The rotavirus nonstructural protein NS53 (NSVP1), the product of genome segment 5, possesses RNA-binding activity and contains a highly conserved cysteine-rich motif located in the amino-terminal half of the protein. The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (F. Hundley, B. Biryahwaho, M. Gow, and U. Desselberger, Virology 143, 88-103, 1985). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. The duplication begins at nucleotide (nt) 1454, which is 53 nt upstream from the end of the ORF for NS53. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 amino acids (aa) (31 kDa), approximately one-half the size of the authentic 491-aa NS53 (58 kDa). Examination of lysates from brvA-infected cells by Western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Despite the truncation, analysis of the gene A product suggested that it, like full-length NS53, accumulated in association with the cytoskeleton of the infected cell, thus providing evidence that the subcellular localization signal in NS53 resides in the amino terminal half of the protein. Given that brvA is a viable, nondefective mutant, these results demonstrate that the carboxyl-terminal 233 aa of NS53 are not required for rotavirus replication in vitro.