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. 2001 Mar;75(5):2076-86.
doi: 10.1128/JVI.75.5.2076-2086.2001.

Effect of intragenic rearrangement and changes in the 3' consensus sequence on NSP1 expression and rotavirus replication

J T Patton  1 , Z Taraporewala, D Chen, V Chizhikov, M Jones, A Elhelu, M Collins, K Kearney, M Wagner, Y Hoshino, V Gouvea
Affiliations

Effect of intragenic rearrangement and changes in the 3' consensus sequence on NSP1 expression and rotavirus replication

J T Patton et al. J Virol. 2001 Mar.

Abstract

The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3' termini ended with 5'-UGAACC-3' instead of the 3' consensus sequence 5'-UGACC-3', which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3' consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3' consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3' end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3' consensus sequence.

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Figures

FIG. 1
FIG. 1
Genome segments of wild-type viruses 30-19 and 4F and variants 30-1A and 5S. RNAs were recovered from the viruses by phenol-chloroform extraction and ethanol precipitation, resolved by SDS-PAGE, and detected by staining with ethidium bromide. The arrows denote the positions of aberrant genome segments for 30-1A and 5S.
FIG. 2
FIG. 2
Alignment of the predicted amino acid sequences of NSP1 encoded by the gene 5 dsRNAs of wild-type viruses 30-19 and 4F and variants 30-1A and 5S. Sequence identity is indicated with dots.
FIG. 3
FIG. 3
Sequence organization of aberrant gene 5 dsRNAs of rotavirus. The lengths of the aberrant gene 5 dsRNAs and their NSP1 products are given, as well as the corresponding values for wild-type gene 5 dsRNAs and their NSP1 products (in parentheses). Regions representing the 5′ and 3′ UTRs of the wild-type dsRNA are shown with diagonal lines. The sizes and positions of duplicated sequences in the gene 5 dsRNAs of SA11 30-1A, SA11-5S, brvE, brvA, and IGV-80-3-re and deleted sequences in the gene 5 dsRNAs of UK-P9Δ5 and A5-16 and the locations of nonsense mutations in the gene 5 dsRNAs of brvA and A5-10 are indicated. The accession numbers for the gene 5 homologs are AF290882 (SA11 30-1A), AF290884 (SA11-5S), Z24735 (brvE, junction sequence only), L12248 (brvA), AF190169 (IGV-80-3-re), Z24736 (UK-P9Δ5), D38149 (A5-16), and D38147 (A5-10).
FIG. 4
FIG. 4
Junction of the duplicated sequences in the gene 5 dsRNAs of SA11 30-1A and SA11-5S. The junction of the duplicated sequences includes one (30-1A) or three (5S) nontemplated T residues. Aligned with each junction are the sequences of the wild-type gene 5 dsRNAs of SA11 30-19 or SA11-4F. The alignment indicates the potential sites where the viral RdRP and nascent RNA dissociated from the wild-type RNA template and then, at an upstream site on the same template, reassociated and reinitiated RNA synthesis using the nascent RNA as a primer. The arrowed line illustrates the movement of the RdRP along the wild-type sequence and the synthesis of the nontemplated T residues required to generate the junction sequence during plus-strand synthesis. Repeated sequences near the site where the RdRP is proposed to have dissociated from the wild-type template are overlined. Identity between the 3′-terminal sequence of the nascent transcript and the sequence near where the RdRP reinitiated transcription is also shown.
FIG. 5
FIG. 5
Expression of truncated NSP1 in cells infected with the variants 30-1A and 5S and wild-type viruses 30-19 and 4F. Mock- or virus-infected MA104 cells which had been maintained in medium containing 35S-labeled amino acids were lysed at 8 h p.i. with Triton X-100, and the cytosol and cytoskeleton fractions of the cell lysates were recovered. (A) 35S-labeled proteins in the fractions were resolved by SDS-PAGE and detected by autoradiography. The suspected positions of wild-type (30-19 and 4F) and C-truncated (30-1A and 5S) NSP1 are indicated with dots. (B) The cytosol fractions described in panel A were resolved by SDS-PAGE, and the proteins were blotted onto nitrocellulose. The blot was probed with antiserum raised against a peptide corresponding to the last 19 aa of SA11 NSP1. Molecular masses were determined by coelectrophoresis of prestained protein markers.
FIG. 6
FIG. 6
Effect of the UGACC→UGAACC mutation on the efficiency of minus-strand synthesis. SA11 gene 5 mRNAs were prepared that ended with the 3′ consensus sequence UGACC (GACC) or the atypical sequence UGAACC (GAACC) and used as templates for the synthesis of minus-strand RNA in the open-core replication system. Reaction mixtures contained various amounts of template RNA (A) or open cores (B) and included [32P]UTP to radiolabel RNA products. 32P-labeled dsRNA products were detected by SDS-PAGE and autoradiography, and the relative levels of dsRNA products were quantified with a PhosphorImager. The percent GAACC/GACC was calculated by dividing the amount of dsRNA product made in reactions containing template RNA ending with UGAACC by the amount of that ending with UGACC and multiplying the result by 100.
FIG. 7
FIG. 7
Isolation of His-tagged rNSP3 expressed in bacteria. Protein markers (lane 1) and His-tagged rNSP3, purified with a Ni-nitrilotriacetic acid agarose column (lane 2), were resolved by SDS-PAGE and detected by staining with Coomassie blue R-250.
FIG. 8
FIG. 8
Impact of the UGACC→UGAACC mutation on the RNA-binding activity of NSP3. Complexes formed by incubating purified rNSP3 with the 32P-labeled RNA probes v40-GACC and v40Δ3′-11 (A) or v40-GACC and v40-GAACC (B) were resolved by electrophoresis on a nondenaturing 6% polyacrylamide gel and detected by autoradiography. The intensity of bands representing rNSP3-probe complexes and free probe were quantified with a PhosphorImager. The values were used to calculate the percentage of probe binding to rNSP3.
FIG. 9
FIG. 9
Effect of the UGACC→UGAACC mutation on protein expression in infected cells. The chimeric reporter RNAs g6-Fluc-UGACC and -UGAACC and glo/g6-Fluc-UGACC and -UGAACC were separately cotransfected with nv-Rluc into SA11-infected MA104 cells at 1 h p.i. At 9 h p.i., the levels of firefly and Renilla luciferases per milligram of cell lysate were determined and the expression of firefly luciferase was normalized to the expression of Renilla luciferase. To ease the comparison of values, the expression of firefly luciferase for the g6-Fluc-UGACC and glo/g6-Fluc-UGACC RNAs was set to 100%.
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