Poliovirus requires a precise 5' end for efficient positive-strand RNA synthesis

Abstract

Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5' end, which are deleted during replication to generate authentic viral genomes. We removed those 5'-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5' ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5' end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.

FIG. 1

A poliovirus-specific cis-acting hammerhead ribozyme. (A) Predicted secondary structure of the cis-active hammerhead ribozyme attached to the 5′ end of the poliovirus genome. Core residues of the ribozyme are shown in bold; poliovirus sequences are in italic. Numbering refers to the first poliovirus nucleotide U as 1. The active ribozyme contains a cytosine at position −1; the inactive form has a guanosine. (B) Schematic presentation of the in vitro transcripts used throughout this study. Either poliovirus type 1 (Mahoney) transcripts (XpA) or luciferase-expressing replicon RNA (RLuc) were used. Constructs containing an active ribozyme at the 5′ end are referred to as rib(+); those containing the inactive form are indicated as rib(−). The parental constructs contain two extra guanosine residues at the 5′ end upon transcription. (C) 5′-end analysis of in vitro transcripts by primer extension. Radiolabeled oligonucleotide IX was hybridized to virion RNA or in vitro-transcribed RNA and extended with reverse transcriptase. The reaction products were analyzed on a denaturing 6% polyacrylamide gel. Lanes 1 to 4, sequencing reaction using the plasmid prib(+)XpA as a template and oligonucleotide IX as sequencing primer; lanes 5 to 8, primer extension using virion RNA (lane 5), rib(+)XpA-RNA (lane 6), rib(−)XpA-RNA (lane 7), or XpA-RNA (lane 8) as the template.

FIG. 2

Replication of in vitro transcripts in tissue culture cells. (A) Luciferase expression in replicon RNA-transfected 293 cells. rib(+)RLuc-RNA, rib(−)RLuc-RNA, or RLuc31-RNA was transfected into 293 cells, and the luciferase activity (relative light units [RLU]) corresponding to 2.5 × 10⁴ cells was measured every hour for 6 h. The cells were kept either in the presence (open symbols) or absence (closed symbols) of 2 mM guanidinium hydrochloride (GuHCl). RNA transfections were repeated at least twice. Error bars have been omitted since the range of variation was smaller than the symbol size. (B) Accumulation of positive-strand RNA in transfected 293 cells. rib(+)XpA-RNA, rib(−)XpA-RNA, or XpA-RNA was transfected into 293 cells, and poly(A)⁺ RNA was isolated from 10⁵ cells every hour for 6 h. The RNA was separated by electrophoresis in denaturing agarose gels. The gels were dried and hybridized with oligonucleotide IX. (C) Negative-strand RNA synthesis in radiolabeled RNA-transfected 293 cells. ³³P-labeled (10⁷ cpm/μg) rib(+)XpA-RNA (lanes 2 and 4) or rib(−)XpA-RNA (lanes 3 and 5) was transfected into 293 cells, and poly(A)⁺ RNA was isolated after 1 (lanes 2 and 3) and 2 (lanes 4 and 5) h. The RNA was analyzed by electrophoresis in native agarose gels and detected by autoradiography after exposure for 2 days (lanes 1 to 5) or 7 days (lanes 6 to 9). Lane 1, radiolabeled rib(+)XpA-RNA (10⁴ cpm).

FIG. 3

RNA replication of in vitro transcripts in alternative replication systems. (A) RNA replication in X. laevis oocytes. Virion RNA (lanes 1 to 4), rib(+)XpA-RNA (lanes 5 to 8), rib(−)XpA-RNA (lanes 9 to 12), or XpA-RNA (lanes 13 to 16) was injected into X. laevis oocytes together with [α-³³P]UTP, and total RNA was isolated at 3, 6, 9, and 12 h postinjection. After poly(A) selection, the RNA was analyzed on native agarose gels and detected by autoradiography. (B) RNA replication in translation-replication extracts. rib(+)XpA-RNA (lanes 1 to 4) and rib(−) XpA-RNA (lanes 5 to 8) were used to program a cell extract. After 4 h of incubation at 30°C in the presence of 2 mM guanidinium hydrochloride, preinitiation complexes were isolated by centrifugation at 15,000 × g. Preinitiation complexes were resuspended in labeling mix containing [α-³³P]UTP, and total RNA was prepared at 15, 30, 45, and 60 min. The RNAs were separated on native agarose gels and detected by autoradiography.