERLINK \l "_Figure_16._" Effects of Viroporin Inhibitors on Intracellular Vesicular pH. 67
Figure 17: HYPERLINK \l "_Figure_17._" p7 Channel Activity within Infected Cells 69
TOC \f Figure 18: HYPERLINK \l "_Figure_18.__1" Effects of Viroporin Inhibitors on Infectious Virus Production 77
Figure 19: HYPERLINK \l "_Figure_19.__1" Bafilomycin A1 Rescue of Infectious Virus Production. 80
TOC \f Figure 20: HYPERLINK \l "_Figure_20._Intracellular_1" Intracellular Infectious Virus is Acid Sensitive 83
Figure 21: HYPERLINK \l "_Figure_21.__1" Influenza M2 Protein Can Trans-complement an HCV p7 Channel Mutant 86
Figure 22: HYPERLINK \l "_Figure_22._" Stages where p7 May Function During the HCV Lifecycle 95
Chapter 1: Introduction
Hepatitis C Virus Course of Disease and Therapy
In the seventies, an unknown, post-transfusion hepatitis causing agent, initially referred to as non-A, non-B hepatitis, was observed in many countries ADDIN EN.CITE Feinstone200130730730717Feinstone, S. M.Kapikian, A. Z.Purcell, R. H.Alter, H. J.Holland, P. V.Transfusion-associated hepatitis not due to viral hepatitis type A or B. 1975Rev Med VirolRev Med Virol3-8; discussion 8-91112001/03/10Antibodies, Viral/*bloodBlood Transfusion/adverse effectsCytomegalovirus/immunologyHepatitis A AntibodiesHepatitis Antibodies/bloodHepatitis B Antibodies/bloodHepatitis B Surface Antigens/immunologyHepatitis, Viral, Human/blood/*history/virologyHerpesvirus 4, Human/immunologyHistory, 20th CenturyHumansMicroscopy, Immunoelectron2001Jan-Feb1052-9276 (Print)
1052-9276 (Linking)11241798http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1124179810.1002/rmv.304 [pii]eng[1] and not until 1989 did this infectious agent become discovered. It turned out to be a virus of the Flaviviridae family termed hepatitis C virus (HCV) and soon after its discovery, it was quickly established that HCV infection was the cause of more than 90% of the previously termed non-A, non-B hepatitis. HCV infection occurs largely through the transfer of blood or blood-derived products including during dialysis and organ transplantation.
HCV remains a major health concern affecting an estimated 200 million individuals worldwide. An important characteristic of HCV is its high tendency to establish a chronic infection in the host. As many as 50% of the newly infected individuals fail to clear the virus and become chronic carriers ADDIN EN.CITE Thimme200685858517Thimme, R.Lohmann, V.Weber, F.Department of Medicine II, University Hospital Freiburg, D-79106 Freiburg, Germany.A target on the move: innate and adaptive immune escape strategies of hepatitis C virusAntiviral ResAntiviral Res129-416932006/01/18Hepacivirus/*immunology/*pathogenicity/physiologyHepatitis C/*immunologyHepatitis C Antibodies/biosynthesisHumansImmunity, InnateInterferons/biosynthesis/metabolismKiller Cells, Natural/immunologyMutationT-Lymphocytes/immunology2006Mar0166-3542 (Print)16413618http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16413618S0166-3542(05)00260-3 [pii]
10.1016/j.antiviral.2005.12.001eng[2]. The mechanisms responsible or viral persistence are unclear yet several factors are thought to contribute including host related factors and genetic variability of the HCV genome. Primary infection with HCV is predominantly asymptomatic; however individuals with chronic infection develop progressive hepatic fibrosis leading to an increased risk of cirrhosis, liver failure and hepatocellular carcinoma (HCC).
Antiviral therapy for HCV is only partially successful and of the treated individuals, the likelihood of a sustained response varies depending on the infecting genotype ADDIN EN.CITE ADDIN EN.CITE.DATA [2,3]. The molecular basis for the varying responses in HCV genotypes is unknown; however investigation has identified that the infecting genotype is a major predictor of response to antiviral therapy ADDIN EN.CITE ADDIN EN.CITE.DATA [4,5]. HCV is highly heterogeneous. This genotypic diversity has shown to account for differences in their virulence, pathogenicity and responsiveness to antiviral therapy ADDIN EN.CITE ADDIN EN.CITE.DATA [3,4,5]. Genotype 1 is the most common throughout the world and individuals infected with genotype 1 have a relatively poor response to antiviral therapy. Standard HCV therapy, consisting of interferon and ribavirin, aims to enhance the natural host immune response to the virus. Because recovery from viral infections often depends on an intricate relationship between virus and host, new antiviral targets aimed to directly target specific virus-host interactions offer the possibility of improved therapy.
Genome Organization
HCV is a small RNA virus belonging to the Flaviviridae family which also includes Pestivirus and Flavivirus. Based on sequence variability, the HCV strains are divided into at least six genotypes and a large group of subtypes. The viral genome is represented as a single-stranded, positive-sense RNA molecule, composed of approximately 9600 nucleotides (reviewed in ADDIN EN.CITE ADDIN EN.CITE.DATA [6,7,8]). The viral RNA contains a single, large open reading frame encoding a polyprotein of ~3,000 amino acids, which is flanked by non-translated regions at the 5- and 3-ends. The HCV polyprotein is cleaved co- and post-translationally by cellular and viral proteinases into ten different products. The structural components of the virion (core, E1 and E2) are located in the amino-terminal one-third of the polyprotein. The non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) coordinate the intracellular processes of the virus life cycle. The p7 protein is located at the junction between the structural and non-structural proteins and it is currently unknown if it is a structural component of the virion (reviewed in ADDIN EN.CITE ADDIN EN.CITE.DATA [6,7,8]).
