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. 1998 Dec;72(12):9897-905.
doi: 10.1128/JVI.72.12.9897-9905.1998.

Formation and amplification of a novel tombusvirus defective RNA which lacks the 5' nontranslated region of the viral genome

B Wu  1 , K A White
Affiliations

Formation and amplification of a novel tombusvirus defective RNA which lacks the 5' nontranslated region of the viral genome

B Wu et al. J Virol. 1998 Dec.

Abstract

Defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) are small, subgenomic, helper-dependent replicons that are believed to be generated primarily by aberrant events during replication of the plus-sense RNA genome. Prototypical TBSV DI RNAs contain four noncontiguous segments (regions I through IV) derived from the 5' nontranslated region (NTR) (I), an internal section (II), and the 3'-terminal portion (III and IV) of the viral genome. We have studied the formation of these molecules by using engineered precursor DI RNA transcripts and report here the consistent accumulation of a novel defective RNA species, designated RNA B. Northern blot, primer extension, and sequence analyses indicated that, unlike prototypical DI RNAs, RNA B lacks region I. In vitro transcripts corresponding to the region II-III-IV structure of RNA B were amplified when coinoculated with helper, indicating that the 5' NTR of the genome does not harbor cis-acting replication elements essential for viral RNA replication. Region I is, however, important for DI RNA fitness, since molecules lacking it accumulated to significantly lower levels ( approximately 10-fold reduction). Analysis of the minus-strand sequence of region I led to the identification of an RNA undecamer sequence, arranged in tandem, at its very 3' terminus. Additional variants of the undecamer motif were also identified at internal positions in region I and in the negative strands of regions II, III, and IV. Features of the undecamer motif, the consensus of which is (-)3'-CCCAAAGAGAG, are consistent with a role as a cis-acting replication element. It is proposed that the ability of RNA B to be amplified is due, in part, to compensatory effects of a strategically positioned undecamer motif in region II. Possible replicase-mediated mechanisms for the generation of this novel viral RNA are also presented.

