| RNA-dependent RNA polymerases (RdRPs) are central components in the life cycle of RNA viruses. The replication of the plus-strand RNA viral genome consists of two steps:(1) synthesis of complementary minus-strand RNA with the plus-strand genomic RNA as a template; and (2) subsequent synthesis of the progeny RNA with the minus-strand RNA as a template. Thus, the cis-acting sequences required for the replication of the viral genome generally thought to be located at the 3'end of both plus-and minus-strand RNAs and to act as promoters for the initiation of RNA synthesis by RdRp. At present, it has been shown that positive strand RNA viruses initiate RNA synthesis by either of two major mechanisms:primer-dependent RNA synthesis and primer-independent de novo RNA synthesis.Ectropis obliqua picorna-like virus (EoPV) is an insect RNA virus causing a lethal granulose infection in larvae of the tea looper (Ectropis oblique); it is the first identified insect picorna-like virus in China. The genome of EoPV is ssRNA, polyadenylated, contains a single, large open reading frame (nt 391-9351) encoding a polyprotein of 2987 aa, structural and nonstructural proteins, located at the N-and C-terminal regions, respectively.5'UTR is 390nt in length and 3'UTR is 43nt in length. EoPV has been classified as a member of the genus Iflavirus in family Iflaviridae.Alignment of the C-terminal region of the EoPV polyprotein indicates that it contains consensus motifs that are shared by RdRps of the Picornaviridae that have been identified previously, including core motif (YGDD) for nucleotide binding and other conservative motifs. Prediction of its three dimensional structure showed that it is analogous with the RdRp of Rhinovirus, which contains three subdomain, palm subdomain, thumb subdomain and finger subdomain.To better understanding the initiation mechanism of EoPV RNA replication, we first cloned the RNA polymerase coding region by RT-PCR from the purified virus, and then expressed this protein in Escherichia coli BL21 (DE3). The protein was purified on a Ni-chelating HisTrap affinity column. Furthermore, an in vitro RdRp replicate system was built for examining the initiation of minus RNA by Northern-blot and RT-PCR. Results showed that EoPV RNA polymerase habors the activity of synthesizing minus RNA when using EoPV RNA 3'terminal region as the template and artificially synthesized oligo (U) 20 as the primer. The GDD mutant protein abrogated RdRp activity. We also comfirmed that poly (A) was a prerequisite for the RdRp activity.EoPV 3Dpol RdRp activity was found to depend on the presence of Mg2+and Mn2+. It showed a broad optimum for Mg2+concentration. The optimal Mn2+ concentration was 1.0-2.0 mM, but higher concentrations tended to be inhibitory. The optimum K+ concentration for the replicase reaction was 100-200 mM, but higher concentrations were inhibitory to RdRp activity. The optimum temperature was between 30-35℃.Our previous study have confirmed that EoPV 5'UTR possesses IRES (Internal ribosome entry site) activity in vivo and in vitro. In the present study, three different cell lines (Sf9, S2, Cos-7) were co-transfected by bicistronic construct containing EoPV 5'UTR and plasmids harboring RNA polymerase. The results showed that RNA polymerase could down-regulate IRES activity. The expression of the protein led to a decrease in IRES activity of approximately 50% in Sf9 and 20% in S2, but had slight decrease in Cos-7. Our results also demonstrated that the inhibited effect was increased as the expression of RNA polymerase accumulated in cell. Furthermore, deletions within the C terminal amino acid had a slight impact on inhibiting IRES activity; in contrary, deletion within the N terminal amino acid cause a remarkable impact on IRES activity. In conclusion, our study showed that EoPV RNA polymerase inhibited EoPV IRES-dependent translation in a dose-dependent manner. In contrast, it had no detectable effect on cap-dependent translation. All these results suggest, for the first time, that EoPV RNA polymerase negatively modulates IRES activity in a specific manner.Furthermore, we constructed EoPV full-length cDNA clone successfully. Five pairs of primers were designed according to the published sequence of EoPV to amplify 5 overlapping fragments covering EoPV genome. These fragments were subcloned to pMD18-T vector and assembled. The full-lenth cDNA clone p-EoPV was clone to pET-28a and proved by sequencing and restrictive digestion. Comparing to the published sequence, this clone possessed 10 mutations and 3 deletions in amino acid (aa). We are making efforts to seek sensitive cell line for EoPV.
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