Core, E1, E2, and p7 are processed from the polyprotein by cellular proteases. Viral protein function is illustrated in Fig. 1. The integral protein NS2 together with the N-terminal domain of NS3, forms the zinc-stimulated metalloprotease responsible for the NS2-NS3 autocleavage. NS3 consists of two domains, the N-terminal serine protease and the C-terminal ATPase/helicase. NS4A is a small hydrophobic sequence acting as a membrane anchor and a co-factor to NS3, enabling the NS3-NS4A protease processing of non-structural proteins NS3 through NS5B. NS4B is an integral membrane protein involved in the induction of the membrane alterations seen in viral infection. The NS5B is the RNA dependent RNA polymerase. It is important to note that the HCV viral proteins are multifunctional and involved in several viral and cellular processes.
HCV Lifecycle
HCV circulates in association with low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL). Its primary target is the hepatocyte, but there is strong evidence that it can also replicate in peripheral blood mononuclear cells, B- and T-cell lines ADDIN EN.CITE Esteban199832232232217Esteban, J. I.Cordoba, J.Sauleda, S.Department of Internal Medicine, Hospital General Universitari Vall d'Hebron, Barcelona, Spain.The clinical picture of acute and chronic hepatitis CCurr Stud Hematol Blood TransfusCurr Stud Hematol Blood Transfus102-18621998/03/21Acute DiseaseClinical Trials as TopicDisease ProgressionHepatitis C/diagnosis/epidemiology/*therapyHepatitis C, Chronic/diagnosis/epidemiology/*therapyHumansTreatment Outcome19980258-0330 (Print)
0258-0330 (Linking)9507806http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9507806eng[9], epithelial cells in the gut ADDIN EN.CITE ADDIN EN.CITE.DATA [10], and in the central nervous system ADDIN EN.CITE Forton200433233233217Forton, D. M.Thomas, H. C.Taylor-Robinson, S. D.Hepatology Section, Division of Medicine A, Faculty of Medicine, Imperial College, London, United Kingdom. d.forton@imperial.ac.ukCentral nervous system involvement in hepatitis C virus infectionMetab Brain DisMetab Brain Dis383-91193-42004/11/24Cognition Disorders/diagnosis/physiopathology/*virologyDisease ProgressionEncephalitis, Viral/diagnosis/physiopathology/*virologyFatigue/physiopathology/virologyHepacivirus/geneticsHepatic Encephalopathy/physiopathology/virologyHepatitis C/*complicationsHumansMagnetic Resonance SpectroscopyVasculitis, Central Nervous System/physiopathology/virology2004Dec0885-7490 (Print)
0885-7490 (Linking)15554429http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15554429eng[11]. HCV has a high rate of replication and due to the lack of proofreading by the HCV RNA polymerase, it has an extremely high mutation rate whose estimated evolution rate is 1-3 x 103 nucleotide substitutions per viral genome per year ADDIN EN.CITE ADDIN EN.CITE.DATA [12,13].
A schematic of the HCV lifecycle is shown in Fig. 2. Viral entry occurs via clathrin-mediated endocytosis involving the viral envelope proteins E1 and E2 and several cell surface receptors including CD81, scavenger receptor B1, claudin-1, occludin and low-density lipoprotein receptor ADDIN EN.CITE Cocquerel200635735735717Cocquerel, L.Voisset, C.Dubuisson, J.CNRS-UMR8161, Institut de Biologie de Lille, Institut Pasteur de Lille, France.Hepatitis C virus entry: potential receptors and their biological functionsJ Gen VirolJ Gen Virol1075-8487Pt 52006/04/11Antigens, CD/metabolism/physiologyAntigens, CD36/metabolism/physiologyGlycosaminoglycans/metabolismHepacivirus/*physiologyHepatocytes/virologyHumansLectins, C-Type/metabolismReceptors, LDL/metabolismReceptors, Virus/metabolism/*physiologyViral Envelope Proteins/metabolismVirus Replication2006May0022-1317 (Print)
0022-1317 (Linking)16603507http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1660350787/5/1075 [pii]
10.1099/vir.0.81646-0eng[14]. Successful infection depends on the acidification of the endosomal compartment, which triggers a fusion of the viral and
Figure 1. Genetic organization, polyprotein processing and membrane association of hepatitis C virus (HCV). (A) Schematic representation of the 9.6-kb positive-strand RNA genome is shown at the top; the translation products are given below. Internal ribosome entry site-mediated translation yields a polyprotein precursor that is processed into the mature structural and non-structural proteins. Cleavage sites of the polyprotein precursor by the endoplasmic reticulum signal peptidase are denoted by solid diamonds. The open diamond indicates further processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by the HCV NS23 and NS34A proteases. Dots in E1 and E2 indicate the glycosylation of the envelope proteins. (B) Schematic representation of the structures and membrane association of the HCV proteins are shown. Scissors indicate cleavages by the endoplasmic reticulum (ER) signal peptidase. The cyclic arrow denotes cleavage by the NS23 protease while black arrows indicate processing by the NS34A protease complex. Known protein structures are shown as ribbon diagrams. Adapted from Moradpour et al., Nature Reviews Microbiology. 2007 ADDIN EN.CITE Moradpour200792092092017Moradpour, D.Penin, F.Rice, C. M.Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland. darius.moradpour@chuv.chReplication of hepatitis C virusNat Rev MicrobiolNat Rev Microbiol453-63562007/05/10Hepacivirus/genetics/*physiologyHepatitis C/virologyHumansViral Proteins/chemistry/physiologyVirus Cultivation/methodsVirus Internalization*Virus Replication2007Jun1740-1534 (Electronic)
1740-1526 (Linking)17487147http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17487147nrmicro1645 [pii]
10.1038/nrmicro1645eng[7].
cellular membranes. This releases the positive-stranded RNA HCV genome into the cytoplasm to be transported to the endoplasmic reticulum (ER) where it is translated. The RNA first functions as messenger RNA for the viral protein synthesis at the ER. The non-structural proteins then form together with the viral RNA to form a replication complex which resides in vesicular membrane structures and is termed the membranous web. Virions presumably form by budding directly into the ER, where they associate with lipids, and leave the cell through the secretory pathway ADDIN EN.CITE ADDIN EN.CITE.DATA [8,15].
