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[摘要]：Polioviruses (PVs) carrying a reporter gene are useful tools for studies of virus replication, particularly if the viral chimeras contain the polyprotein that provides all of the proteins necessary for a complete replication cycle. Replication in HeLa cells of a previously constructed poliovirus expressing the gene for Renilla luciferase (RLuc) fused to the N terminus of the polyprotein H2N-RLuc-P1-P2-P3-COOH (P1, structural domain; P2 and P3, nonstructural domains) led to the deletion of RLuc after only one passage. Here we describe a novel poliovirus chimera that expresses Gaussia luciferase (GLuc) inserted into the polyprotein between PI and P2 (N2H-P1-GLuc-P2-P3-COOH). This chimera, termed PV-GLuc, replicated to 10% of wild-type yield. The reporter signal was fully retained for three passages and then gradually lost. After six passages the signal was barely detectable. On further passages, however, the GLuc signal reappeared, and after eight passages it had reached the same levels observed with the original PV-GLuc at the first passage. We demonstrated that this surprising observation was due to coevolution of defective interfering (D1) particles that had lost part or all of the capsid coding sequence (Delta P1-GLuc-P2-P3) and wild-type-like viruses that had lost the GLuc sequence (P1-P2-P3). When used at low passage, PV-GLuc is an excellent tool for studying aspects of genome replication and morphogenesis. The GLuc protein was secreted from mammalian cells but, in agreement with published data, was not secreted from PV-GLuc-infected cells due to poliovirus-induced inhibition of cellular protein secretion. Published evidence indicates that individual expression of enterovirus polypeptide 3A, 2B, or 2BC in COS-1 cells strongly inhibits host protein secretion. In HeLa cells, however, expression of none of the poliovirus polypeptides, either singly or in pairs, inhibited GLuc secretion. Thus, inhibition of GLuc secretion in PV-infected HeLa cells is likely a result of the interaction between several viral and cellular proteins that are different from those in COS-1 cells. |
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