Requirements at the 3' end of the sindbis virus genome for efficient synthesis of minus-strand RNA

Abstract

The 3'-untranslated region of the Sindbis virus genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3' CSE) immediately preceding the 3'-poly(A) tail. The 3' CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3' CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3' CSE, (iii) deletion or substitution of the 3' 13 nucleotides of the 3' CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3' sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3' sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3' component of the minus-strand promoter is the wild-type 3' CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.

FIG. 1.

Requirement for the 3′ CSE for efficient minus-strand RNA synthesis. (A) Diagram of the wt(+) genome analog RNA showing the regions of the SIN genome present in this RNA. UTR, untranslated region; sub-gen pro, subgenomic RNA promoter. (B) Results of in vitro minus-strand RNA synthesis from the wt(+) RNA (lanes 1, 2, and 3), the wt(+) RNA lacking the 3′ CSE and poly(A) tail (Δ−20 no A, lanes 4 and 5), and wt(+) RNA lacking the 3′ CSE (Δ−20 + A, lanes 6 and 7). Template RNA used for reactions yielding the products in lanes 3, 5, and 7 was treated with sodium metaperiodate prior to use in order to remove the 3′ hydroxyl group. Following 1 h of incubation at 30°C, RNA from the reactions was phenol-chloroform extracted and ethanol precipitated. Precipitated RNA was denatured and separated by agarose-phosphate gel electrophoresis. Products were visualized by autoradiography.

FIG. 2.

Analysis of in vivo minus-strand RNA synthesis. (A) SIN replicon RNA and the experimental RNA were cotransfected into BHK-21 cells and incubated at 37°C for 5 h. Cells were harvested into a detergent buffer for lysis, nuclei were removed, and RNA was extracted from the lysate. First-strand cDNA synthesis was performed using ImPromII reverse transcriptase (Promega) and an oligonucleotide primer corresponding directly to a region in the luciferase gene of the experimental RNA (nt 1905 to 1926 of 5′SIN3′SIN). PCR amplification of the cDNA was done using Taq DNA polymerase (Brinkman-Eppendorf), the primer described above, and an oligonucleotide complementary to nucleotides 2671 to 2691 of the 5′SIN3′SIN RNA. Samples were taken from the PCR mixture after 10, 15, and 20 cycles of amplification, run on a TAE/agarose gel, and stained with ethidium bromide. (B) The procedure described above was followed for cells transfected with SIN replicon RNA and 5′SIN3′SIN RNA (lanes 2 to 4), SIN replicon RNA alone (lanes 5 to 7), SIN replicon RNA plus 5′SIN3′SINΔ−20 RNA (lanes 11 to 13), or SIN replicon RNA plus 5′SIN3′SINΔ−20+A RNA (lanes 14 to 16). The result of a control (con) RT-PCR using 25 ng of purified SIN5′/SIN3′ template RNA is shown in lane 8. Lanes 1, 9, and 10 show a 100-bp DNA ladder (New England Biolabs).

FIG. 3.

In vitro minus-strand RNA synthesis (synth.) from template RNA with poly(A) tails of various lengths. RNA templates were generated by in vitro transcription from BsgI-digested pwt(+) and pMini1(+) or PCR products encoding a SIN genome analog giving rise to RNA templates with 3′ poly(A) tails varying in size from 0 to 34 adenylate residues. These RNA templates were used in an in vitro minus-strand synthesis assay. Products of the reaction were analyzed by agarose-phosphate gel electrophoresis. An autoradiograph of a typical gel is shown. The numbers above the lanes refer to the size of the poly(A) tail. The percentage of wt minus-strand RNA synthesis is shown below each lane. Quantitation was performed by phosphorimaging, and the numbers shown are the average of three independent experiments. The degree of experimental variability is shown in parentheses under each lane.

FIG. 4.

Minus-strand RNA synthetic activity of template RNA with interrupted poly(A) tails. (A) Diagram of RNA templates generated with 25 A residues interrupted at specific points with three C, three U, or three G residues. Residues introduced into the poly(A) tail are shown in bold lowercase letters. (B) Levels of minus-strand RNA synthesized from templates with interrupted poly(A) tails. The numbers above the lanes indicate the number of A residues immediately following the 3′ CSE and preceding the insertion. The RNA templates were used in an in vitro minus-strand RNA synthesis assay, and products were analyzed by agarose-phosphate gel electrophoresis. An autoradiograph of a gel from a representative experiment is shown. Products were quantitated by phosphorimaging and expressed as a percentage of minus-strand synthesis from a wt template (lane 8). The amount of minus-strand synthesis is shown below each lane as a percentage of that synthesized from the wt(+) template (average of results from three independent experiments; the degree of experimental variability is shown in parentheses under each lane).

FIG. 5.

Minus-strand RNA synthesis from template with uridylate insertions between a mutated 3′ CSE and the poly(A) tail. (A) 3′ sequences of RNA templates. Dashes indicate deletions in the 3′ CSE, and bold lowercase letters indicate insertions. (B) RNA templates possessing a deletion of the −1 residue followed by insertions of 2, 3, 4, 5, or 6 uridylate residues prior to the poly(A) tail were examined for their ability to program minus-strand RNA synthesis in vitro. Products were analyzed and quantitated as previously described.