The HCV p7 Protein and the Role of Viroporins
HCV p7 is a member of the viroporin class of viral ion channels. Viroporins are small, highly hydrophobic, virus-encoded proteins that interact with membranes modifying the cells permeability to ions or other small molecules ADDIN EN.CITE ADDIN EN.CITE.DATA [16,17]. They typically consist of less than 100 amino acids and contain one or two highly hydrophobic domains that are able to form an amphipathic - h e l i x . V i r o p o r i n p r o t e i n s w i l l o l i g o m e r i z e o n c e t h e y h a v e i n s e r t e d i n t o c e l l u l a r m e m b r a n e s , t y p i c a l l y f o r m i n g a h y d r o p h i l i c p o r e . T h e y m a y a l s o d i r e c t l y i n t e r a c t w i t h c e l l u l a r m e m b r a n e s t o d i s t u r b t h e o r g a n i z a t i o n o f t h e l i p i d b i l a y e r a n d t h e y m a y c o n t ain a stretch of basic amino acids that act to destabilize the membrane. The main function of a viroporin is to aid in the release of progeny virus from infected cells and they often achieve this through the dissipation of ions across cellular membranes ADDIN EN.CITE ADDIN EN.CITE.DATA [16,17]. This dissipation can lead to several outcomes which include the direct permeabilization of cellular and plasma membranes, the inhibition of host cell translation or the alteration in host cell pH regulation. A number of viroporins have been reported and include HIV-1 Vpu, Dengue M protein, Influenza A M2 protein, and poliovirus 2B; all of which contain similar characteristics, structures and functions.
Figure 2. Lifecycle of HCV. The virus binds to a complex of receptors at the cell surface, which leads to endocytosis and internalization of the particle (a). Fusion between the viral envelope and an endosomal membrane leads to the release of HCV genome into the cytoplasm (b).The positive strand RNA is directly translated and the viral proteins are simultaneously produced and processed (c). Expression of the HCV proteins induces the formation of a membranous web which is the site of RNA replication (d). Accumulation of HCV genomic RNA and the structural proteins leads to the assembly of a nucleocapsid which then acquires an ER-derived envelope (e). The viral particle is then secreted presumably through the secretory pathway (f). Adapted from Moradpour et al., Nature Reviews Microbiology. 2007 ADDIN EN.CITE Moradpour200792092092017Moradpour, D.Penin, F.Rice, C. M.Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland. darius.moradpour@chuv.chReplication of hepatitis C virusNat Rev MicrobiolNat Rev Microbiol453-63562007/05/10Hepacivirus/genetics/*physiologyHepatitis C/virologyHumansViral Proteins/chemistry/physiologyVirus Cultivation/methodsVirus Internalization*Virus Replication2007Jun1740-1534 (Electronic)
1740-1526 (Linking)17487147http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17487147nrmicro1645 [pii]
10.1038/nrmicro1645eng[7].
HIV-1Vpu
Vpu is an 81 amino acid, integral membrane phophoprotein that is unique to HIV-1. It is comprised of a single transmembrane hydrophobic stretch of residues distributed along its N-terminal domain and a hydrophilic carboxy terminus which is oriented toward the cytoplasm ADDIN EN.CITE ADDIN EN.CITE.DATA [18,19]. These two domains are connected by a short stretch of basic amino acid residues. Structural analysis of Vpu ion channels suggest that the channel likely forms a pentamer, but higher or lower order oligomers may also be formed ADDIN EN.CITE ADDIN EN.CITE.DATA [19,20,21]. Ion channel activity of Vpu has been shown and purified Vpu will form cation-selective (Na+) ion channels in planar lipid bilayers that are sensitive to amiloride derivatives. Vpu is thought to be a gated channel whereby the gating mechanism is controlled by a conformational change that takes places within the peptide. Vpu has several domain-specific roles in the HIV-1 life cycle. The N-terminal transmembrane domain is critical for efficient virus particle release while the C-terminal domain is required to promote the degradation of CD4 in host cells via ubiquitination ADDIN EN.CITE ADDIN EN.CITE.DATA [19,22]. Vpu is localized to the ER and the Golgi complex and is excluded from the virus particle. It impairs normal trafficking of membrane proteins by inducing modifications within compartments of the secretory pathway as well as increasing cell membrane permeability. HIV-1 genomes carrying defective Vpu are able to replicate however a Vpu-defective virus will accumulate virions at the cell surface and within endosomes.
Poliovirus 2B
The poliovirus 2B protein contains approximately 100 amino acids and contains two hydrophobic transmembrane spanning regions ADDIN EN.CITE ADDIN EN.CITE.DATA [16,17]. It is comprised of two domains: an N-terminal amphipathic stretch of amino acids that has cationic channel activity and a C-terminal hydrophobic domain. Structural analysis of 2B shows that it oligomerizes to form dimers and tetramers containing a hydrophilic pore. The 2B protein interacts with cellular membranes and is found to mostly localize at intracellular membranes ADDIN EN.CITE ADDIN EN.CITE.DATA [23,24]. Membrane permeabilization due to 2B leads to increased cytoplasmic calcium levels as well as inhibition of protein secretion. In addition to enhancing membrane permeability, 2B is also able to induce intracellular membrane remodeling and disassembly of the Golgi complex ADDIN EN.CITE ADDIN EN.CITE.DATA [23,24]. This results in disruption of the vesicular system as well as glycoprotein trafficking. This is required for virus propagation, thus a poliovirus genome lacking the 2B gene is not viable.
Influenza Virus M2
M2 is an integral membrane phosphoprotein of 96 amino acids. It is comprised of a single transmembrane domain which is extended on each end by an N-terminal extracellular domain and a C-terminal cytoplasmic tail ADDIN EN.CITE ADDIN EN.CITE.DATA [16,25,26]. The M2 protein homo-oligomerizes to form a tetramer leading to increased permeability towards ions. M2 is a H+-selective ion channel that acts at two stages of the influenza lifecycle. During virus entry, it promotes the passage of H+ into virions to initiate virus uncoating within the endosome. The M2 ion channel is also activated during virion egress through the exocytic pathway where it equilibrates the acidic pH of the trans Golgi network with the cytoplasm ADDIN EN.CITE ADDIN EN.CITE.DATA [25,26,27]. This serves several purposes including the protection of virions from premature conformation changes in hemagglutinin (HA) which would otherwise inactivate the virus. It also alters glycoprotein trafficking as well as inhibits the secretion of cellular proteins. M2 is not entirely essential for virus entry but an M2-deficient variant will have significantly decreased virus production. This is consistent with other viruses lacking their respective viroporin gene.
