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. 2008 Sep;14(9):1791-813.
doi: 10.1261/rna.993608. Epub 2008 Jul 30.

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication

Byung-Hak Song  1 , Sang-Im Yun, Yu-Jeong Choi, Jeong-Min Kim, Chan-Hee Lee, Young-Min Lee
Affiliations

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication

Byung-Hak Song et al. RNA. 2008 Sep.

Abstract

Tertiary or higher-order RNA motifs that regulate replication of positive-strand RNA viruses are as yet poorly understood. Using Japanese encephalitis virus (JEV), we now show that a key element in JEV RNA replication is a complex RNA motif that includes a string of three discontinuous complementary sequences (TDCS). The TDCS consists of three 5-nt-long strands, the left (L) strand upstream of the translation initiator AUG adjacent to the 5'-end of the genome, and the middle (M) and right (R) strands corresponding to the base of the Flavivirus-conserved 3' stem-loop structure near the 3'-end of the RNA. The three strands are arranged in an antiparallel configuration, with two sets of base-pairing interactions creating L-M and M-R duplexes. Disrupting either or both of these duplex regions of TDCS completely abolished RNA replication, whereas reconstructing both duplex regions, albeit with mutated sequences, fully restored RNA replication. Modeling of replication-competent genomes recovered from a large pool of pseudorevertants originating from six replication-incompetent TDCS mutants suggests that both duplex base-pairing potentials of TDCS are required for RNA replication. In all cases, acquisition of novel sequences within the 3'M-R duplex facilitated a long-range RNA-RNA interaction of its 3'M strand with either the authentic 5'L strand or its alternative (invariably located upstream of the 5' initiator), thereby restoring replicability. We also found that a TDCS homolog is conserved in other flaviviruses. These data suggest that two duplex base-pairings defined by the TDCS play an essential regulatory role in a key step(s) of Flavivirus RNA replication.

