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. 2001 Jul;75(14):6719-28.
doi: 10.1128/JVI.75.14.6719-6728.2001.

Essential role of cyclization sequences in flavivirus RNA replication

A A Khromykh  1 , H Meka, K J Guyatt, E G Westaway
Affiliations

Essential role of cyclization sequences in flavivirus RNA replication

A A Khromykh et al. J Virol. 2001 Jul.

Abstract

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

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Figures

FIG. 1
FIG. 1
Computer-generated secondary-structure analysis of the interaction between the genomic plus-strand RNA at the 5′ and 3′ ends for four mosquito-borne flaviviruses. The predicted secondary structures of the proposed CS and some flanking sequences connected by a poly(A) insert were produced using version 3.0 of the M-fold program (31, 55). The conserved putative CS are boxed. The arrows indicate insertion points for the stuffer poly(A) sequence. The AUG initiation codon and the conserved pentanucleotide loop [5′-CACAG(A/U)-3′] in the 3′-terminal stem-loop are shown in bold. Nucleotides are numbered from the 5′ and 3′ termini. (A) KUN (12, 25); (B) Japanese encephalitis virus (45); (C) yellow fever virus vaccine strain 17D (40); (D) DEN-2 (15). The relevant GenBank accession numbers are shown in Table 1.
FIG. 2
FIG. 2
Nucleotide sequence and secondary-structure analysis of wild-type and mutant KUN replicon RNAs. (A) Interaction between 5′ and 3′ ends of the putative cyclization motif (shown in boxes). Mutated nucleotides are shown in bold. Dashes indicate deleted nucleotides. The ΔG values shown are for base-pairing of the boxed CS. (B) Computer-generated secondary-structure analysis of the interaction between the genomic plus-strand RNA at the 5′ and 3′ ends of wild-type and mutant KUN replicon RNAs. The predicted secondary structures of the proposed CS and some flanking sequences connected by a poly(A) insert were produced using the M-fold program as for Fig. 1. The boxes enclose either the conserved cyclization motifs or the relevant mutated sequences. The arrows indicate insertion points for the stuffer poly(A) sequence. The AUG initiation codon and the conserved pentanucleotide loop [5′-CACAC(A/U)-3′] in the 3′-terminal stem-loop are shown in bold. Nucleotides are numbered from the 5′ and 3′ termini.
FIG. 2
FIG. 2
Nucleotide sequence and secondary-structure analysis of wild-type and mutant KUN replicon RNAs. (A) Interaction between 5′ and 3′ ends of the putative cyclization motif (shown in boxes). Mutated nucleotides are shown in bold. Dashes indicate deleted nucleotides. The ΔG values shown are for base-pairing of the boxed CS. (B) Computer-generated secondary-structure analysis of the interaction between the genomic plus-strand RNA at the 5′ and 3′ ends of wild-type and mutant KUN replicon RNAs. The predicted secondary structures of the proposed CS and some flanking sequences connected by a poly(A) insert were produced using the M-fold program as for Fig. 1. The boxes enclose either the conserved cyclization motifs or the relevant mutated sequences. The arrows indicate insertion points for the stuffer poly(A) sequence. The AUG initiation codon and the conserved pentanucleotide loop [5′-CACAC(A/U)-3′] in the 3′-terminal stem-loop are shown in bold. Nucleotides are numbered from the 5′ and 3′ termini.
FIG. 3
FIG. 3
Detection of replication and expression of the wild-type and mutated KUN replicon RNAs by IF analysis. BHK cells were electroporated with ∼5 to 10 μg of in vitro-transcribed wild-type and mutated replicon RNAs as described previously (26) and assayed for expression of the NS3 protein by IF analysis with anti-NS3 antibodies (51) at 24, 36, and 48 h after electroporation. Panels 1 to 3 show the results of IF analysis of the wild-type (pC17) RNA, and panels 4 to 6 show the corresponding results for cells transfected with RNA containing simultaneous compensatory mutations in the 5′ and 3′ CS (pC17-5′&3′mut). Transfection with RNAs containing mutations only in the 5′- and 3′-terminal regions (pC17-5′mut and pC17-3′mut, respectively) (Fig. 2A) did not result in the detection of NS3-positive cells.
FIG. 4
FIG. 4
Northern blot showing effects of mutations in the cyclization motifs on replication of KUN replicon RNA. BHK cells were electroporated with ∼5 to 10 μg of in vitro-transcribed wild-type and mutated replicon RNAs as described previously (26), and total cellular RNA was harvested with Trizol (Gibco BRL) at 24, 36, and 48 h postelectroporation. Fifteen micrograms of total cellular RNA was separated electrophoretically in a 1% agarose gel under fully denaturing conditions and transferred to nylon (Hybond-N; Amersham), and the blot was probed simultaneously with two 32P-labeled cDNA fragments encompassing either the entire KUN 3′ UTR or 291 nucleotides of human β-actin sequence. The upper panel was exposed to X-ray film for 22 h; the arrow indicates the position of RNA of ca. 9 kb. The lower panel was exposed for 2 h, and it indicates the relative abundance of the β-actin transcript in each RNA sample.
FIG. 5
FIG. 5
Computer-generated secondary structures of the interaction between the RNA at the 5′ and 3′ ends of TBE virus (29) and of CFA (6). The predicted secondary structures of the proposed CS and some flanking sequences connected by a poly(A) insert were produced using the M-fold program as for Fig. 1. The putative CS are boxed. The arrows indicate insertion points for the stuffer poly(A) sequence. The AUG initiation codon and the conserved pentanucleotide loop [5′-CACAG(A/U)-3′] in the 3′-terminal stem-loop are shown in bold. Nucleotides are numbered from the 5′ and 3′ termini.
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