Evidence for p7 Channel Activity
The first demonstration that p7 possesses ion channel activity came in 2003 when it was shown that a GST-p7, when inserted into black lipid bilayers (BLM), displayed transient conductance fluctuations, indicating the presence of short-lived ion channel open states ADDIN EN.CITE Griffin200393939317Griffin, S. D.Beales, L. P.Clarke, D. S.Worsfold, O.Evans, S. D.Jaeger, J.Harris, M. P.Rowlands, D. J.School of Biochemistry and Molecular Biology, University of Leeds, Division of Microbiology Old Medical School, Thoresby Place, Leeds LS2 9JT, UK.The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, AmantadineFEBS LettFEBS Lett34-85351-32003/02/01Amantadine/*pharmacologyAntiviral Agents/*pharmacologyCarcinoma, Hepatocellular/metabolismHumansIon Channels/chemistry/*drug effects/metabolismLipid Bilayers/chemistryMembranes, ArtificialMicroscopy, ElectronRecombinant Fusion Proteins/biosynthesis/chemistryTumor Cells, CulturedViral Proteins/*chemistry/*metabolism/ultrastructure2003Jan 300014-5793 (Print)12560074http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12560074S0014579302038516 [pii]eng[28]. When p7 was examined without a fusion tag, it dramatically increased this channel activity and in addition, these were cation-selective channels with a preference for Ca2+ over K+. A similar study using a chemically synthesized p7 tagged to biotin confirmed the channel activity of p7 ADDIN EN.CITE Pavlovic200313513513517Pavlovic, D.Neville, D. C.Argaud, O.Blumberg, B.Dwek, R. A.Fischer, W. B.Zitzmann, N.Department of Biochemistry, University of Oxford, United Kingdom.The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivativesProc Natl Acad Sci U S AProc Natl Acad Sci U S A6104-8100102003/04/30Amino Acid SequenceCarbohydrates/pharmacology*HepacivirusImines/*pharmacologyIon Channels/antagonists & inhibitors/drug effects/*physiologyLipid BilayersMembrane Potentials/drug effects/physiologyMolecular Sequence DataPeptides/chemical synthesis/chemistryViral Proteins/chemistry/*physiology2003May 130027-8424 (Print)12719519http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1271951915633310.1073/pnas.1031527100
1031527100 [pii]eng[29]. However when inserted into BLMs, variable amplitude single channel events were observed, with channels having conductances as low as 14 pS and an average conductance of approximately 100 pS. The varied conductance levels of p7 have been proposed to arise from p7 aggregates which create a larger channel or the non-uniform opening of several p7 channels. Similar multi-channel bursts have been seen elsewhere using a FLAG-p7 ADDIN EN.CITE ADDIN EN.CITE.DATA [30]. Several studies also showed that amantadine completely abrogates the single channel conductance of p7 in BLMs ADDIN EN.CITE ADDIN EN.CITE.DATA [28,30]. A feature common to all viroporins is their ability to drastically alter specific ion gradients within cells. By examining the reversal potential of p7-induced currents, it was found that these channels were permeable to K+, Na+ and Ca2+ ADDIN EN.CITE ADDIN EN.CITE.DATA [28,29]. Further investigation revealed the p7 displays cation selective characteristics with a preferred stoichometry of approximately 8 K+ to 1 Cl- ADDIN EN.CITE ADDIN EN.CITE.DATA [30]. The cation-selective nature of p7 has suggested several proposed functions of the protein pertaining to its role in the HCV lifecycle. Evidence of a finite Ca2+ permeability has suggested that p7 may modulate the flow of intracellular Ca2+.
Methods other than single channel recording have confirmed an ion channel like activity for p7. Using a cell-based assay originally designed to measure the activity of M2, it was found that, similar to M2, p7 was able to function to allow the influenza HA protein to reach the cell surface. Also similar to M2, p7s function in this assay and was inhibited by amantadine ADDIN EN.CITE ADDIN EN.CITE.DATA [31]. This system relied on the fact that M2 is required to prevent the accumulation of H+ within the exocytic vesicles containing haemagglutinin (HA), which would otherwise undergo a premature fusogenic conformational change at low pH. Therefore, when maintained at a more alkaline pH, HA can be transported to the cell surface and this transport can thus be used as a measure of M2 channel function. Substituting p7 for M2 in this assay resulted in HA being successfully transported to the cell surface indicating that p7 can shunt the pH of exocytic vesicles. Moreover, the p7-induced HA transport was sensitive to amantadine. All p7 sequences share a conserved set of basic residues which separate the two transmembrane domains. The strict conservation of K33 and R35 suggests that these are important for p7 function. A p7 mutant whereby the K33 and R35 are exchanged to alanine, was unable to successfully transport HA to the cell surface indicating that these residues are required for a functional ion channel.
Another system used to determine p7 function relied on the ability of p7 to initiate the release of carboxyfluorescein (CF) from liposomes. When present at high concentration within the liposome, CF is self-quenched. Thus, release or leakage from the liposome will eliminate the quenching of the released material resulting in a fluorescence signal. Purified FLAG-tagged p7 once incorporated into CF-containing artificial liposomes promoted a rapid release of CF as observed by a rapid increase in fluorescence of the extra-liposomal environment ADDIN EN.CITE ADDIN EN.CITE.DATA [32]. The CF release could be blocked by several viroporin inhibitors including amantadine and rimantadine. To further define the channel formation within the liposome, the release of various molecular weight fluorescent dextrans was monitored. The pores formed by p7 were able to efficiently release fluorescent dextrans of 4 kDa. However, the release of a 10 kDa dextran was significantly reduced indicating the formation of defined channel structures.
