Functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses

Abstract

The linear, positive-stranded RNA genome of flaviviruses is thought to adopt a circularized conformation via interactions of short complementary sequence elements located within its terminal regions. This process of RNA cyclization is a crucial precondition for RNA replication. In the case of mosquito-borne flaviviruses, highly conserved cyclization sequences (CS) have been identified, and their functionality has been experimentally confirmed. Here, we provide an experimental identification of CS elements of tick-borne encephalitis virus (TBEV). These elements, termed 5'-CS-A and 3'-CS-A, are conserved among various tick-borne flaviviruses, but they are unrelated to the mosquito-borne CS elements and are located at different genomic positions. The 5'-CS-A element is situated upstream rather than downstream of the AUG start codon and, in contrast to mosquito-borne flaviviruses, it was found that the entire protein C coding region is not essential for TBEV replication. The complementary 3'-CS-A element is located within the bottom stem rather than upstream of the characteristic 3'-terminal stem-loop structure, implying that this part of the proposed structure cannot be formed when the genome is in its circularized conformation. Finally, we demonstrate that the CS-A elements can also mediate their function when the 5'-CS-A element is moved from its natural position to one corresponding to the mosquito-borne CS. The recognition of essential RNA elements and their differences between mosquito-borne and tick-borne flaviviruses has practical implications for the design of replicons in vaccine and vector development.

FIG. 1.

Secondary structures in the 5′- and 3′-terminal regions of TBEV and putative long-range interactions between the two regions of the strain Neudoerfl sequence (numbering corresponding to the wild-type sequence, GenBank accession no. U27495). The stem-loop structures (5′-SL1, -2, -3, and -4 and 3′-SL1, -2, -3, -4, and -5) are shown as predicted for the linear genome, i.e., without consideration of potential long-range interactions between 5′- and 3′-proximal sequence motifs. Predicted ΔG values of individual SL structures are given for the tick-borne consensus sequence (no ΔG value is shown for 5′-SL1 due to considerable variation among different tick-borne flavivirus sequences). Base-pair assignment corroborated by compensatory mutations in other tick-borne flavivirus genomes are indicated by circles. Base pairs that are absent in one or two of the other tick-borne sequences are marked by asterisks. The AUG start codon in 5′-SL3 is marked with “Start.” Potential long-range interactions of putative cyclization elements (CS) are highlighted in color and are indicated by connecting lines. Solid lines depict long-range interactions predicted by the algorithm used in the present study (see Materials and Methods), whereas dashed lines refer to previously predicted interactions not confirmed in the present analysis.

FIG. 2.

Organization and characterization of replicon C17. (A) Schematic drawing of C17 (not to scale). The nucleotide sequence between positions 183 and 2386 (corresponding to amino acid residue 18 of protein C and 471 of protein E) of the wild-type TBEV genome was replaced by an artificial sequence that includes an MCS with recognition sequences for the restriction enzymes PacI, SnaBI, and NotI and a sequence coding for a potential cleavage site of the viral protease NS2B/3. (B) Immunofluorescence analysis of cells transfected with RNA of replicon C17, the prM-cleavage-deficient TBEV mutant prM(ΔR88) (positive control), and the replication-deficient mutant ΔNS5 (negative control). Immunofluorescence staining was performed 3 days posttransfection with a monoclonal antibody recognizing protein NS1. (C) Replication kinetics of C17 and control RNAs. RNA isolated from 2,000 cells was quantified at various time points after transfection by real-time PCR. Logarithmic means from two experiments are shown (the error bars indicate the maximum and minimum values). ΔNS5 (no ep.) refers to control experiments in which cells were incubated with RNA but not electroporated to quantify the number of RNA molecules that remain attached to the cell surface. This value dropped below the cutoff limit of 10¹ (indicated by a dashed line) at 24 h posttransfection. (D) Average increase or decrease in RNA copies in percent per hour between the individual measurements of the experiments.

FIG. 3.