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Figures

FIGURE 1.
FIGURE 1.
Schematic diagram outlining the identification and characterization of a complex RNA motif, the TDCS, required for the replication of JEV genomic RNA.
FIGURE 2.
FIGURE 2.
A TDCS is required for the replication of JEV genomic RNA. (A) Schematic presentation of the putative JEV TDCS (shaded box). The 5′-end nucleotide of the genome is numbered +1, and bases extending downstream are assigned (blue) positive numbers; the 3′-end nucleotide is numbered −1, and bases extending upstream are assigned (red) negative numbers. The translation initiation codon (AUG) and stop codon (UAG) are shown in boldface type. The three RNA elements of the flavivirus-conserved primary sequence and the secondary structure required for RNA replication are indicated (Markoff 2003): (I) 5′-AG and CU-3′; (II) the 3′ stem–loop (3′SL), subdivided into four stem regions (Stem1 to Stem4); (III) a pair of cyclization sequences (5′CS, 5′-UCAAUAUG; 3′CS, 5′-CAUAUUGA). (Inset) Relative locations of the three strands of the TDCS, left (L), middle (M), and right (R), and their potential base-pairing patterns are highlighted. (B) Eight mutations introduced into the TDCS of JEV, and the base-pairing potentials disrupted by each mutation. (Shaded boxes) The altered nucleotides are indicated in boldface type. (C,D) (C) Specific infectivities of the synthetic RNA transcripts derived from JEV wild-type (WT) and eight TDCS-mutant cDNAs and (D) representative focus morphologies. The WT and eight TDCS-mutant cDNA templates, as indicated, were used for SP6 polymerase runoff transcription. (C) The specific infectivities of the synthetic RNA transcripts were estimated by infectious center assays, in which naive BHK-21 cells were electroporated with 2 μg of the synthetic RNA transcripts, as described in Materials and Methods. (ND) Not detected. (D) For focus morphology, the cells were immunostained with a mouse JEV-specific hyperimmune antiserum and a peroxidase-conjugated goat anti-mouse IgG.
FIGURE 3.
FIGURE 3.
Functional significance of 17 novel sequences discovered in LMM-derived pseudorevertants for M-R duplex base-pairing and genome RNA replication. (A) Novel sequences (LMM/Rev1 to 17) acquired in LMM-derived pseudorevertants, and (shaded boxes) predicted complex motifs of the TDCS formed by WT, LMM, and LMM derivatives containing one of the 17 novel sequences. Nucleotides that were acquired in LMM-derived pseudorevertants are shown in black boldface type. Fractions in parentheses are the number of clones discovered that contain the particular sequence/the total number of independently picked clones that were sequenced. The 17 LMM derivatives were classified into five groups, I to V, as described in Results. (B,C) Specific infectivities and representative focus morphologies obtained 4 d after transfection of BHK-21 cells with the synthetic RNAs transcribed from the reconstructed JEV cDNAs, each containing (B) one of the 17 novel sequences, and (C) their levels of JEV RNA production and JEV NS1 protein accumulation, estimated at 20 and 24 h post-transfection (hpt), respectively. Naive BHK-21 cells were mock-transfected (Mock) or transfected with 2 μg of WT, LMM, or one of 17 LMM-derivative RNAs transcribed from each cDNA, as indicated. (B) At 4 d after transfection, the specific infectivities of the synthetic RNA transcripts were estimated by infectious center assays (Infectivity); the infectious centers of foci were visualized by immunostaining of RNA-transfected cells with anti-JEV antiserum (Representative foci). (C) Changes in JEV RNA levels at 20 h post-transfection, relative to those at 6 h post-transfection, were quantitated by real-time quantitative RT-PCR, using a JEV-specific probe complementary to nucleotides 5837-5856 in NS3 (JEV RNA). JEV NS1 protein accumulation at 24 h post-transfection was analyzed by immunoblotting with anti-JEV NS1 antiserum (Anti-NS1). (NT) Not tested. (D) One-step growth kinetics. BHK-21 cells were infected at a multiplicity of infection of 8 FFU/cell with WT or one of 17 LMM-derivative viruses obtained from the corresponding RNA-transfected cells. Culture supernatants were harvested at the indicated hour post-infection (hpi), and virus titers were determined on naive BHK-21 cells. The curve shows cumulative virus titers.
FIGURE 4.
FIGURE 4.
Functional significance of six novel sequences discovered in MLR-derived pseudorevertants for M-R duplex base-pairing and genome RNA replication. (A) Novel sequences (MLR/Rev1 to 6) acquired in MLR-derived pseudorevertants, and predicted complex motifs of the TDCS formed by WT, MLR, and MLR derivatives containing one of the six novel sequences. The nucleotides acquired in MLR-derived pseudorevertants are shown in black boldface type. Fractions in parentheses are the number of clones discovered that contain the particular sequence/the total number of independently picked clones that were sequenced. (B) Specific infectivities and representative focus morphologies obtained 4 d after transfection of BHK-21 cells with the synthetic RNAs transcribed from the reconstructed JEV cDNAs, each containing one of the six novel sequences; and (C) their levels of JEV RNA production and JEV NS1 protein accumulation at 20 and 24 h post-transfection, respectively. (D) One-step growth kinetics of the recombinant viruses originating from the corresponding synthetic RNAs, as indicated. Experimental procedures were performed as described for Figure 3. (NT) Not tested.
FIGURE 5.
FIGURE 5.
