Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD) through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

Annexin A2 is involved in the formation of hepatitis C virus replication complex on the lipid raft.

The hepatitis C virus (HCV) RNA replicates in hepatic cells by forming a replication complex on the lipid raft (detergent-resistant membrane [DRM]). Replication complex formation requires various viral nonstructural (NS) proteins as well as host cellular proteins. In our previous study (C. K. Lai, K. S. Jeng, K. Machida, and M. M. Lai, J. Virol. 82:8838-8848, 2008), we found that a cellular protein, annexin A2 (Anxa2), interacts with NS3/NS4A. Since NS3/NS4A is a membranous protein and Anxa2 is known as a lipid raft-associated scaffold protein, we postulate that Anxa2 helps in the formation of the HCV replication complex on the lipid raft. Further studies showed that Anxa2 was localized at the HCV-induced membranous web and interacted with NS4B, NS5A, and NS5B and colocalized with them in the perinuclear region. The silencing of Anxa2 decreased the formation of membranous web-like structures and viral RNA replication. Subcellular fractionation and bimolecular fluorescence complementation analysis revealed that Anxa2 was partially associated with HCV at the lipid raft enriched with phosphatidylinositol-4-phosphate (PI4P) and caveolin-2. Further, the overexpression of Anxa2 in HCV-nonsusceptible HEK293 cells caused the enrichment of HCV NS proteins in the DRM fraction and increased the colony-forming ability of the HCV replicon. Since Anxa2 is known to induce the formation of the lipid raft microdomain, we propose that Anxa2 recruits HCV NS proteins and enriches them on the lipid raft to form the HCV replication complex.

Hepatitis C virus egress and release depend on endosomal trafficking of core protein.

Hepatitis C virus (HCV) assembly is known to occur in juxtaposition to lipid droplets, but the mechanisms of nascent virion transport and release remain poorly understood. Here we demonstrate that HCV core protein targets to early and late endosomes but not to mitochondria or peroxisomes. Further, by employing inhibitors of early and late endosome motility in HCV-infected cells, we demonstrate that the movement of core protein to the early and late endosomes and virus production require an endosome-based secretory pathway. We also observed that this way is independent of that of the internalization of endocytosed virus particles during virus entry.

Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A.

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.

Hepatitis C virus NS3/4A protein interacts with ATM, impairs DNA repair and enhances sensitivity to ionizing radiation.

Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Nonstructural protein 3 (NS3) of HCV possesses serine protease, nucleoside triphosphatase, and helicase activities, while NS4A functions as a cofactor for the NS3 serine protease. Here, we show that HCV NS3/4A interacts with the ATM (ataxia-telangiectasia mutated), a cellular protein essential for cellular response to irradiation. The expression of NS3/4A caused cytoplasmic translocation of either endogenous or exogenous ATM and delayed dephosphorylation of the phosphorylated ATM and gamma-H2AX following ionizing irradiation. As a result, the irradiation-induced gamma-H2AX foci persisted longer in the NS3/4A-expressing cells. Furthermore, these cells showed increased comet tail moment in single-cell electrophoresis assay, indicating increased double-strand DNA breaks. The cells harboring an HCV replicon also exhibited cytoplasmic localization of ATM and increased sensitivity to irradiation. These results demonstrate that NS3/4A impairs the efficiency of DNA repair by interacting with ATM and renders the cells more sensitive to DNA damage. This effect may contribute to HCV oncogenesis.

Hepatitis C virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to DNA damage and STAT3 activation.

Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Previously, we reported that HCV infection causes cellular DNA damage and mutations, which are mediated by nitric oxide (NO). NO often damages mitochondria, leading to induction of double-stranded DNA breaks (DSBs) and accumulation of oxidative DNA damage. Here we report that HCV infection causes production of reactive oxygen species (ROS) and lowering of mitochondrial transmembrane potential (DeltaPsi(m)) in in vitro HCV-infected cell cultures. The changes in membrane potential could be inhibited by BCL-2. Furthermore, an inhibitor of ROS production, antioxidant N-acetyl-L-cysteine (NAC), or an inhibitor of NO, 1,400W, prevented the alterations of DeltaPsi(m). The HCV-induced DSB was also abolished by a combination of NO and ROS inhibitors. These results indicated that the mitochondrial damage and DSBs in HCV-infected cells were mediated by both NO and ROS. Among the HCV proteins, core, E1, and NS3 are potent ROS inducers: their expression led to DNA damage and activation of STAT3. Correspondingly, core-protein-transgenic mice showed elevated levels of lipid peroxidation and oxidatively damaged DNA. These HCV studies thus identified ROS, along with the previously identified NO, as the primary inducers of DSBs and mitochondrial damage in HCV-infected cells.

Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

BACKGROUND: Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. RESULTS: The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation). CONCLUSIONS: Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.