Abstract
Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784–820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784–820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.
Highlights
We showed that rSA11Wa and rSA11DS-1 were efficiently rescued and exhibited no detectable growth defects
We found that restriction determinants map to the SR within the AU-1 NSP2 gene ORF
It is important to note that the SR is not the only region containing restriction determinants; there are certainly other regions that contributed to the lack of rescuable rSA11AU-1 virus
Summary
A plasmid expressing the full-length, shRNA-resistant NSP2 gene from simian RV strain SA11 (pBS-SA11g8R) was obtained from Dr John Patton (University of Maryland, College Park)[31]. To construct similar plasmids expressing the full-length NSP2 genes from human RV strains Wa (pBS-Wag8R), DS-1 (pBS-DS-1g8R), or AU-1 (pBS-AU-1g8R) RT-PCR and molecular cloning was used. The cloned Wa and DS-1 NSP2 gene sequences are identical to what is reported in GenBank (accession numbers RO1NS35F and EF672580, respectively). The AU-1 NSP2 gene sequence is identical to what is reported in GenBank (accession number DQ490534) with the exception of a single G-to-A nucleotide change at position 40 in the 5′ UTR and the 6 nucleotide changes in the shRNA target site (nucleotides 655–673). To engineer the chimeric NSP2 genes, PCR was used to amplify specified regions (Fig. 3a) from the pBS-SA11g8R and pBS-AU-1g8R plasmids.
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