Structural Analysis of p7
Each p7 monomer consists of 63 amino acids, most of which are hydrophobic and this makes structural determination via crystal structural analysis difficult. Electron microscopy and computer modeling studies aimed at defining the oligomerization state of the p7 channel have suggested that monomers assemble into either hexamers or heptamers in lipid bilayers ADDIN EN.CITE ADDIN EN.CITE.DATA [28,33]. The first hint of p7 structure came in 2002 when possible membrane sequences as well as structural and functional motifs of p7, sequence alignments and comparisons among various HCV isolates were examined ADDIN EN.CITE Carrere-Kremer200290909017Carrere-Kremer, S.Montpellier-Pala, C.Cocquerel, L.Wychowski, C.Penin, F.Dubuisson, J.CNRS-FRE2369, Institut de Biologie de Lille/Institut Pasteur de Lille, 59021 Lille Cedex, France.Subcellular localization and topology of the p7 polypeptide of hepatitis C virusJ VirolJ Virol3720-307682002/03/22Amino Acid SequenceCell LineHepacivirus/chemistry/genetics/growth & development/*metabolismHepatitis C/virologyHumansMembrane Proteins/metabolismMolecular Sequence DataProtein Sorting SignalsSequence Analysis, DNASubcellular Fractions/*metabolismViral Proteins/*chemistry/genetics/*metabolism2002Apr0022-538X (Print)11907211http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11907211136108eng[34]. Sequence and hydropathy analysis of the specific residues of p7 revealed a strong conservation of p7 structure and organization amongst HCV genotypes. Using several computer modeling techniques, the two long hydrophobic stretches were predicted to be transmembrane regions of the protein with the first transmembrane segment comprising residues 19 to 32 and possibly extending as far as position 10. The second transmembrane segment consists of residues 36 to 58 and is suggested to be - h e l i c a l i n n a t u r e . T h e t w o t r a n s e m e m b r a n e r e g i o n s a r e c o n n e c t e d b y a s h o r t b a s i c s e g m e n t ( r e s i d u e s 3 3 t o 3 5 ) a n d t h i s s e g m e n t , w h i l e n o t w i t h i n t h e m e m b r a n e , h a d b e e n i n i t i a l l y p r o p o s e d t o a i d i n t h e t r a n s m e m b r a n e d o m a i n p o s i t i o n i n g w i t h i n t h e m e m b r a n e . Further dissection of an expanded loop region corresponding to amino acids 25 to 42 showed that this region binds strongly to phospholipids and has a high tendency to oligomerize in the presence of phospholipids ADDIN EN.CITE Perez-Berna200812812812817Perez-Berna, A. J.Guillen, J.Moreno, M. R.Bernabeu, A.Pabst, G.Laggner, P.Villalain, J.Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, Alicante, Spain.Identification of the membrane-active regions of hepatitis C virus p7 protein: biophysical characterization of the loop regionJ Biol ChemJ Biol Chem8089-101283132008/01/17Amino Acid SequenceCell Membrane/drug effects/*metabolismHepacivirus/*chemistry/geneticsMagnetic Resonance SpectroscopyMolecular Sequence DataPeptide Fragments/chemistry/pharmacologyThermodynamicsViral Proteins/*chemistry/genetics/*metabolism2008Mar 280021-9258 (Print)18198177http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18198177M709413200 [pii]
10.1074/jbc.M709413200eng[35]. This loop region is also sufficient to facilitate membrane permeabilization.
Further in vivo evidence showed that p7 oligomerizes when expressed in HepG2 cells ADDIN EN.CITE Griffin200393939317Griffin, S. D.Beales, L. P.Clarke, D. S.Worsfold, O.Evans, S. D.Jaeger, J.Harris, M. P.Rowlands, D. J.School of Biochemistry and Molecular Biology, University of Leeds, Division of Microbiology Old Medical School, Thoresby Place, Leeds LS2 9JT, UK.The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, AmantadineFEBS LettFEBS Lett34-85351-32003/02/01Amantadine/*pharmacologyAntiviral Agents/*pharmacologyCarcinoma, Hepatocellular/metabolismHumansIon Channels/chemistry/*drug effects/metabolismLipid Bilayers/chemistryMembranes, ArtificialMicroscopy, ElectronRecombinant Fusion Proteins/biosynthesis/chemistryTumor Cells, CulturedViral Proteins/*chemistry/*metabolism/ultrastructure2003Jan 300014-5793 (Print)12560074http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12560074S0014579302038516 [pii]eng[28]. This demonstration required cross linking of the cell lysates and after treatment with DSP, a larger molecular weight complex of 42 kDa appears, suggesting that p7 may form a hexamer although heptameric bundles have also been suggested. Further investigation revealed that a HIS-p7 formed ring-like structures observed by transmission electron microscopy in unilamellar vesicles and computational modeling of this structure suggests that the ring-like structure is stabilized by the C-terminal region of one monomer interacting with the N-terminal region of the adjacent monomer ADDIN EN.CITE Griffin200393939317Griffin, S. D.Beales, L. P.Clarke, D. S.Worsfold, O.Evans, S. D.Jaeger, J.Harris, M. P.Rowlands, D. J.School of Biochemistry and Molecular Biology, University of Leeds, Division of Microbiology Old Medical School, Thoresby Place, Leeds LS2 9JT, UK.The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, AmantadineFEBS LettFEBS Lett34-85351-32003/02/01Amantadine/*pharmacologyAntiviral Agents/*pharmacologyCarcinoma, Hepatocellular/metabolismHumansIon Channels/chemistry/*drug effects/metabolismLipid Bilayers/chemistryMembranes, ArtificialMicroscopy, ElectronRecombinant Fusion Proteins/biosynthesis/chemistryTumor Cells, CulturedViral Proteins/*chemistry/*metabolism/ultrastructure2003Jan 300014-5793 (Print)12560074http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12560074S0014579302038516 [pii]eng[28]. A deeper look into p7 structure came about using a more global computational approach and favors a model in which p7 oligermizes into a hexamer with the histidine at position 17 facing the interior of the pore ADDIN EN.CITE Chew200811911191119117Chew, C. F.Guy, A.Biggin, P. C.Department of Biochemistry, University of Oxford, Oxford OX13QU, United Kingdom.Distribution and dynamics of adamantanes in a lipid bilayerBiophys JBiophys J5627-3695122008/10/07AbsorptionAdamantane/chemistry/*metabolismCell Membrane/chemistry/metabolismLipid Bilayers/*chemistry/*metabolismModels, MolecularMolecular ConformationProtons2008Dec 151542-0086 (Electronic)
0006-3495 (Linking)18835906http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=188359062599851S0006-3495(08)81981-9 [pii]
10.1529/biophysj.108.139477eng[36]. The ability of copper to inhibit p7 channel activity in black lipid membranes further suggests that His-17 lines the p7 pore and suggests a possible functional role as a proton channel. The interior positioning of the histadine residue is seen with other proton channels including M2 of influenza ADDIN EN.CITE ADDIN EN.CITE.DATA [37]. The His-37 of M2 proton channel plays a role in pH detection, gating and proton conduction.