Functional significance of three novel sequences discovered in LRM-derived pseudorevertants for base-pairing between the M strand of 3′M-R duplex and an alternative 5′L (5′LAlt) strand and for genome RNA replication. (A) Novel sequences (black boldface type) acquired in LRM-derived pseudorevertants. Also illustrated schematically are predicted complex motifs of the TDCS formed by each RNA in the presence (WT, LRM, LRM/Rev1, LRM/Rev2, and LRM/Rev3) or absence (WT/ΔLAlt, LRM/ΔLAlt, LRM/Rev1/ΔLAlt, LRM/Rev2/ΔLAlt, and LRM/Rev3/ΔLAlt) of the 5′LAlt strand (shown as a box outlined by a solid line). Fractions in parentheses are the number of clones discovered that contain the particular sequence/the total number of independently picked clones that were sequenced. (B) Specific infectivities and representative focus morphologies obtained 4 d after transfection of BHK-21 cells with the synthetic RNAs transcribed from each cDNA, as indicated, and (C) their levels of JEV RNA production and JEV NS1 protein accumulation at 20 and 24 h post-transfection, respectively. (D) One-step growth kinetics of the recombinant viruses derived from the corresponding synthetic RNAs, as indicated. Experimental procedures were performed as described for Figure 3. (ND) Not detected; (NT) not tested.
FIGURE 6.
FIGURE 6.
Functional significance of two point mutations discovered in MMR-derived pseudorevertants for base-pairing between the M strand of 3′M-R duplex and an alternative 5′L (5′L1Alt or 5′L2Alt) strand and for genome RNA replication. (A) Point mutations (black boldface type) acquired in MMR-derived pseudorevertants. Also illustrated schematically are predicted complex motifs of the TDCS formed by each RNA in the presence (WT, MMR, MMR/Rev1, and MMR/Rev2) or absence (two sets of three: WT/ΔL1Alt, MMR/ΔL1Alt, and MMR/Rev1/ΔL1Alt, and WT/ΔL2Alt, MMR/ΔL2Alt, and MMR/Rev2/ΔL2Alt) of either the (box outlined with a solid line) 5′L1Alt or (box outlined with a dotted line) 5′L2Alt strand. Fractions in parentheses are the number of clones discovered that contain the particular sequence/the total number of independently picked clones that were sequenced. (B) Specific infectivities and representative focus morphologies obtained 4 d after transfection of BHK-21 cells with the synthetic RNAs transcribed from each cDNA, as indicated, and (C) their levels of JEV RNA production and JEV NS1 protein accumulation at 20 and 24 h post-transfection, respectively. (D) One-step growth kinetics of the recombinant viruses derived from the corresponding synthetic RNAs, as indicated. Experimental procedures were performed as described for Figure 3. (ND) Not detected; (NT) not tested.
FIGURE 7.
FIGURE 7.
Functional significance of an identical novel 5-nt sequence in the replication of both LLR and LRR genomic RNAs. (A, black boldface type) An identical novel sequence of five nucleotides (U−7U−6U−5U−4A−3) acquired in the R strand of the TDCS in two pseudorevertants originating from LLR (LLR/Rev) and LRR (LRR/Rev). Also presented are predicted complex motifs of the TDCS formed by each RNA in the presence or absence of the 5′LAlt strand (shown as a box outlined with a solid line). (B) Specific infectivities and representative focus morphologies obtained 4 d after transfection of BHK-21 cells with the synthetic RNAs transcribed from each cDNA, as indicated, and (C) their levels of JEV RNA production and JEV NS1 protein accumulation at 20 and 24 h post-transfection, respectively. (D) One-step growth kinetics of the recombinant viruses derived from the corresponding synthetic RNAs, as indicated. Experiments were performed as described for Figure 3. (ND) Not detected; (NT) not tested.
FIGURE 8.
FIGURE 8.
Phylogenetic conservation in the TDCS of JEV. (A) GenBank accession numbers of 34 full-length JEV genomes used in the sequence alignments. (B) Schematic presentation of the JEV TDCS. The nucleotide sequences and relative locations of three (L, M, and R) strands of the TDCS in the genome are shown. Also presented are potential base-pairings between the L and M strands and between the M and R strands that can occur in other JEV strains. Sequence alignments of the 34 full-length JEV genomes were produced using ClustalX (Thompson et al. 1997), and the resulting output file was analyzed to predict the effect of each variant on TDCS formation. Sequences that differ from the consensus are circled.
FIGURE 9.
FIGURE 9.
JEV TDCS is highly conserved among other flaviviruses, including West Nile virus (WNV), yellow fever virus (YFV), and dengue virus serotype 4 (DV-4). The predicted nucleotide sequences and relative locations of the three (L, M, and R) strands of the TDCS in the various viral genomes are shown, as described in Figure 2. (Shaded boxes) Also highlighted are the potential base-pairing patterns of the TDCSs. The translation initiation codon (AUG) and stop codon (UAG for JEV, UAA for WNV and DV-4, and UGA for YFV) are shown in boldface type. The yellow fever virus genome is predicted to form two alternative forms (YFVL1 and YFVL2) of a TDCS homolog, analogous to that of JEV, depending on the choice of L strand: either L1 (G+110A+111C+112C+113A+114) or L2 (A+106A+107C+108U+109G+110). The GenBank accession numbers of the four flaviviruses analyzed are AY585243 (JEV CNU/LP2), M12294 (WNV), X03700 (YFV 17D), and M14931 (DV-4).
FIGURE 10.
FIGURE 10.
Two working hypotheses (A,B) for the TDCS-mediated cyclization and replication of JEV genomic RNA. The predicted stem–loop structures for the (blue) 5′-terminal (5′SL) and (red) 3′-terminal (3′SL) regions of the genome. Also indicated are the relative locations of the L (5′-A+87A+88G+89A+90U+91), M (5′-A−82U−81C−80U−79U−78), and R (5′-A−7G−6G−5A−4U−3) strands of the TDCS and the potential base-pairing patterns. (Boldface type) The translation initiation codon (AUG) and stop codon (UAG). The TDCS brings the flavivirus-conserved 3′SL upstream of the translation initiator AUG adjacent to the 5′-end and renders the 3′SL capable of communicating with the flavivirus-conserved 5′SL. See the text for a detailed explanation.
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