The Role Of Non-structural And Structural Proteins In HCV Replication And Infection

Hepatitis C virus first officially separated in 1989 is difficult to culture in vitro for it low viral titer in human blood and it is a big impediment for HCV related research and screening anti-drugs. This obstacle has recently been overcome by using JFH1-based full-length genomes that allows production of infectious viruses both in vitro and in vivo. JFH1 strain separated from a fulminant hepatitis C virus belongs to HCV 2a genetype. Although it is a milestone in the history of HCV research, the unique character of JFH1 can not be completely representative of HCV biology and there is an urgent demand for cell culture systems of all HCV genotypes especially HCV 1b a main genotype in China.People has gained a consensus through the genomic replicon that NS5A protein is an important factor involved in replication since the cell model system of HCV was first described in 1999. NS5A interacts with other proteins participated in replicating and forms a activate replication complexes initialing the replication process. Non-structural genes of HCV replicon may have the phenomenon of adaptive mutations cultured in vitro cell and introduction of cell-cultured adaptive mutations will increase the efficiency of colony formation (ECF). Adaptive mutations generally appear in the NS3, NS4B and NS5A districts, some mutations have synergistic effects. Especially, NS5A adaptive mutations resulted in highly phosphorylated forms of NS5A protein (p58) reduction and contributing to HCV replication. Other related work model is accelerating the formation of replication complexes and promoting the replicon replication.The amino acid of core is highly conservative in all genotypes of HCV and homology sequences reach 95%. Protein core plays a key role in wrapping viral RNA, maintaining its inherently external morphology, and causing persistent infection. The interaction between structural and non-structural proteins is essential for producing cell-cultured HCV viral particles. Protein core is an important factor in HCV infection and particles packaging. Some researches have shown that the process of replication will be inhibited to some extent by blocking the expression of core protein, but the specific mechanism is remain to be seen and related scientific researches about protein core are rarely few.The firstly important step for viral invasion is binding to the corresponding receptors located in cell membrane and forming a ligand - receptor complexes. Envelope glycoprotein E of HCV is such a receptor-binding protein. It binds to receptors in host cell membrane such as CD81, SR-B1, claudin1, occludin or other molecules with dimer form, and then mediates the invading process. Just like HCV, Japanese encephalitis virus (JEV) also belongs to yellow fever virus and has a similar gene structure. JEV is one of single, positive strand RNA viruses. Glycoprotein E of JEV is a structural protein, and also have the effect on mediating the process of invasion. JEV can infect not only people body, but also mouse. It is reported that JFH1 strain can replicate in mouse cell, but can't produce and package infectious viral particles.Therefore, in this study we take an attempt to replace the NS5A, core, E gene of HCV with corresponding districts of homologous or different virus belonging to yellow fever family. The ultimate aim is to set up a cell or small animal model supporting HCV replication and producing cell-cultured viral particles. A chimeric full-length HCV replicon FL-J6JFH/J4NS5A was constructed by replacing the NS5A of wild-type FL-J6JFH1 with the corresponding gene of J4. We compared the ability to replicate and produce cell-cultured infectious viral particles of FL-J6JFH/J4NS5A with wild-type FL-J6JFH1. The next, we deliberately changed three NS5A amino acids (S2197P, S2201del, S2204I) of FL-J6JFH/J4NS5A by artificial site-directed mutagenesis and analysised the replication capacity after mutation. The third part of my research was to replace the core gene of FL-J6JFH1 with the corresponding area of J4 by gene-swapping method, and compared the differences about HCV replication and infection. The secondary section of my paper was about HCV cell-tropsim. Japanese encephalitis virus (JEV) belongs to yellow fever virus, we constructed a chimeric HCV/JEV replicon by substituting the envelop (E) gene of HCV with the homologous gene. We take an attempt to obtain a cross-species spread model of HCV by changing the cell-tropism.1.Influence of JFH1 NS5A on the replication and infectivity of FL-J6JFH1Chimeric FL-J6JFH/J4NS5A, FL-J6JFH1 and the replication-defective NS5B negative control FL-J6JFH (GND) were linearized and transcribed with T7 RNA polymerase in vitro.The genomic RNA transcripts