Recently a 3-dimensional analysis of the p7 structure was revealed. Cross-linked p7 monomers were negatively stained and analyzed by single particle electron microscopy as well as immunoelectron microscopy. The analysis revealed that p7 adopts a conically shaped hexameric orientation with protruding petal-like N- and C-terminal ends which orient luminally ADDIN EN.CITE Luik200917817817817Luik, P.Chew, C.Aittoniemi, J.Chang, J.Wentworth, P., Jr.Dwek, R. A.Biggin, P. C.Venien-Bryan, C.Zitzmann, N.Department of Biochemistry and Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopyProc Natl Acad Sci U S AProc Natl Acad Sci U S A12712-6106312009/07/11Imaging, Three-DimensionalMicroscopy, ElectronMicroscopy, ImmunoelectronModels, MolecularViral Proteins/*chemistry2009Aug 41091-6490 (Electronic)19590017http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1959001727223410905966106 [pii]
10.1073/pnas.0905966106eng[38] (Fig. 3). The broad nature of the termini provides potential interaction sites for both cellular and viral proteins. Typical interaction points of ion channels and oligomeric integral membrane proteins lie within the N and C terminal regions where one such domain lines the pore of the channel and the other acts to stabilize the oligomeric structure within the membrane. Similar connection points have been proposed for the p7 monomers however, the recent single particle resolution of immuno-labeled p7 monomers shows that interaction points are restricted to the bottom half of the protein. This is consistent with the finding that the loop region of p7 was sufficient to oligomerize in lipid bilayers ADDIN EN.CITE Perez-Berna200812812812817Perez-Berna, A. J.Guillen, J.Moreno, M. R.Bernabeu, A.Pabst, G.Laggner, P.Villalain, J.Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, Alicante, Spain.Identification of the membrane-active regions of hepatitis C virus p7 protein: biophysical characterization of the loop regionJ Biol ChemJ Biol Chem8089-101283132008/01/17Amino Acid SequenceCell Membrane/drug effects/*metabolismHepacivirus/*chemistry/geneticsMagnetic Resonance SpectroscopyMolecular Sequence DataPeptide Fragments/chemistry/pharmacologyThermodynamicsViral Proteins/*chemistry/genetics/*metabolism2008Mar 280021-9258 (Print)18198177http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18198177M709413200 [pii]
10.1074/jbc.M709413200eng[35].
Figure 3. The 3D structure of a hexameric HCV p7 channel. Chemically synthesized p7 monomers were solubilized in detergent and the resultant oligomeric channels were ultimately analyzed using single particle reconstruction. The p7 oligomer forms a conically shaped channel with protruding N- and C-termini. Adapted from Luik et al., PNAS. 2009 ADDIN EN.CITE Luik200917817817817Luik, P.Chew, C.Aittoniemi, J.Chang, J.Wentworth, P., Jr.Dwek, R. A.Biggin, P. C.Venien-Bryan, C.Zitzmann, N.Department of Biochemistry and Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopyProc Natl Acad Sci U S AProc Natl Acad Sci U S A12712-6106312009/07/11Imaging, Three-DimensionalMicroscopy, ElectronMicroscopy, ImmunoelectronModels, MolecularViral Proteins/*chemistry2009Aug 41091-6490 (Electronic)19590017http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1959001727223410905966106 [pii]
10.1073/pnas.0905966106eng[38].
Biology of p7 in Virus Production
Studies of HCV sub-genomic replicons show that p7 is not critical for RNA replication and in support, several p7 mutants display no defect in replication. Yet, the role of p7 in the virus life cycle has remained elusive. Before the establishment of a fully infectious cell culture system, the direct examination of p7 in culture was hampered. However intrahepatic transfection of chimpanzees using mutagenized HCV cDNA clones provided insight into the role of p7. Various p7 mutations were introduced into the infectious genotype 1a cDNA clone and RNA transcripts of each mutant were tested for infectivity in chimpanzees ADDIN EN.CITE Sakai200318118118117Sakai, A.Claire, M. S.Faulk, K.Govindarajan, S.Emerson, S. U.Purcell, R. H.Bukh, J.Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequencesProc Natl Acad Sci U S AProc Natl Acad Sci U S A11646-51100202003/09/25AnimalsCell LineGenetic VectorsGenotypeHepacivirus/genetics/*pathogenicityHumansMutagenesisPan troglodytesViral Proteins/genetics/*physiologyVirulence2003Sep 300027-8424 (Print)14504405http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1450440520881210.1073/pnas.1834545100
1834545100 [pii]eng[39]. Mutants with deletions of all or part of p7 were not viable and were unable to establish infection demonstrating that p7 is critical for infectivity. Also not viable were p7 mutants with substitutions of the two conserved residues in the cytoplasmic loop ADDIN EN.CITE Sakai200318118118117Sakai, A.Claire, M. S.Faulk, K.Govindarajan, S.Emerson, S. U.Purcell, R. H.Bukh, J.Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequencesProc Natl Acad Sci U S AProc Natl Acad Sci U S A11646-51100202003/09/25AnimalsCell LineGenetic VectorsGenotypeHepacivirus/genetics/*pathogenicityHumansMutagenesisPan troglodytesViral Proteins/genetics/*physiologyVirulence2003Sep 300027-8424 (Print)14504405http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1450440520881210.1073/pnas.1834545100
1834545100 [pii]eng[39].
