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. 1999 Aug;73(8):6415-23.
doi: 10.1128/JVI.73.8.6415-6423.1999.

Initiation of genomic plus-strand RNA synthesis from DNA and RNA templates by a viral RNA-dependent RNA polymerase

K Sivakumaran  1 , C C Kao
Affiliations

Initiation of genomic plus-strand RNA synthesis from DNA and RNA templates by a viral RNA-dependent RNA polymerase

K Sivakumaran et al. J Virol. 1999 Aug.

Abstract

In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.

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Figures

FIG. 1
FIG. 1
Synthesis of plus-strand RNA by using DNA templates. (A) Template d(−1/13) containing the sequence complementary to nt 1241 to 1252 of BMV RNA3 are shown with the initiation cytidylate indicated by an arrow. Changes of the 3′ ends of d(−1/13), the +2 adenylate, the +3 uridylate, and the +4 adenylate were used for RNA synthesis by BMV RdRp. The changes are indicated above the autoradiogram of the RdRp products. The positions of the 13- and 14-nt products are shown on the left. The reaction products were separated by denaturing PAGE (12% polyacrylamide) and visualized by autoradiography. (B) Summary of the effect of nucleotide changes focusing on the 3′ end of the initiation site. All results presented were from at least three independent trials. (C) Summary of the effect of nucleotide changes at positions +2, +3, and +4 in d(−1/13). (D) Effect of changes to a guanylate in the first six positions in d(−1/13) on plus-strand RNA synthesis.
FIG. 2
FIG. 2
Effect of nucleotide changes in d(−1/13) on the ability of the resultant DNA template to compete for RdRp. An RNA template, r(−20/15), directing the synthesis of a 15-nt product, was used as a reference. The amounts of RNA synthesis generated from r(−20/15) in the presence of the different competitors are listed as percentages relative to synthesis in the absence of competitor. DNA competitors were used at 5- and 10-fold molar excess with respect to r(−20/15). All results were from at least three independent trials. ND, not determined.
FIG. 3
FIG. 3
Concentration of DNA templates needed to reduce RNA synthesis from r(−20/15) by 50%. The percentage of synthesis from r(−20/15) directing the synthesis of a 15-nt product was measured in the presence of increasing amounts of competitor DNA. The IC50s are given within the boxed region. The results for d(−1/13)dT were not plotted, to reduce the complexity of the figure.
FIG. 4
FIG. 4
Initiation of genomic plus-strand RNAs directed by minus-strand endscripts. (A) Comparison of the 3′ sequences of BMV and CMV minus-strand RNAs. The nontemplated guanylate added to each template is shown in bold type. The initiation cytidylate is denoted by an arrow. (B) Predicted secondary structures of the 3′ ends of BMV RNA1, RNA2, and RNA3, i.e., B1(−)58G, B2(−)46G, and B3(−)51G, respectively. The structure predictions were generated by the MFOLD program (10). (C) Initiation of genomic plus-strand RNAs from minus-strand endscripts. RdRp reaction products were separated by denaturing PAGE (12% polyacrylamide) and visualized by autoradiography. The amounts of RNA synthesized from various templates relative to B2(−)46G (% Syn) are shown at the bottom of the autoradiogram. The results presented are an average from three independent trials. The sizes of the RNA products are indicated on the side of the autoradiogram. The symbol φ represents the products of a control reaction with no added template. Endscripts that are initiation competent are indicated by +, while initiation-incompetent endscripts are indicated by −. C2(−)G and C3(−)G are endscripts of CMV RNA2 and CMV RNA3, respectively. (D) Synthesis of a 200-nt genomic plus-strand RNA. The endscript with a guanylate replacing the +1 cytidylate is indicated by +1c/g. −G denotes a 200-nt endscript without the designed nontemplated guanylate; +G denotes a 200-nt endscript with a guanylate at the 3′ end of the RNA. RNA synthesis from B2(−)200+G, B2(−)200Δ-1, and B2(−)200+1C/G was 100, 27, and 0% respectively. The results presented are an average from three independent trials. M denotes a reaction designed to produce a molecular mass marker of 203 nt.
FIG. 5
FIG. 5
The effects of nucleotide changes near the initiation cytidylate on RNA synthesis. (A) Effect of nucleotide changes near the initiation cytidylate. Changes from B2(−)46G (top sequence) are indicated in bold type. − indicates the absence of the −1 nucleotide. (B) Effects of the identity of the 3′ nontemplated nucleotide on genomic plus-strand initiation. The initiation cytidylate is indicated by an arrow. Substitutions of the 3′ nontemplated nucleotide is indicated in bold type. The synthesis directed by the different endscripts is given as a percentage relative to B2(−)46G. The results presented are from three independent trials.
FIG. 6
FIG. 6
Effect of multiple initiation sites on RNA synthesis. The authentic initiation site is indicated by an arrow in the first construct marked 1. Additional initiation sites added to the 3′ end of B2(−)46G are indicated by arrows 2 and 3. The three initiation sites should generate products of 46, 49, and 52 nt. RNA synthesis directed by the different initiation sites from their respective templates are presented relative to initiation from cytidylate 1 in B2(−)46G. Products initiated from the three potential initiation sites are indicated on the right as 1, 2, and 3 respectively.
FIG. 7
FIG. 7
Requirements for plus-strand RNA synthesis. (A) The predicted secondary structure of B2(−)46G with the stems (A1 and A2) and loops (L1 and L2) indicated by brackets. The initiation cytidylate is denoted by an arrow. (B) Analysis of the sequence in the predicted stem-loop region. The templates used in the specified reactions are indicated at the top of the autoradiogram. Endscripts that have a +1 are indicated by +, while endscripts with changes of the initiation cytidylate to a guanylate are indicated by −. The sizes of the RdRp products are denoted on the right. The amounts of plus-strand synthesis directed by B2(−)Δ3–11 and B2(−)Δ17–26 were 200 and 70%, respectively, compared to synthesis directed by B2(−)46G after correcting for the number of CMP units incorporated. The results presented are an average from three independent trials. (C) Additional analysis of sequences required for efficient RNA synthesis. The templates used and whether they can direct the initiation of RNA synthesis (+ or −) are indicated at the top of the autoradiogram. The sizes of the RNA products are denoted on the right of the autoradiogram. The amounts of synthesis, after adjusting to the number of radiolabelled CMP units incorporated, from B2(−)26G, B2(−)26TV, B2(−)22G, and B2(−)16G were 100, 17, 22, and 5% respectively. (D) Alignment of the sequences in B2(−)26G, B2(−)Δ3–11 (deletion of A2 stem region), B2(−)Δ17–26 (deletion of L1 loop region), and B2(−)26TV (transversion of nt 17 to 24). The two guanylates as well as the two adenylates putatively required for efficient synthesis are shown in bold type.
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