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Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

ABSTRACT: Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body.The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system.Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-? was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-? than the other ratios (1:50 and 2:50).Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.

SUBMITTER: Sabet S

PROVIDER: S-EPMC5528829 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

Sabet Salwa S George Marina A MA El-Shorbagy Haidan M HM Bassiony Heba H Farroh Khaled Y KY Youssef Tareq T Salaheldin Taher A TA

PloS one 20170726 7

<h4>Background</h4>Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body.<h4>Aim of work</h4>The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery sys ...[more]

PMID: 28746382

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Project description:UnlabelledRecombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1orig mutant termed SA13/JFH1Core-NS5B, containing 13 amino acid changes (R114W and V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, and I2340T [NS5A]; A2500S and V2841A [NS5B]), displayed fitness comparable to that of the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication but increased HCV assembly and specific infectivity as early as 24 h posttransfection. Infectious coculture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1orig and SA13/JFH1Core-NS5B, with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens.ImportanceHepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high-titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary.

Project description:Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

Dataset Information

Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

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Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

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