The 3' sequences required for incorporation of an engineered ssRNA into the Reovirus genome

Abstract

Background: Understanding how an organism replicates and assembles a multi-segmented genome with fidelity previously measured at 100% presents a model system for exploring questions involving genome assortment and RNA/protein interactions in general. The virus family Reoviridae, containing nine genera and more than 200 members, are unique in that they possess a segmented double-stranded (ds) RNA genome. Using reovirus as a model member of this family, we have developed the only functional reverse genetics system for a member of this family with ten or more genome segments. Using this system, we have previously identified the flanking 5' sequences required by an engineered s2 ssRNA for efficient incorporation into the genome of reovirus. The minimum 5' sequence retains 96 nucleotides and contains a predicted sequence/structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79-81 that are essential for the incorporation of in vitro generated ssRNAs into new reovirus progeny viral particles. The work presented here builds on these findings and presents the results of an analysis of the required 3' flanking sequences of the s2 ssRNA.

Results: The minimum 3' sequence we localized retains 98 nucleotides of the wild type s2 ssRNA. These sequences do not interact with the 5' sequences and modifications of the 5' sequences does not result in a change in the sequences required at the 3' end of the engineered s2 ssRNA. Within the 3' sequence we discovered three regions that when mutated prevent the ssRNA from being replicated to dsRNA and subsequently incorporated into progeny virions. Using a series of substitutions we were able to obtain additional information about the sequences in these regions. We demonstrate that the individual nucleotides from, 98 to 84, 68 to 59, and 28 to 1, are required in addition to the total length of 98 nucleotides to direct an engineered reovirus ssRNA to be replicated to dsRNA and incorporated into a progeny virion. Extensive analysis using a number of RNA structure-predication software programs revealed three possible structures predicted to occur in all 10 reovirus ssRNAs but not predicted to contain conserved individual nucleotides that we could probe further by using individual nucleotide substitutions. The presence of a conserved structure would permit all ten ssRNAs to be identified and selected as a set, while unique nucleotides within the structure would direct the set to contain 10 unique members.

Conclusion: This study completes the characterization and mapping of the 5' and 3' sequences required for an engineered reovirus s2 ssRNA to be incorporated into an infectious progeny virus and establishes a firm foundation for additional investigations into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus. As researchers build on this work and apply this system to additional reovirus genes and additional dsRNA viruses, a complete model for genome assortment and replication for these viruses will emerge.

Figure 1

Survey of the minimum 3' terminal s2 ssRNA nucleotides required to direct a ssRNA into the reovirus genome using 50-nucleotide deletions, single-nucleotide deletions and nucleotide deletions using ssRNAs with extended 5' sequences. At the top, are the sequences of the 5' nucleotides of the ssRNAs produced using the T7 RNA polymerase promoter and cDNA template pS2CAT198 and pS2CAT96. The top two sequences include the first 18 nucleotides of the CAT coding sequence but do not show the 3' end of the ssRNA. The 3' sequence of these ssRNAs is shown in its entirety from the CAT stop codon to the authentic reovirus 3' terminus directly below the 5' ssRNA sequences. A line connects the last retained s2 nucleotide in the displayed sequence to the named cDNA plasmid, below which are displayed autoradiogram panels. Within each panel, the ssRNA, dsRNA and CAT dsRNA panels are Northerns analyzing RNA extracted from cells lipofected 12 hours earlier with 9 wildtype ssRNAs and the ssRNA obtained following transcription of the indicated cDNA template. The fourth and bottom panel of each set is an autoradiogram generated by in vivo labeling with ³²P of the dsRNA genome segments of an isolated progeny virus containing the indicated mutated-S2 dsRNA. Progeny virus generated following lipofection was first triple-plaque purified. Deletions were initially made in blocks of 50 nucleotides. Based on the sequences required to incorporate a ssRNA into a reovirus using the ssRNAs generated from these templates, additional cDNA templates were constructed deleting ten, five and individual nucleotides until the minimal 3' sequence had been determined. Left and center panels. To test the possibility that increasing the 5' s2 sequence from 96 to 198 nucleotides might reduce the length of the 3' sequence required, a number of the 3' deleted cDNA templates were altered to include a the 198 5' sequence and retested. The ability to incorporate these ssRNAs into the genomes of reoviruses is summarized in the right panel. As described in the Materials and Methods, all ssRNAs were sequenced/analyzed to confirm the 5' and 3' ends of these RNAs.

Figure 2

Detection of virus-generation intermediates using the ssRNAs generated from the sequence-substitution cDNA templates outlined in Table 2, using the reovirus infectious RNA system. The ssRNA (in the top panel), the dsRNA (in the next panel down) and the CAT dsRNA (the third panel down) are shown utilizing Northerns analyzing RNA extracted from cells lipofected 12 hours earlier with 9 wildtype ssRNAs and the ssRNA obtained following transcription of the indicated cDNA template. The fourth and bottom panel is an autoradiogram following SDS-PAGE generated by in vivo labeling with ³²P of the dsRNA genome segments of an isolated progeny virus containing the indicated mutated-S2 dsRNA. Progeny virus generated following lipofection was first triple-plaque purified.

Figure 3

Ball and stick representation of three secondary structures predicted, using FOLDALIGN^®, to exist in the 3' terminal 100 nucleotides of all 10 reovirus ssRNAs. Individual nucleotides present at each position in each of the 10 reovirus ssRNAs are shown in the table below the figure.

Figure 4

Three regions, nucleotides 98 to 84, 68 to 59, and 28 to 1, that when coupled with an additional 96 s2 5' terminal nucleotides are required to direct the incorporation of this RNA into an infectious reovirus.