Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication - PubMed (original) (raw)
Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication
Mark Tilgner et al. J Virol. 2004 Aug.
Abstract
Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3'-terminal 6 nucleotides (nt) (5'-GGAUCU(OH)-3') of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3'-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i). The flavivirus-conserved terminal 3' U is optimal for WN virus replication. Replacement of the wild-type 3' U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3' A or G mutants, with only partial reversion. (ii). The flavivirus-conserved 3' penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3'-terminal CU(OH), are absolutely essential for viral replication. (iii). The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv). The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v). Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3' flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.
Figures
FIG. 1.
Enhancement of replication by the engineering of an HDVr at the 3′ end of WN virus replicons. (A) A stem-loop structure formed by the 3′-terminal sequence of the WN virus genome. The base pairs involved in a putative pseudoknot interaction (58) are indicated by dotted lines. The 3′-terminal 6 nt and their potential base pairs are shaded. An HDVr is engineered immediately at the 3′ end of the WN virus replicon RNA to yield a precise 3′ end through the HDVr-mediated cleavage. The RNA sequence is numbered from the 3′ end. (B, top) WN replicons with (Rep-HDVr) and without (Rep) the 3′ HDVr. All replicons contained an in-frame deletion from nt 190 to 2379 (GenBank accession no. AF404756) spanning three structural genes, C-prM-E (dotted open box). (B, bottom) IFA of BHK cells transfected with Rep or Rep-HDVr at 72 h posttransfection. WN virus immune mouse ascites fluid and Texas red-conjugated goat anti-mouse immunoglobulin G antibody were used as the primary and secondary antibodies for IFA, respectively.
FIG. 2.
The Rluc-reporting replicon containing an HDVr at its 3′ end can be used to monitor viral translation and RNA replication. (A) Rluc-reporting replicon containing a 3′ HDVr (RlucRep-HDVr). Rluc reporter is fused in-frame with the open reading frame of the replicon in the position where the structural region was deleted. RlucRep-NS5mt-HDVr contains a single-nucleotide frameshift upstream of the active site of the RdRp domain of NS5. (B) Time course of the Rluc activity in BHK cells transfected with RlucRep-HDVr or RlucRep-NS5mt-HDVr. (C) IFA of BHK cells transfected with RlucRep-HDVr or RlucRep-NS5mt-HDVr at 72 h posttransfection.
FIG. 3.
Mutagenesis of the 3′-terminal nucleotide (A) and the penultimate nucleotide (B) of the WN virus genome in the RlucRep-HDVr system. Depicted are replicons containing single or double mutations within the 3′-terminal 6 nt (numbered 1 to 6 from the 3′ end of the genome) and their putative base-pairing nucleotides (nt 74 to 79). Specific base pairs analyzed in panels A and B are shaded in the wild-type (WT) structure. For mutant replicons, the mutated nucleotides are indicated as solid ovals. The mutant replicons are named after their mutated position, mutated nucleotides (underlined), blockage of base pair (indicated by an “x”), and potential base pair (indicated by a —). BHK cells transfected with wild-type or mutant replicons were monitored for Rluc activity at 2.5 and 72 h posttransfection. The Rluc activities of mutant replicons are presented as percentages of the corresponding wild-type replicon signal. Representative results of IFAs, performed at 72 h posttransfection, are also presented. Results of one of three representative experiments are shown.
FIG. 4.
Sequencing of viable replicons by 3′ RACE. Replicon RNA recovered at 72 h posttransfection was polyadenylated and amplified through RT-PCR. A 3′ adapter primer containing a 12-T tract was used for RT, and virus-specific primer F-WN10501 and adapter-specific primer 1 were used for PCR. The resulting RT-PCR products were directly subjected to DNA sequencing by a virus-specific primer, F-WN10729. ORF, open reading frame.
FIG. 5.
Mutagenesis of the 3′-terminal 3rd (A) and 4th (B) nucleotides of the WN virus genome in viral translation and replication. See the legend to Fig. 3 for details.
FIG. 6.
Mutagenesis of the 3′-terminal 5th (A) and 6th (B) nucleotides of the WN virus genome in viral translation and replication. See the legend to Fig. 3 for details.
FIG. 7.
In vitro comparison of the HDVr-mediated cleavage efficiencies among wild-type and mutant replicons. (A) Diagram of the HDVr cleavage assay. RNA representing the 3′-terminal 908 nt of the RlucRep-HDVr is in vitro transcribed. The HDVr-mediated cleavage processes the 908-nt RNA precursor into the 712- and 196-nt RNA products. (B) Sequencing gel analysis of the HDVr cleavage assay. 32P-labeled RNAs containing the specified mutations were compared in terms of their cleavage efficiencies by HDVr. The expected sizes of the RNA precursor and products were observed as depicted in panel A. An 890-nt RNA derived from the WN virus sequence (75) was used as an RNA marker. The cleavage efficiency was quantified through a PhosphorImager and is presented as the percentage of the sum of the intensities of the cleaved RNA bands divided by the intensities of the sum of the cleaved and uncleaved RNA bands.
FIG. 8.
Comparison of the 3′ sequences and helix structures formed by various flavivirus genomic RNAs. (A) Summary of nucleotide sequence and base-pairing structure essential for WN virus RNA replication (shaded). (B) 3′-terminal helix structures formed by genomic RNAs of KUN virus (29), MVE virus (GenBank accession no. NC_000943), JE virus (GenBank accession no. AF486638), four serotypes of DEN virus (63), YF virus (54), and TBE virus (43, 44). Essential elements identified in WN virus are shaded in equivalent locations within each helix structure.
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