were delivered to Huh-7.5.1 cells by liposome-mediated transfection. The transfected cells were cultured and passed. At the day5, 8, 12 postransfection, cellular total RNA was isolated from the transfected Huh-7.5.1 cells, and the levels of HCV RNA were determined by HCV-specific FQ RT-PCR. The HCV RNA level first detected at the 5th day was about 1.1×106 GE/μg RNA in FL-J6JFH1 transfected cell, then decreased subsequently to minimum level of 9.5×105 GE/μg RNA at the day8 posttransfection. Intracellular HCV RNA level reached 4.6×106GE/μgRNA at the day12 posttransfection. Chimeric FL-J6JFH/J4NS5A maintained a low and stable HCV level above 104 GE/μg RNA. The replication incompetent RNA genome, FL-J6JFH (GND) showed a rapid disappearance after transfection. When the cells were passed, some were collected and examined for HCV proteins by immunofluorescence staining with serum from patient of hepatitis C, and HCV-specific proteins were detectable both in FL-J6JFH1 and FL-J6JFH/J4NS5A at days3,5,8,1 2 posttransfetion, but not in FL-J6JFH(GND). The Fluorescence intensity of FL-J6JFH/J4NS5A was lower than FL-J6JFH1 at the three different days. These results indicated that both FL-J6JFH/J4NS5A and FL-J6JFH1 can efficiently replicate in Huh7.5.1 cell, but the FL-J6JFH/J4NS5A had a lower replication potential comparing with FL-J6JFH1. The naive Huh-7.5.1 cells were then inoculated with the supernatants collected at days3, 5, 8, 12 posttransfection. HCV proteins were detected by immunofluorescence day3 after inoculation. The cells infected with FL-J6JFH1 and FL-J6JFH/J4NS5A transfected cell supernatants resulted in positive immunostaining. But the Fluorescence intensity of FL-J6JFH/J4NS5A was lower than FL-J6JFH1 in all four times detections. These results suggested that Huh-7.5.1 cells transfected with FL-J6JFH/J4NS5A were not only able to support HCV replication but also produce infectious HCV particles, although lower than wild type FL-J6JFH1. We also observed the growth kinetics of cells transfected with both chimeric and wild type FL-J6JFH. After day9 posttransfection, high numbers of non-adherent cells were noted continuously in the culture transfected with the wild type. In contrast, the chimeric RNA transfected cells failed to show a cytopathic effect (CPE), which also reflected that virus production was much less than the wide type. The above data showed that, when JFH1 NS5A domain was replaced with NS5A from 1b genotype J4, the replication level, the expression of protein and the production of HCVcc were enormously decreased, which suggested that JFH1 NS5A may play an important role in replication of HCV and the production of HCVcc. FL-J6JFH/J4NS5A not having the CPE may be closer to the natural HCV viral particles and it provided an ideal experimental platform for continuing to study the function of NS5A.2.Site-directed mutagenesis can supply a compensation for decrease of HCV replication potential Adopting deliberately site-directed mutagenesis method, we changed the three amino acids of NS5A in FL-J6JFH/J4NS5A, 2197-position(Ser-Pro), 2201-position serine missing deletion(del),2204-position (Ser-Ile),and then constructed the four HCV mutation replicon FL-J6JFH/J4NS5A(S2197P),FL-J6JFH/J4NS5A(S2201del),FL-J6JFH/J4NS5A(S2197P+S2201del)和FL-J6JFH/J4NS5A(S2197P+S2204I).The four mutation-containing HCV replicons and FL-J6JFH/J4NS5A were separately transfected with Huh7.5.1 cell after transcription in vitro. The cellular total RNA of transfected Huh7.5.1 cell was extracted at days5, 10 and the levels of HCV RNA were quantified by HCV-specific FQ RT-PCR. First we carried on qualitative detection of HCV RNA by HCV 5'UTR special primers at days5, 10, and all the five replicons were positive results. Semi-FQ RT-PCR found that the RNA levels of FL-J6JFH/J4NS5A(S2201del) and FL-J6JFH/J4NS5A(S2197P+S2204I) were increased. Cellular total RNA was isolated from the transfected Huh-7.5.1 cells at days5, 8 posttransfection, and the levels of HCV RNA were quantified by HCV-specific FQ RT-PCR. The HCV RNA highest level detected at the 8th day was about 5.3×105GE/ug RNA in FL-J6JFH/J4NS5A(S2197P+S2204I), nearly equivalent 150 times of FL-J6JFH/J4NS5A. The lowest HCV RNA level transfected with FL-J6JFH/J4NS5A(S2197P+S2201del) at day8 was 3.3×103 GE/ug RNA, as low as 1/160 of the highest HCV RNA level. When the cells were passed, some were collected and examined for HCV proteins by immunofluorescence staining method. We found that the number of HCV-special cells in FL-J6JFH/J4NS5A (S2197P) and FL-J6JFH/J4NS5A (S2197P+2201del) was lower than FL-J6JFH/J4NS5A. Especially the FL-J6JFH/J4NS5A, positive cell was hardly visible after transfection. The consequence of FL-J6JFH/J4NS5A (S2201del) was equal with FL-J6JFH/J4NS5A.The highest number of HCV special protein