The establishment of a fully infectious cell culture system has allowed for a more systematic approach to investigate the biological function of p7 in the replication cycle of HCV. Using a monocistronic reporter virus, the necessity of p7 was examined and it was found that p7 is essential for infectious virus production in culture ADDIN EN.CITE Jones200712112112117Jones, C. T.Murray, C. L.Eastman, D. K.Tassello, J.Rice, C. M.Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virusJ VirolJ Virol8374-8381162007/06/01Cells, CulturedConserved SequenceGenes, Reporter/geneticsHepacivirus/genetics/*growth & development/physiologyHumansIon Channels/genetics/*metabolismLuciferases/analysis/geneticsMutationViral Nonstructural Proteins/genetics/*metabolismViral Proteins/genetics/*metabolism2007Aug0022-538X (Print)17537845http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=175378451951341JVI.00690-07 [pii]
10.1128/JVI.00690-07eng[40]. A p7 deleted mutant, J6/JFH-1 p 7 , f a i l s t o p r o d u c e i n f e c t i o u s v i r i o n s u n l i k e t h e w i l d - t y p e J 6 / J F H - 1 w h i c h y i e l d e d n e a r l y 1 0 6 R L U . T h i s d i r e c t l y c o r r e l a t e d w i t h t h e l a c k o f c o r e p r o t e i n r e l e a s e d i n t o t h e s u p e r n a t a n t i n d i c a t i n g t h a t m u t a t i o n s i n p 7 c a u s e a g e n e r a l b l o c k i n v i r i o n r e l e a se ADDIN EN.CITE Jones200712112112117Jones, C. T.Murray, C. L.Eastman, D. K.Tassello, J.Rice, C. M.Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virusJ VirolJ Virol8374-8381162007/06/01Cells, CulturedConserved SequenceGenes, Reporter/geneticsHepacivirus/genetics/*growth & development/physiologyHumansIon Channels/genetics/*metabolismLuciferases/analysis/geneticsMutationViral Nonstructural Proteins/genetics/*metabolismViral Proteins/genetics/*metabolism2007Aug0022-538X (Print)17537845http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=175378451951341JVI.00690-07 [pii]
10.1128/JVI.00690-07eng[40]. Interestingly, no intracellular virus was produced in the presence of these p7 mutations, indicating that p7 acts at an early stage of morphogenesis and assembly prior to the formation of infectious intracellular virus.
Several residues within p7 are highly conserved amongst genotypes and thus thought to be involved in p7 function. These residues include histidine (H31), tyrosine (Y42) and tryptophan (W30) and similar residues are essential for the structure and function of several other viroporins including Vpu and M2 ADDIN EN.CITE ADDIN EN.CITE.DATA [37]. When the histidine at position 31 within a p7 consensus sequence was mutated to tyrosine, phenylalanine or leucine, virus production remained unchanged ADDIN EN.CITE Steinmann200712312312317Steinmann, E.Penin, F.Kallis, S.Patel, A. H.Bartenschlager, R.Pietschmann, T.Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.Hepatitis C virus p7 protein is crucial for assembly and release of infectious virionsPLoS PathogPLoS Pathoge103372007/07/31AnimalsApe Diseases/*virologyBase SequenceBiological Products/metabolism/physiologyDisease Models, Animal*Gene Expression Regulation, ViralGenotypeHepacivirus/*pathogenicity/physiologyMolecular Sequence Data*Pan troglodytesViral Proteins/*physiologyVirus Assembly/geneticsVirus Replication/genetics2007Jul1553-7374 (Electronic)17658949http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17658949192487007-PLPA-RA-0033 [pii]
10.1371/journal.ppat.0030103eng[41]. However, the mutations W30F and Y42F in the p7 sequence of JFH-1 severely impaired virus production suggesting that these residues are involved in the function of p7. Besides this, mutation of the basic loop which eliminates channel activity, KR33/35AA, significantly impaired virus production, and only after 72 h is a modest amount, if any at all, of virus produced. Interestingly, a genotypic-dependent effect in virus inhibition with the various p7 mutants is also seen ADDIN EN.CITE Steinmann200712312312317Steinmann, E.Penin, F.Kallis, S.Patel, A. H.Bartenschlager, R.Pietschmann, T.Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.Hepatitis C virus p7 protein is crucial for assembly and release of infectious virionsPLoS PathogPLoS Pathoge103372007/07/31AnimalsApe Diseases/*virologyBase SequenceBiological Products/metabolism/physiologyDisease Models, Animal*Gene Expression Regulation, ViralGenotypeHepacivirus/*pathogenicity/physiologyMolecular Sequence Data*Pan troglodytesViral Proteins/*physiologyVirus Assembly/geneticsVirus Replication/genetics2007Jul1553-7374 (Electronic)17658949http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17658949192487007-PLPA-RA-0033 [pii]
10.1371/journal.ppat.0030103eng[41] . W h i l e t h e "p 7 w a s u n a b l e t o y i e l d i n f e c t i o u s v i r u s i n a n y o f t h e g e n o t y p e s t e s t e d , c h a n n e l i n a c t i v a t i n g m u t a t i o n s h a v e a v a r y i n g e f f e c t . I n t h e c o n t e x t o f t h e J F H - 1 a n d C o n - 1 g e n o m e s , t h e R R 3 3 / 3 5 A A m u t a t i o n c o m p l e t e l y b l o c k e d v i r u s p r o d u c t i o n . H o w e v e r in the context of the Jc1 (genotype 2a/2a) chimera, the same p7 mutation had, although significant, only a 2-log reduction in virus production. Further investigation hinted that this mutation in Jc1 resulted in an increase in intracellular virus and thus it was concluded that p7 acts at a very late state of virus production, namely release. However, examination of the published data shows that the RR33/35AA mutation reduced extracellular virus yield by 99% and intracellular virus by 97%. If p7 were to function only at the late stage of infectious virus release, one would expect an accumulation of intracellular virus, however the drastic reduction in both intra- and extracellular virus production does not suggest that p7 functions in late release but rather p7 acts early in infectious virus particle assembly.
