Alphavirus RNA genome repair and evolution: molecular characterization of infectious sindbis virus isolates lacking a known conserved motif at the 3' end of the genome

Abstract

The 3' nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3'CSE). The 3'CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3'CSEs were studied in detail to evaluate the role of the 3'CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3'CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3' terminal motifs of the SIN genome; iii) the 5' 1-kb regions of all SIN genomes, regardless of the differences in 3' terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3'CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3' terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3'CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.

FIG. 1

(A) Organization of the SIN genome. NS, coding region for nonstructural proteins; S, coding region for structural proteins; 5′CSE-1, the 44-nt 5′ conserved sequence element at the 5′ end; 5′CSE-2, the 51-nt 5′ conserved sequence element located within the 5′ translatable region; JN-CSE, the 21-nt-long conserved sequence element located at the junction of the NS and S coding regions that serves as a promoter for subgenomic RNA synthesis; 3′NTR, the 0.3-kb 3′ nontranslated region that carries several repeat sequence elements; 3′CSE, the 19-nt conserved sequence element located at the 3′ end adjoining the poly(A) tail. The drawing is not according to scale. (B) Sequences of the 3′NTR domain of eight SIN isolates. These eight SIN isolates, which were recovered from individual plaques (46), were used to infect BHK cells to generate virus stocks. The virus-specific RNAs obtained from infected BHK cells were reverse transcribed with 18TSac− and amplified by PCR using the primers T11200 and 18TSac− as previously described (46). The single species of PCR product obtained for each of the isolates was purified and sequenced. Each isolate is identified on the left of each sequence. The sequence 1101-RR corresponds to the SIN derived from the parental clone Toto 1101 (39, 47, 51). The control isolate S3-7 was generated from the SIN clone Tapa (51). Since the Tapa plasmid was derived from Toto 1101, it was expected to carry identical protein-coding regions. The Tapa plasmid carries an intact 3′NTR and an ApaI restriction site at the 3′ end of the S-coding region. The location of the ApaI site (gggccc) and the stop codon (tga) are shown. Notations used: lowercase letters, original sequence of the template used for virus production; underline, bases corresponding to the 3′CSE; uppercase letters, bases inserted during genome repair in vivo; dots, base deletions; hyphens, base identity; back-slashes, discontinuity in sequence used for drawing purposes.

FIG. 2

Plaque sizes of SIN isolates. Confluent Vero cultures were infected with p-1 stocks of the eight virus isolates, overlaid with agar, and incubated at 37°C. Around 50 to 54 h p.i., plaques were fixed with paraformaldehyde, stained with crystal violet, and photographed. Note that the plaques of isolates S3-7 and S3-23 were larger, whereas those of S3-10 and S3-20 were smaller. The plaques of isolate S3-10 were clear, but the plaques of all other isolates were somewhat diffused.

FIG. 3

Time course of virus release. Duplicate BHK cell cultures were infected with 6 PFU of p-3 SIN isolates/cell and incubated at 37°C for 1 h, the inoculum was removed, and the cells were replenished with 1 ml of medium containing 2% fetal bovine serum. At 3, 5, 7, 9, and 11 h p.i., culture supernatants were completely removed, and 1 ml of fresh medium was added to the cells. The amount of infectious virus found in the recovered medium was determined by plaque assay. Each value represents the average of two experiments. In the two experiments, the isolate S3-20 showed very similar growth kinetics.

FIG. 4

Levels of virus-specific RNA expressed by SIN isolates. BHK cell cultures were infected with 6 PFU of each virus isolate/cell and labeled with [³H]uridine between 5 and 9 h p.i. as previously described (46, 47). In brief, infected cells were replenished with 0.6 ml of minimal essential medium containing 3 μg of dactinomycin per ml at 6 h p.i. Twenty minutes later, 50 μCi of [³H]uridine (Dupont-NEN) was added to each plate, and the infection was continued at 37°C for an additional 3 h. At the end of the labeling period, cells were harvested in phosphate-buffered saline and disrupted with 1% NP-40, and the cytoplasmic supernatants were recovered by centrifugation. Cytoplasmic RNA was purified by phenol-chloroform extraction and precipitation with two volumes of ethanol (46, 47). The amount of RNA recovered was determined by UV spectrophotometry. Six micrograms of the isolated RNA was denatured with glyoxal, analyzed on a 1.25% agarose gel, and then fluorographed as described previously (46, 47). The radioactivity corresponding to each of the bands was recovered, solubilized in a biodegradable solvent (BCS; Amersham) and quantitated. For comparison, the intensity of the RNA bands was also determined by densitometry using the Gel-doc 2000 apparatus and Quantity One image analysis software (Bio-Rad). The top of each lane indicates the identity of the isolate. UI, uninfected; g, genomic RNA, 11.7 kb; sg, subgenomic RNA, 4.2 kb; g/sg, ratio of genomic RNA to subgenomic RNA. The values were determined from two experiments. RNA samples derived from each experiment were loaded twice on the same gel for densitometry and determination of radioactivity.

FIG. 5

Evolution of the 3′ proximal region of SIN isolates. Infected cell cytoplasmic RNA obtained from the indicated passages of all virus isolates was reverse transcribed with 18TSac− and amplified by PCR with T11200 and 18TSac−. The PCR products were purified and sequenced using the primer T11200. The 3′CSE (−1 to −19) and its remnants are identified by underlining. The bases newly added during the repair process are identified by uppercase letters. The hyphens denote nucleotide identity. Slashes and vertical lines denote discontinuity in sequence, used for drawing purposes.

FIG. 6

Physical map of the AU-rich RSEs. Each RSE found within the AU-rich terminal motif of isolates S3-23 (A) and S3-4 (B) was arranged and identified by the same letters as indicated in Table 2. Bars of the same kind indicate the same RSE. For example, bars denoting the motif x (panel A) occur very close to each other, whereas those denoting p (panel B) are well separated.