expressed cell was FL-J6JFH/ J4NS5A (S2197P+S2204I) approaching 60% and the number of FL-J6JFH/ J4NS5A was 5%.These results showed that serine position 2197, 2202, 2204 in NS5A of J4 played a very important role in HCV replication and protein products generation and determined HCV replication and protein translation capacity to some extent.3.Influence of J4 core on the replication and infectivity of FL-J6JFH1Utilizing gene swapping method, we constructed a chimeric HCV replicon FL-J6JFH/J4core by replacing the core gene of wild type FL-J6JFH1 with the corresponding district of J4. FL-J6JFH/J4core, FL-J6JFH/J4NS5A and FL-J6JFH1 were linearized by enzyme Xbal and transcribed with T7 RNA polymerase in vitro. The genomic RNA transcripts were delivered to Huh-7.5.1 cells by liposome-mediated transfection. The transfected cells were cultured and passed. At the day5 postransfection, cellular total RNA was isolated from the transfected Huh-7.5.1 cells, and the levels of HCV RNA were determined by HCV-specific FQ RT-PCR. The HCV RNA level of FL-J6JFH/J4core was 5.6×105GE/ug RNA equal with FL-J6JFH1. On the contrary, the RNA level of FL-J6JFH/J4NS5A was 4.3×104GE/ugRNA. Immunofluorescence staining was used to detecte the HCV-specific proteins expressing cell at day8 posttransfection with serum from patient of hepatitis C. The results showed that ratio of positive cell in FL-J6JFH1 approached 50%, and the number of FL-J6JFH/J4core was approximately 30% and both were higher than FL-J6JFH/J4NS5A less than 5%. The possible reason maybe that the interactions between structured and non-structured proteins products affect the level of HCV replication.The naive Huh-7.5.1 cells were then inoculated with the supernatants collected at days 8 posttransfection. HCV proteins were detected by immunofluorescence day3 after inoculation. The cells infected with all three RNA transfected cell supernatants resulted in positive immunostaining. But the Fluorescence intensity of FL-J6JFH/J4core was lower than FL-J6JFH1 and the lowest was FL-J6JFH/J4NS5A.4.Influence of HCV envelop E1/E2 replacement on cell tropsim and the mechanismBHK-21 cell had conspicuously cytopathic effect after infected with attenuated JEV SA14-14-2 strain. We collected the infectious cell culture supernatant and incubated with naive BHK-21 cell, the similar CPE was observed. The naive BHK-21 cells were then inoculated with the supernatants containing attenuated JEV SA14-14-2 strain. JEV E proteins were detected by immunofluorescence after72h. The ratio of positive cell reached 70%. All of these mean BHK-21 is highly susceptible for JEV. There are special receptors proteins on the surface of BHK-21 cell and effectively mediate the viral invasion. We first collected the JEV viral particles, extracted the RNA and then amplified the prM plus E fragment by RT-PCR method. Last, we constructed a chimeric HCV/JEV replicon by replacing the E gene of HCV with the corresponding district of JEV. HCV/JEV and GND/JEV were delivered to BHK-21 cells by liposome-mediated transfection after transcription in-vitro. The RNA replication and proteins expression were confirmed by RT-PCR and immunofluorescence assay respectively.The level of chimeric virus RNA replication and protein expression was significantly lower than J6JFH1. Whether HCV / JEV can produce infectious virus in BHK-21 will require further experiments.Summary:1. FL-J6JFH/J4NS5A has a lower replication capability than wild type FL-J6JFH. It indicates that NS5A is a very important factor affecting HCV replication. FL-J6JFH/J4NS5A not having the CPE maybe closer to the natural HCV viral particles and it provides an ideal experimental platform for continuing to study the function of NS5A.2. Adopting deliberate site-directed mutagenesis method, we changed the three amino acids of NS5A in FL-J6JFH/J4NS5A, 2197-position(Ser-Pro), 2201-position serine missing deletion(del),2204-position (Ser-Ile),and then constructed the four HCV mutation replicon FL-J6JFH/J4NS5A(S2197P),FL-J6JFH/J4NS5A(S2201del),FL-J6JFH/J4NS5A(S2197P+S2201del) and FL-J6JFH/J4NS5A(S2197P+S2204I). We obtained a efficiently HCV mutation replicon (S2197P+S2204I) by comparing the replication ability. It shows that some key amino acid mutagenesis can supply a compensation for decrease of HCV replication potential.3. Utilizing gene swapping method, we constructed a chimeric HCV replicon FL-J6JFH/J4core by replacing the entire core gene of wild type FL-J6JFH1 with the corresponding district of J4. And then we discussed the level of replication, translation of chimeric HCV in Huh7.5.1 cell.4. We constructed a chimeric HCV/JEV replicon by replacing the entire E gene of HCV with the corresponding district of JEV and we will take a new attempt to establish a cross-species cell and animal work model of HCV.