JVI Accepts, published online ahead of print on 10 December 2014 J. Virol. doi:10.1128/JVI.02982-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Phosphatidylserine-Specific Phospholipase A1 is involved in Hepatitis C Virus
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Assembly through NS2 Complex Formation
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Min Guoa, b, Rongjuan Peia*, Qi Yanga, b, Huang Caoa, b, Yun Wanga, Chunchen Wua,
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Jizheng Chena, Yuan Zhoua, Xue Hua, Mengji Luc, Xinwen Chena*
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a. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy
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of Sciences, Wuhan, 430071，China,
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b. University of Chinese Academy of Sciences, Beijing, China
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c. Department of Infectious Disease, University hospital Essen, University of
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Duisburg-Essen, Essen, Germany
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Running title, role of PLA1A in HCV assembly
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Corresponding author:
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Prof. Dr. Xinwen Chen
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State Key Laboratory of Virology,
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Wuhan Institute of Virology, Chinese Academy of Sciences,
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Wuhan 430071, People’s Republic of China
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Phone&Fax: 86-027-87199106;
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E-mail:
[email protected]. cn
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Dr. Rongjuan Pei
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State Key Laboratory of Virology,
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Wuhan Institute of Virology, Chinese Academy of Sciences, 1
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Wuhan 430071, People’s Republic of China
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Phone: 86-027-87197575;
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E-mail:
[email protected]. cn
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2
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ABSTRACT
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Several members of the phospholipase family have been reported to be involved in
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hepatitis C virus (HCV) replication. Here, we identified another phospholipase,
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phosphatidylserine-specific phospholipase A1 (PLA1A), as a host factor involved in
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HCV assembly. PLA1A was upregulated by HCV infection; and PLA1A knockdown
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significantly reduced J399EM (genotype 2a) HCV propagation at the assembly but
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not the entry, RNA replication, and protein translation steps of the life cycle. Protein
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localization and interaction analysis further reveal a role of PLA1A in the interaction
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of NS2-E2 and NS2-NS5A, as the formation of the NS2-E2 and NS2-NS5A
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complexes was weakened in the absence of PLA1A. In addition, PLA1A stabilized
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the NS2/NS5A-dotted structure during infection. These data suggest that PLA1A
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plays an important role of bridging the membrane-associated NS2-E2 complex and
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the NS5A-associated replication complex via its interaction with E2, NS2 and NS5A,
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which leads to a coordinating interaction between the structural and non-structural
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proteins and facilitates viral assembly.
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Importance
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Hepatitis C virus (HCV) genomic replication is driven by the replication complex and
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occurs at the membranous web, while the lipid droplet is the organelle in which virion
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assembly is initiated. In this study, we identified phosphatidylserine-specific
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phospholipase A1 (PLA1A), a member of phospholipase A 1 family, as a novel host
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factor involved in the assembly process of HCV. PLA1A which is induced by HCV 3
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infection at late infection stage interacts with HCV E2, NS2 and NS5A proteins and
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enhance and stabilize the NS2-E2 and NS2-NS5A complex formation, which is
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essential for viral assembly. Thus, PLA1A is an important host factor which is
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involved in the initiation of the viral assembly in close proximity to Core-decorated
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lipid droplet through bringing together the HCV replication complex and envelope
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complex.
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4
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Introduction
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Hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting
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approximately 185 million people worldwide (42). HCV is a positive single-stranded
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RNA virus belonging to the Flaviviridae family. The HCV 9.6 kb genome contains a
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large open reading frame encoding a single polyprotein that is processed into its
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structural proteins (Core, E1 and E2) and nonstructural (NS) proteins (p7, NS2, NS3,
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NS4A, NS4B, NS5A and NS5B) by host and viral proteinases (24). The structural
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proteins are components of the virion, while the non-structural proteins NS3 to NS5B
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compose the minimal viral replicase governing RNA replication (4, 25).
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The overall HCV life cycle has been well defined since the development of an
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infectious HCV cell culture system (23, 49, 51). HCV genomic replication is driven
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by the replication complex (RC) and occurs at the membranous web, a rearranged
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membrane structure induced by virus infection (9, 12, 43). Recent progress regarding
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the study of the assembly process demonstrates that the lipid droplet (LD) is the
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organelle in which virion assembly is initiated (29) and that, in addition to the
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structural proteins, almost all the nonstructural proteins and many host factors are
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involved in this process (22). The roles of NS2 and P7 in virion assembly attract
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attention because these molecules are not components of the virion or the replicase.
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NS2 is a polytopic transmembrane protein containing 3 putative transmembrane
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segments (17) and is suggested to serve as the scaffold for virus assembly by
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interacting with both structural and nonstructural proteins such as E1/E2, NS3/4A and
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NS5A (26, 37, 44). P7 is a small protein with 63 amino acids (aa) harboring ion 5
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channel activity. Its role in HCV morphogenesis has been studied by mutation
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analysis and has been shown to be important for capsid assembly and envelopment (6,
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19, 45). NS5A is another critical factor in the assembly process and is recruited to the
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Core-decorated LD through the interaction between its domain II and Core, bringing
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HCV RNA to the assembly site (27).
