Abstract
Three variants of group A rotavirus with large changes in their gene 5 structures have been analyzed at the molecular level. The first of these, P9 delta 5, was obtained during plaque purification undertaken as part of the biological cloning of a field isolate of virus. The gene 5 homolog in this isolate migrated just ahead of the normal segment 6 RNA, giving an estimated size of 1,300 bp. Molecular cloning and sequencing of this homolog revealed it to have a single 308-bp deletion in the center of the normal gene 5 sequence extending between nucleotides 460 and 768 of the normal gene sequence. This deletion caused a frameshift in the gene such that a stop codon was encountered 8 amino acids downstream of the deletion point, giving a predicted size for the protein product of this gene of 150 amino acids compared with the 490 amino acids of its normal-size counterpart. Attempts to detect this shortened protein in virus-infected cells were not successful, indicating that it was much less stable than the full-length protein and/or had suffered a large change in its antigenicity. The second two variants, brvA and brvE, were generated in an earlier study following the high-multiplicity passage of the UKtc strain of bovine rotavirus. Polyacrylamide gel electrophoresis analysis of these nondefective variants showed that brvA had a gene 5 homolog approximately equal in size to the normal RNA segment 2 (approximately 2,700 bp) and that brvE had a size of approximately 2,300 bp. Both variants showed changes in their gene 5 protein products, with brvA mimicking P9 delta 5 in failing to produce a detectable product whereas brvE produced a new virus-specific protein approximately 80 kDa in size. Full-length cDNA clones of the brvE gene 5 homolog were isolated, and analysis of their structure revealed a head-to-tail concatemerization of the normal gene 5 sequence with the first copy of the concatemer covering nucleotides 1 to 808 and the second covering nucleotides 92 to 1579, giving a total length of 2,296 bp. Sequencing across the junction region of the two copies of the gene showed that they were joined in frame to give a predicted combined open reading frame of 728 amino acids with the amino-terminal region consisting of amino acids 1 to 258 fused at the carboxy terminus to amino acids 21 to 490.(ABSTRACT TRUNCATED AT 400 WORDS)