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Figures

FIG. 1
FIG. 1
(A) Schematic representation of the TBSV RNA genome and various defective viral RNAs. The wild-type TBSV genome is shown at the top as a thick horizontal line, with coding regions depicted as open boxes and the approximate molecular weights (in thousands) of the encoded proteins indicated (12). The regions corresponding to the two sg mRNAs are shown as arrows above the genome. Below, a prototypical DI RNA and various precursor DI RNAs are depicted, with shaded boxes representing regions of the genome retained in these molecules and thin lines corresponding to segments which are absent. DI-72 is composed of four noncontiguous regions (I through IV), the lengths of which are indicated (in nucleotides) (43). Various artificially constructed precursor DI RNAs are shown below DI-72, and the positions of engineered XbaI (X) and PstI (P) sites in DI-82XP are indicated. The rightward-pointing arrowheads in LA1 and its derivatives represent the inserted 191-nt segment, which is complementary to an existing upstream sequence (leftward-pointing arrowhead). (B) Proposed replicase-mediated model for generation of prototypical DI RNAs from precursor LA1 containing complementary segments (note: the stem-loop structure depicted is not to scale) (45). During minus-strand synthesis (broken arrow) the replicase is able to traverse the base of the strong secondary structure and resume copying on the other side. Synthesis of a complementary plus strand (solid arrow) generates a prototypical DI RNA.
FIG. 2
FIG. 2
Northern blot analysis of progeny viral RNAs isolated from cucumber protoplasts inoculated with various combinations of viral RNA transcripts. (A) Coinoculation of precursor DI-82XP and CNV-K2/M5 helper transcripts. (B) Coinoculations of precursor LA1 (2 μg [lane 5] or 5 μg [lane 6]), gel-purified LA1 (GP-LA1) (2 μg [lane 3] or 5 μg [lane 4]), and CNV-K2/M5 helper transcripts. The RNA transcripts used in the inoculations are indicated at the top, and the positions of the genome RNA (gRNA), sg mRNAs (sgRNA1 and sgRNA2), and defective RNAs (RNAs B and B′) are shown on the left. The predicted position for prototypical DI RNAs is indicated with an asterisk. Total nucleic acids were isolated from approximately 4 × 105 protoplasts after a 24-h incubation and were separated in a 4.5% polyacrylamide gel in the presence of 8 M urea, transferred to a nylon membrane, and hybridized with a 32P-end-labeled oligonucleotide probe (P9) complementary to the 3′-terminal 23 nt of the TBSV and CNV genomes.
FIG. 3
FIG. 3
(A) Northern blot analysis of progeny viral RNAs isolated from cucumber protoplasts inoculated with CNV-K2/M5 helper and various precursor DI RNA transcripts. The RNA transcripts used in the inoculations are shown at the top, and the positions of the genome RNA (gRNA), sg mRNAs (sgRNA1 and sgRNA2), and defective RNAs (RNAs B, B′, and BX) are indicated. Total nucleic acids were isolated and analyzed as described in the legend to Fig. 2. (B) Analysis of products generated from digestion of RNA transcripts of various precursor DI RNAs with RNase T1. The identity of the precursor analyzed is shown at the top, with the total number of units of RNase T1 present in each reaction indicated. HpaII-digested pUC19 is separated in lane M, and the sizes (in base pairs) of relevant fragments are indicated to the left. At right, an asterisk denotes the positions of full-length precursor transcripts and an arrowhead indicates products resistant to digestion. The samples were separated in a nondenaturing 2% agarose gel and then stained with ethidium bromide.
FIG. 4
FIG. 4
Northern blot analysis of progeny viral RNAs isolated from cucumber protoplasts coinoculated with helper and precursor DI RNA transcripts containing different-sized complementary segments and intervening sequences. See Table 1 for additional information on the structures of the precursors. The RNA transcripts used in the inoculations are indicated at the top, and the positions of the genome RNA (gRNA), sg mRNAs (sgRNA1 and sgRNA2), and RNA B are shown on the left. Total nucleic acids were isolated and analyzed as described in the legend to Fig. 2.
FIG. 5
FIG. 5
Analysis of the structure of RNA B. (A) Schematic representation of LA1, with the relative positions of various complementary oligodeoxyribonucleotides indicated (note: the stem-loop structure depicted is not to scale). (B) Northern blot analysis of progeny viral RNAs isolated from cucumber protoplasts coinoculated with helper and precursor LA1. The RNA transcripts used in the inoculations are indicated at the top, except for lanes labeled LA1, where ∼70 ng of the LA1 transcript was analyzed in the gels. The positions of the LA1 transcripts and RNA B are shown on the left, and the oligonucleotide probes used for detection are indicated at the bottom. Total nucleic acids were isolated and analyzed as described in the legend to Fig. 2 except that blots were hybridized with 32P-end-labeled oligonucleotide probes complementary to various segments of LA1.
FIG. 6
FIG. 6
Mapping of the 5′ termini of RNA B. (A) Primer extension analysis of RNA B. The sources of the nucleic acids which were analyzed by primer extension are identified above lanes 5 to 8, and the corresponding sequencing ladder of LA1 is identified above lanes 1 to 4. Major termination sites, along with their corresponding positions in the plus-strand sequence, are indicated on the right by arrowheads. Products were separated in an 8% polyacrylamide gel in the presence of 8 M urea. (B) Schematic representation of an internal segment of LA1 showing the relative positions of the mapped 5′ termini of RNA B. The arrows below the sequence indicate the predicted 5′ termini, whereas the arrow above the sequence defines the 5′ terminus of region II. Sequence corresponding to the 3′-terminal region of the inserted 191-nt segment is in boldface type, and the engineered XbaI site is italicized. The relative position of oligonucleotide PB22, used for primer extension analysis, is indicated.
FIG. 7
FIG. 7
Northern blot analysis of progeny viral RNAs showing the kinetics of accumulation of RNA B. The RNA transcripts used in the inoculations are indicated at the top, and the positions of the genome RNA (gRNA), sg mRNAs (sgRNA1 and sgRNA2), and RNA B are shown on the left. The times after inoculation at which the nucleic acid samples were isolated are indicated (in hours) above each lane. Total nucleic acids were isolated and analyzed as described in the legend to Fig. 2.
FIG. 8
FIG. 8
(A) Alignment of the predicted sg RNA2 promoter (Psg2) sequence (15) with the sequence in LA1 corresponding to the mapped 5′ termini of RNA B. The termini mapped for the two major RNA B species are in boldface type and underlined in the LA1 sequence, and the initiating nucleotide for sg mRNA2 synthesis (12) is in boldface type and indicated by an arrow. Identical nucleotides between Psg2 and LA1 are indicated by asterisks. The coordinates of the 3′-most residues in the sequences are shown in parentheses to the right and correspond to the numbering of the TBSV genome (12). (B) Replicase-mediated mechanism proposed to explain how secondary structure could facilitate the generation of RNA B from precursor LA1 (note: the stem-loop structure depicted is not to scale). Synthesis of a minus strand (broken arrow) complementary to LA1 is stalled at the strong secondary structure. The prematurely terminated minus strand is then copied to generate RNA B (solid arrow), which is amplified further via replication. (C) Alignment of selected conserved undecamer sequences present in the minus strand of DI-72 (Fig. 1), a prototypical TBSV DI RNA (43). The DI RNA regions from which the sequences were derived are indicated on the left, and individual sequences are numbered consecutively. The numbers in parentheses on the right indicate the coordinates of the 5′-most residues of the sequences shown and correspond to the numbering of the TBSV genome (12). Residues in sequences 1 through 16 which conform (see below) to the undecamer consensus sequence (boxed) are in boldface type and shaded. The undecamer consensus represents the most prevalent nucleotides at the respective positions. The sequences shown contain a minimum of 6 of the 11 consensus residues. For comparison, the consensus sequence of a carmovirus motif implicated in plus-strand synthesis is also shown (8). (D) 3′-terminal sequences of the minus strands of region I and RNAs B1 and B2. The terminal and adjacent undecamer motifs in the region I sequence are over- and underlined, respectively, and those in RNAs B1 and B2 are underlined. The boxes indicate nucleotides in the more 5′ undecamer in region I which are identical to those in RNAs B1 and B2. Terminal RNA B segments identical to the region I terminus are in boldface type and doubly underlined. Gaps were introduced into the RNA B sequences to maximize the alignment of 3′-terminal nucleotides with identical residues in region I.
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