Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

Huang, Yanping

doi:10.3748/wjg.v11.i36.5659

Cited by 18 publications

(14 citation statements)

References 37 publications

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“…Whereas the majority of the described interactions are for bacterial proteins, there are a few viral proteins that have been shown to interact with human VN, including the HIV gp120/160 protein (34,35) and the hepatitis C virus (HCV) F protein (36). This is the first study to report the interaction of DENV NS1 with VN as a complement evasion strategy.…”

Section: Discussionmentioning

confidence: 90%

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

Conde

Silva

Allonso

et al. 2016

J Virol

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Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV).The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system. D engue constitutes a major public health problem in tropical and subtropical countries. According to current estimates, at least 390 million cases of dengue occur annually, of which approximately 100 million are symptomatic (1). The infection is caused by dengue virus (DENV), a member of the Flaviviridae family that cocirculates in nature as four distinct antigenic serotypes (DENV1 to -4). DENV infection in humans is generally asymptomatic, but symptomatic cases can vary from a mild and self-limited fever to a potentially fatal hemorrhagic syndrome (2). The DENV genome is composed of a single positive-sense RNA that encodes a single viral polyprotein that is further processed by viral and host proteases into three structural proteins (C, prM/M, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3).NS1 ...

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Section: Discussionmentioning

confidence: 90%

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

Conde

Silva

Allonso

et al. 2016

J Virol

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“…In fact, recent studies indicated that ARFP is an unstable protein that associates with the endoplasmic reticulum [13] and may play a role in the regulation of gene expression, cell signaling and apoptosis [14][15][16][17], as well as in altering immune responses [18]. These potential properties of the ARFP have attracted recently much attention and intensified the studies of its features.…”

mentioning

confidence: 96%

Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein

et al. 2012

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Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polypro tein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1," protein, which is expressed through a ribosomal frame shift within the capsid coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame shifted forward primer exploiting the capsid sequence of the lb subtype as a template. The amplicon was cloned into the pET 24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel nitrilotriacetic acid (Ni NTA) affinity column in a sin gle step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.

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“…These results implicate core11 in functions previously attributed to core protein, in the pathogenesis of chronic infection and cellular transformation. Guo et al also identified genes transactivated by core11/F and involved in metabolism, cell cycle regulation and cell apoptosis (97).…”

Section: Functional Properties Of Core11 Proteinsmentioning

confidence: 99%

“…Dou et al searched for changes in gene expression profile in human hepatoma Huh7 cells stably producing core11/F in microarray and semiquantitative RT-PCR analyses (98), whereas Guo et al screened for genes differentially expressed in the presence and absence of core11/F by suppression subtractive hybridization (97). Guo et al identified 36 interacting partners, 34 of which had known functions closely related to lipid and carbohydrate metabolism, protein synthesis and transport, immunoregulation or the occurrence and development of tumors (97). However, these data remain to be confirmed.…”

Section: Functional Properties Of Core11 Proteinsmentioning

confidence: 99%

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

Vassilaki

Mavromara

2009

IUBMB Life

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Hepatitis C virus (HCV) is an enveloped positive‐strand RNA virus of the Flaviviridae family. It has a genome of about 9,600 nucleotides encoding a large polyprotein (about 3,000 amino acids) that is processed by cellular and viral proteases into at least 10 structural and nonstructural viral proteins. A novel HCV protein has also been identified by our laboratory and others. This protein—known as ARFP (alternative reading frame protein), F (for frameshift) or core+1 (to indicate the position) protein ‐ is synthesized by an open reading frame overlapping the core gene at nucleotide +1 (core+1 ORF). However, almost 10 years after its discovery, we still know little of the biological role of the ARFP/F/core+1 protein. Abolishing core+1 protein production has no affect on HCV replication in cell culture or uPA‐SCID mice, suggesting that core+1 protein is probably not important for the HCV reproductive cycle. However, the detection of specific anti‐core+1 antibodies and T‐cell responses in HCV‐infected patients, as reported by many independent laboratories, provides strong evidence that this protein is produced in vivo. Furthermore, analyses of the HCV sequences isolated from patients with hepatocellular carcinoma and in vitro studies have provided strong preliminary evidence to suggest that core+1 protein plays a role in advanced liver disease and liver cancer. The available in vitro data also suggest that certain core function proteins may depend on production of the core+1 protein. We describe here the discovery of the various forms of the core+1 protein and what is currently known about the mechanisms of their production and their biochemical and functional properties. We also provide a detailed summary of the results of patient‐based research. © 2009 IUBMB IUBMB Life, 61(7): 739–752, 2009

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Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

Cited by 18 publications

References 37 publications

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

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