Recently, a novel trans-complementation system permitting the rescue of genomes containing deleted p7 with expression of p7 in trans has been developed ADDIN EN.CITE ADDIN EN.CITE.DATA [42]. T h i s s y s t e m c o - t r a n s f e c t e d H C V g e n o m e s c a r r y i n g a d e f e c t i v e p 7 w i t h b i c i s t r o n i c J F H 1 - d e r i v e d h e l p e r r e p l i c o n s t h a t e x p r e s s v a r i o u s p 7 s e q u e n c e s i n t h e f i r s t c i s t r o n a n d J F H 1 p r o t e i n s N S 3 t o N S 5 B i n t h e s e c o n d . T h e i n f e c t i v i t y o f J c 1 p 7 h a l f w a s r e s c u e d b y s e v e r a l p 7 - c o n t a i n n g h e l p e r R N A s . H o w e v e r r e s c u e o f t h e J c 1 p 7 f u l l m u t a n t w a s a c h i e v e d o n l y b y t h e c o - e x p r e s s i o n o f E 2 - p 7 - N S 2 s u g g e s t i n g t h a t t h i s d e f e c t m a y a l t e r p r o t e i n p r o c e s s i n g . C o n s i s t e n t w i t h o t h e r f i n d i n g s A D D I N E N . C I T E A D D I N E N . C I T E . D A T A [ 3 9 , 4 0 ] , J c 1 g e n o m e s w i t h e i t h e r a p o i n t m u t a t i o n o f t h e c o n s e r v e d b a s i c r e s i d u e s o f p 7 ( J c 1 - K R 3 3 / 3 5 Q Q ) o r t h e p a r t i a l ( J c 1 p 7 h a l f ) o r c o m p l e t e ( J c 1 p 7 f u l l ) d e l e t i o n o f p 7 v a r i e d i n t h e i r a b i l i t y t o p r o d u c e i n f e c t i o u s v i r u s p a r t i c l e s . W h i l e h a l f o r c o m p l ete deletions of the p7 coding region entirely abolished the production of infectious viruses, the mutation of the basic loop reduced viral titers almost 100-fold. In summary, p7 plays a critical role in the production of infectious virus particles and mutations within various regions of the protein can be deleterious to this function.
HCV Therapy and p7 Inhibitors
Current HCV therapy consists of a combination of pegylated interferon- ( I F N ) a n d t h e n u c l e o s i d e a n a l o g , r i b a v i r i n . U n f o r t u n a t e l y t h i s t r e a t m e n t i s n o t w e l l t o l e r a t e d a n d o f t h o s e w h o q u a l i f y f o r t r e a t m e n t , o n l y 5 0 % w i l l s e e a s u s t a i n e d v i r o l o g i c a l r e s p o n s e . T h e p o o r r e s p o n s e r a t e i s d u e t o t h e f a c t t h a t H C V i s h i g h l y v a r i able and that several HCV genomes are intrinsically resistant to the antiviral action of IFN. The variability of HCV will therefore require a combination of drugs which specifically target both the virus and the host.
Due to their capacity to alter the permeability barrier of cell membranes and their necessity for functional virion production, viroporins are attractive candidates for antiviral drug development. Although the role of HCV p7 in virus life cycle has not been fully elucidated, the ability of p7 inhibitors to block p7 channel activity in BLMs has indicated p7 as a potential target for antiviral therapy. p7 channel inhibitors could add a more virus-specific component to the combination therapy currently used to treat HCV infection. Because p7 is required for viral propagation, inhibiting its channel activity could lead to diminished virus production. Amantadine, rimantadine, hexamethylene amiloride, and long alkyl-chain imino sugar derivatives have been identified as ion channel inhibitors that block viroporin-mediated ion transport.
The amiloride derivative hexamethylene amiloride (HMA) was originally targeted for HIV ADDIN EN.CITE Gage20051190119011906Gage, P. W.Ewart, G. D.Melton, J. V.Premkumar, A.Zouhair Atassi, M.Virus Ion Channels Formed by Vpu of HIV-1, the 6k Protein of Alphaviruses and NB of Influenza B VirusProtein: Viral Membrane Proteins: Structure, Function, and Drug Design12005Springer US[19]. It has been shown to interfere with other viral viroporins and is often used to determine viroporin-like activity. HMA inhibits HIV-1 Vpu activity as well as replication of HIV-1 in cultured human macrophages with similar outcomes shown for other viruses. At similar concentrations of 100 M, HMA will also block p7 channel activity in lipid bilayers ADDIN EN.CITE Premkumar200494949417Premkumar, A.Wilson, L.Ewart, G. D.Gage, P. W.John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia.Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amilorideFEBS LettFEBS Lett99-1035571-32004/01/27Amiloride/*analogs & derivatives/*pharmacologyAmino Acid SequenceIon Channels/*biosynthesis/drug effects*Lipid BilayersMembrane Potentials/drug effectsMolecular Sequence DataPeptide Fragments/chemistry/pharmacologySpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationViral Proteins/chemistry/*pharmacology2004Jan 160014-5793 (Print)14741348http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14741348S0014579303014534 [pii]eng[43]. While, it is unknown if this inhibitor is able to block virus production for HCV, HMA was found to be extremely toxic at concentrations as low as 20 M ADDIN EN.CITE ADDIN EN.CITE.DATA [44].
Another class of antiviral agents used against various viruses are the iminosugar derivatives containing the glucose analogue deoxynojirimycin (DNJ) . T h e s e h a v e b e e n s h o w n t o e x e r t t h e i r a n t i v i r a l e f f e c t s o n t h e E R - a s s o c i a t e d e n z y m e s - g l u c o s i d a s e s I a n d I I A D D I N E N . C I T E <