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In addition to viral proteins, several host factors participate in the HCV assembly
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process by influencing the localization of HCV proteins or by mediating the
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interactions
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diacylglycerol acyltransferase-1 (DGAT1) was first found to recruit the Core protein
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to LD (14). Recently, the interaction between DGAT1 and NS5A was confirmed and
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was shown to be the bridge between Core and NS5A, facilitating HCV assembly (7,
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14). Two additional proteins, Rab18 and TIP47, were shown to interact with NS5A
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and promote the interaction between viral replication sites and LD (36, 38, 48). In
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addition, signal peptidase complex subunit 1 (SPCS1) facilitates the interaction
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between NS2 and E2 and is involved in the early step of the assembly of infectious
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particles (46).
between
HCV
proteins.
The
triglyceride-synthesizing
enzyme
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Several members of the phospholipase A2 family such as PLA2G4A, PLA2G4C and
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PLA2GXIIB are involved in HCV replication via various mechanisms (21, 28, 39, 50),
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implying a role for the phospholipase family in HCV replication. Here, we identified
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another
member
of
the
phospholipase
family,
phosphatidylserine
specific 6
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phospholipase A1 (PLA1A), as a host factor involved in HCV assembly. PLA1A was
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first identified in rat pellets (39), and PLA1A mRNA is present at high levels in
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human liver tissue (2). PLA1A specifically acts on phosphatidylserine (PS) and
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1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position
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of these phospholipids (30). PLA1A is a secreted protein, and its substrate, PS,
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typically localizes to the inner leaflet of the lipid bilayer, and when PS is exposed on
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the surface of the cell membrane, PLA1A exerts its lipase functions. Although the
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lipase activity of PLA1A has been verified, its functions in vivo still remain unknown.
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However, PLAIA was shown to act with its substrate PS and product lysoPS (30),
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which is a lipid mediator that plays a role in the inflammation response (10).
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In the present study, we revealed a role for PLA1A in HCV assembly. PLA1A
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interacts with the HCV E2, NS2 and NS5A proteins, facilitating NS2-E2 and
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NS2-NS5A complex formation during virus assembly.
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7
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Materials and methods
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Cell culture
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Huh7.5.1 (kindly provided by Prof. Frank Chisari) and Huh7 cells were cultured in
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Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) supplemented with 2 mM
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L-glutamine, nonessential amino acids, and 10% fetal bovine serum (FBS)
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(Invitrogen). The subgenomic HCV replicon cell lines (Huh7.5.1-SGR (50) and Con1
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(33) containing the subgenomic genotype 2a and 1b HCV, respectively) were grown
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in the same medium supplemented with 500 μg ml-1 G418.
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Virus production
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The JFH-1 virus used in this study was based on the pJFH-1 plasmid kindly provided
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by Prof. Wakita (49). The J399EM strain was derived from the JFH-1 virus by
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insertion of eGFP into the HCV NS5A region (13). The JFH1-HA virus with an HA
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epitope at the N-terminus of NS2 was constructed as previously described (37). The
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viral titer in the culture supernatants and cell lysates was determined by a modified
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end-point dilution assay as previously described (33, 50).
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Plasmid construction
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The PLA1A (NM_001206960) coding sequence was amplified by PCR and inserted
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into the pXJ40-HA and pXJ40-Flag plasmids. To generate the HA- or Flag-tagged
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HCV protein expression plasmids, the coding sequences of HCV Core, E1, E2, NS2,
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NS3, NS34A, NS4B, NS5A and NS5B of genotype 2a (JFH1, AB047639) were 8
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amplified and inserted into the pXJ40-HA and pXJ40-Flag vectors. The bicistronic
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reporter, pHCV-IRES, was previously described (50).
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RNA interference
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The following siRNAs were used: siRNAs specific to PLA1A (siPLA1A-2,
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D-008411-02-0002; siPLA1A-3, D-008411-03-0003; siPLA1A-4, D-008411-04-0004,
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Thermo Scientific Dharmacon), AllStars Negative Control siRNA (siControl,
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#1027281, QIAGEN) and siRNA specific to HCV (siHCV, target sequence: 5 -
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GGUCUCGUAGACCGUGCAC - 3). The siRNAs were transfected using
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Lipofectamine 2000 (Invitrogen) at a final concentration of 20 nM according to the
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manufacturer’s instructions. To maintain the gene silencing effect from the beginning
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of infection to the last time point analyzed, the cells were split at 24 hours
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post-transfection (hpt) and transfected with the same siRNA again. HCV infection
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was performed 6 h after the second transfection.
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Stable cell line construction
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The coding sequences for short hairpin RNA (shRNA, shPLA1A#4 5’-
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GGTTTCCTTTGCCGATCTTAT - 3’) targeting the PLA1A gene and a negative
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control shRNA (5’- GGTGCAGCAGATTGTGAATACCCATAGTAA - 3’) were
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cloned into the shRNA expression vector pSUPER.retro.neo (OligoEngine, Inc)
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following the manufacturer’s instructions. The retroviruses were produced in 293T
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cells by co-transfection of the shPLA1A or shNC plasmid, pVPack-GP and 9
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pVPack-VSV-G (Stratagene). The retrovirus-containing supernatants were harvested
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at 72 hpt. To generate the PLA1A knockdown cell line, the Huh7.5.1 cells were
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transduced with the retrovirus in the presence of 8 μg ml-1 polybrene (Sigma), and
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stable knockdown pools were isolated by G418 selection and named Huh7.5.1-shNC
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(or shNC) and Huh7.5.1-shPLA1A#4 (or shPLA1A#4).