Analysis of protein expression from deletion mutants derived from replicon C17. (A) Schematic drawing of secondary structure and potential CS elements. For details, refer to the legend to Fig. 1. (B to D) Schematic representation of the 5′- and 3′-terminal regions of various deletion mutants, indicating potential CS elements in color (yellow, CS-a, CS-b1 and CS-b2; red, CS-A; blue, CS-B) and the AUG-containing 5′-SL3 in gray. Deletions are represented by dashed lines. Mutant designations are given on the left, together with the exact boundaries of the deletions, as listed in parentheses (numbers correspond to nucleotide positions on the wild-type genome). On the right, protein expression is shown as determined by anti-NS1 immunofluorescence staining at 3 days posttransfection. (B) Positive and negative controls. The position of the AUG start codon is depicted. (C) Mutants with sequential truncations of the protein C coding region and 5′-SL3. (D) Mutants with deletions of 5′ or 3′ copies of putative CS elements.

FIG. 4.

Synopsis of nucleotide changes introduced to analyze the functionality of CS-A. (A) Schematic drawing of the 5′- and 3′-terminal regions. In a cyclic conformation of the genome, the 5′- and 3′-elements of CS-A are predicted to form 15 bp (as indicated by dotted lines in the central part of the panel). In a noncircularized conformation, 3′-CS-A is assumed to form base-pairs as part of the 3′-SL1 stem structure (indicated by a dotted line). A stretch of four predicted base pairs with the sequence motif 5′-AACA-3′ is indicated by solid lines. (B) Four (red) or seven (blue) nucleotide changes were engineered into the 5′-element and/or the 3′-element of CS-A. In addition, mutants with an altered sequence of the AACA-box (green) were engineered together with a wild-type 3′-CS-A or a mutated 3′-CS-A.

FIG. 5.

Protein expression analysis of various CS-A mutants by immunofluorescence staining 3 days posttransfection. The CS-A sequences, the AACA-box and various mutations therein are depicted as in Fig. 4A. Potential base pairings between 5′-CS-A and 3′-CS-A (dotted lines) and/or between 3′-CS-A and the AACA box (solid lines) are indicated. (A) Positive (a) and negative controls (b); four or seven nucleotide changes were introduced either into 5′-CS-A only (c and f), 3′-CS-A only (d and g), or both CS-A elements to restore complementarity (e and h). (B) The AACA-box was mutated in the context of wild-type CS-A sequences (i), a mutated 5′-CS-A (j and m), a mutated 3′-CS-A (k and n), or both elements of CS-A with compensatory mutations (l and o).

FIG. 6.

Kinetics of RNA replication of various CS-A mutants. C17 and ΔNS5 were used as positive and negative controls, respectively. (A and C) RNA copies isolated from 2,000 cells were quantified at various time points posttransfection by real-time PCR. Logarithmic means from two experiments are shown (the error bars indicate maximum and minimum values). (B and D) Average increase or decrease of RNA copies in percent per hour between the individual measurement points of the experiment. Panels A and B show results obtained with mutants having four nucleotide changes in the 5′-CS-A (5′-4mut), the 3-CS-A (3′-4mut), or both elements of CS-A (5′+3′-4mut) to restore complementarity. Panels C and D show results from mutants with a mutated AACA-box in the context of wild-type CS-A (AACAmut), mutated 5′-CS-A (5′-4mut+AACAmut), mutated 3′-CS-A (3′-4mut+AACAmut), or both elements of CS-A (5′+3′-4mut+AACAmut).

FIG. 7.

Construction and characterization of replicon 5′-CS-A/ORF. (A) Schematic drawing of the construction of 5′-CS-A/ORF from replicon C17 (not to scale). 5′-CS-A was deleted from its original position and used to replace the 5′-CS-B element within the ORF, which was not frame shifted by this manipulation. (B) Immunofluorescence analysis of cells transfected with RNA of replicon C17 (positive control), 5′-CS-A/ORF, or ΔNS5 (negative control); Immunofluorescence staining was performed 3 days posttransfection with a monoclonal antibody recognizing protein NS1. (C) Replication kinetics of 5′-CS-A/ORF and control RNAs. RNA isolated from 2,000 cells was quantified at various time points posttransfection by real-time PCR. The logarithmic means from two experiments are shown (error bars indicate maximum-minimum values). (D) Average increase or decrease of RNA copies in percent per hour between the individual measurement points of the experiment.