Preliminary Study On The Mechanism Of Hepatitis C Virus Regulating Stress Granule Formation

HCV (hepatitis C virus, HCV) is a member of the flavivirus, with a single positive-stranded RNA virus. HCV encodes several structural proteins and non-structural proteins that form replication complex for HCV replication. HCV infection has emerged as a major causative agent of chronic liver disease, which can progress to persistent infection and hepatocellular carcinoma.HCV infection is known to elicit stress granules in cells, stress granules (SGs)are granular aggregates formed in the cytoplasm of eukaryotic cells when exposed to environment stresses such as oxidation and viral infection. SGs mainly regulate the translation of cellular protein under stress, consisting of stalled translation preinitiation complexes, mRNA and RNA binding protein. So far, the mechanism of HCV inducing SGs and SG’s effects on persistent infection are remain unclear, and there There may be a certain relationship between them.In this study,we first confirmed the induction of SGs by important constituent molecular and type of SGs under HCV infection. Immuno-fluoresence assay was perform ed to confirmed that the SGs induced by HCV contain four key molecules (G3BP1, TIA-1, eIF4G, eIF3A), which indicate that the SGs are sound. Further, the SGs induced by HCV infection could be inhibited by low concentration of Cycloheximide (CHX),which indicates that the SGs emerge as typical.Next, we detected the percentage of cells containing SGs in total cells infected by HCV during the period of infection SG’s character of dynamic instability. We observed that SGs are induced in3%of infected Huh7cell at8hr pos-infection.and the proportion of SG-positive cells shoots up to8%at12hr post-infection. Then the percentage reach the peak as13.4%at24hr followed by a decrease to10.1%at48hr,and return to almost the peak as13%. All above Indicate that the percentage of SG-positive cells oscillates at low frequency throughout the72hr observation period post-infection. The result above demonstrated that a small amount of cells(about8%) infected by HCV display SGs during period of infection establishment(within12hr),in contrast with>35%SG-positive cells of cells infected within the same period reported on other RNA virus infection like SFV and MHV,then we seted two groups of Huh7cell with one infected with HCV for8hr and the other uninfected,and treated both with sodium arsenite (NaAsO2, classic SG inducer)to induce SGs. The results show that the persentage of SG-containing cells of infected cells was significantly reduced by42%compared with the uninfected, which indicate that HCV infectoin during early peirod could inhibit the induction of SG.The viral proteins or nucleic acids of some viruses can inhibit the SG formation according to previous studies. We conjectured that HCV viral proteins which set up infection in early time may inhibit the formation of stress granules,so we detected the effects of five main viral proteins (Core, NS3/4A, NS4B, NS5A, NS5B) on SG induction by inducer and found that HCV RNA polymerase NS5B could inhibits SG formation (SG+cell rate downregulated by40%) and collocated with G3BP1which is the key component of SG in SG-negative cells. The NS5B’s inhibitory effect on SG displays in protein-dose dependent. And inhibition and co-localization could be observed in tet-on-NS5B HepaG2cell model.(SG+cell rate downregulated by42%).However, NS5B shows no effects on PKR and eIF2a phosphorylation, which confirms that the inhibition occurs at step of SG assembly. We speculated that NS5B might interact directly with G3BP1to disturb SG assembly. In conclusion, NS5B inhibits SG occurs at the time of SG assembly and co-localizats ith G3BP1,and our previous study showed that G3BP1interacts with NS5B directly with CO-IP. all these suggested that NS5B may interact with G3BP1to inhibit SG formation.In order to figure out the detail of NS5B’s inhibitory effect on SG,we first made clear the domain of NS5B bonding with G3BP1.We performed GST pull-down experiments with G3BP1-GST fusion protein and NS5B and found that the palm domain(96-454aa) of NS5B is responsible for interactions with G3BP1. Next, over-expression of the palm domain (96-454aa) of NS5B in Huh7cells showed the same inhibition of SG formation as full length of NS5B(NS5B,96-454aa, downregulated by35%; NS5B downregulated by38%). The results suggested that the palm domain(96-454aa) of NS5B inhibits SG formation by interacting with G3BP1.Constant stimulation on cells will lead to the decay of mRNA wrapped in SG and damage cells. Studies have shown that the SGs induced by HCV contain a large number of ISG (interferon-stimulated genes) gene mRNA and suppresses the translation of ISG. Based on the above facts,we speculated that NS5B interacts with G3BP1and then inhibits SG formation with two possible physiological explanations::on one hand, to avoid the lack of protein necessary for cell so that HCV could successfully establish infection machinery; On the other hand, up-regulating the expression of ISG protein to help maintained at a low level virus replication and chronic in negative feedback way. The results showed that the palm domain(96-454aa) of NS5B can reverse SG’s translational inhibition effects on interferon-stimulated genes (NS5B FL:ISG20,upregulated by141%;APOBEC3G, upregulated by172%. NS5B96-454aa ISG20,upregulated by112%;APOBEC3G, upregulated by134%.,) but not the housekeeping gene (β-actin,GAPDH)Taken together, this study reported that NS5B inhibited the formation of SGs during early period of HCV infection (within12hours) through interacting and re-localizing G3BP1to obstruct its participation in SGs aggregation. The result indicates that HCV may restore the translation of basic functional protein and ISG protein and maintain cellular homeostasis and infection levels, which remain to be made clear further, all these provides new insight into persistence,low level infection and virus-host interaction of HCV.