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Western blot, co-immunoprecipitation analysis and indirect immunofluorescence
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staining
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Whole cell lysates were prepared and quantified using the Bradford method (BioRad
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#500-0006). Equal amounts of protein samples (30 μg) were subjected to SDS-PAGE
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and transferred onto a nitrocellulose filter membrane (Millipore). After blocking with
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5% non-fat milk in TBST, the membranes were incubated with specific primary
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antibodies and corresponding peroxidase-conjugated secondary antibodies. The
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following antibodies were used for western blot: anti-NS3 (#ab65407, Abcam),
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anti-Core (#ab2740, Abcam), anti-Flag (#F1084, Sigma), anti-HA (#H9658, Sigma),
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anti-PLA1A (produced by Abmart), and anti-beta-actin (#sc-47778, Santa Cruz). The
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proteins were visualized using suitable HRP-conjugated secondary antibodies
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(Jackson Immuno Research) and SuperSignal-Femto chemiluminescent substrate
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(Pierce).
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Co-immunoprecipitation and indirect immunofluorescence staining were performed
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as previously described (50). The Alexa Flour 561/488/633-conjugated secondary 10
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antibodies used for indirect immunofluorescence staining were obtained from
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Invitrogen. LDs were stained with HCS LipidTOXTM RED (Invitrogen), and the
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nuclei were stained using Hoechst33258 (Invitrogen).
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Quantitative real-time RT-PCR
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Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen) and
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digested with RNase-free DNase (Promega) according to the manufacturer’s protocols.
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HCV RNA in the supernatant was prepared using TRIzol LS reagent (Invitrogen).
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Specific mRNAs and HCV RNAs were quantified by one-step real-time RT-PCR
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using the QuantiFast SYBR Green RT-PCR kit (Qiagen). The mRNA and HCV RNA
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levels were normalized against the copy number of human beta-actin mRNAs. The
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forward
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GGAACTGAGAAACAAGGACACC
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AAACTCGGTTGGAAGACTGAAA － 3’, and the primers for HCV and actin
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were previously described (52).
and
reverse
primers
used
to －
amplify
PLA1A 3’
and
were 5’
5’
－ －
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Membrane flotation fractionation
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For cell fractionation, 9 ×107 cells were resuspended in 1 ml of cold hypotonic buffer
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(10 mM Tris-HCl, pH 7.8, 10 mM NaCl, EDTA-free protease inhibitor cocktail
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(Roche)) and mechanically disintegrated by forcing the cell suspension through a
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25-gauge injection needle 30 times. The homogenate was centrifuged at 900 × g for 5
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min to pellet the nuclear fraction, followed by centrifugation at 15,000 × g for 20 min 11
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to pellet the cell membrane fraction. The deposit contained the cytoplasmic fraction,
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and the cell membrane fraction was resuspended in 950 μl hypotonic buffer, NP40
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was added to a final concentration of 1%, and the solution was incubated for 30 min
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on ice.
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Regarding sucrose density gradient centrifugation, 1 ml aliquots treated as above were
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mixed with 1 ml 72% (wt/v) sucrose in Laemmli sample buffer (LSB; 50 mM
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Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2) in an ultracentrifuge tube and overlaid
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with 4 ml 55% sucrose and 1.5 ml of 10% sucrose. The aliquots were then subjected
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to ultracentrifugation (38,000 × g for 16 h), and every 1 ml fraction was collected
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from the top of the gradient, mixed with 4 ml cold methyl alcohol and centrifuged at
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10,000 × g for 10 min to pellet the membrane proteins. The protein samples were
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characterized using SDS-PAGE and immunoblotting.
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Proteolytic digestion protection assay
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HCV-infected cells seeded in 6-well dishes were collected in 170 μl proteinase K
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buffer (50 mM Tris-HCl, pH 8.0, 10 mM CaCl2, 1 mM DTT) and subjected to five
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freeze-thaw cycles. Next, 50 μl of the crude lysate was left untreated, 50 μl was
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treated with 50 μg ml-1 proteinase K (Qiagen) for 20 min on ice, and another 50 μl
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was lysed with 5% (v/v) Triton X-100 prior to proteinase K treatment. Proteinase K
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digestion was terminated by the addition of 5 mM PMSF on ice for 10 min.
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Subsequently, 13 μl of 5 SDS sample buffer was added, and the sample was heated 12
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to 95°C for 10 min. The amount of residual core protein was determined by
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SDS-PAGE and immunoblotting.
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Statistical analysis
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Data were analyzed using a two-tailed unpaired t-test. P-values were calculated, and
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statistical significance was reported as highly significant with *(p
