Mutations in the 5 ' nontranslated region of bovine viral diarrhea virus result in altered growth characteristics

The 5' nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling su ggests that the 5'-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib, Spontaneous and engineered mutations locate d in the genomic region comprising Ia and Ib were characterized by using in fectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5' NTR mut ations carrying between 9 and 26 A residues within the loop region of Ib ha d no detectable influence on specific infectivity and virus growth properti es. After tissue culture passages, multiple insertions and deletions of A r esidues occurred rapidly. In contrast, an engineered mutant carrying 5 A re sidues within the Ib loop was genetically stable during 10 tissue culture p assages. This virus was used as starting material to generate a number of a dditional mutants. The analyses show that (i) deletion of the entire Ib loo p region resulted in almost complete loss of infectivity that mas rapidly r estored during passages in cell culture by insertions of variable numbers o f A residues; (ii) mutations within the 5'-terminal 4 nucleotides of the ge nomic RNA severely impaired virus replication; passaging of the supernatant s obtained after transfection resulted in the emergence of efficiently repl icating mutants that had regained the conserved 5'-terminal sequence; (iii) provided the conserved sequence motif 5'-GUAU was retained at the 5' end o f the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Is and part of Ib were found to support replicatio n, but to a lower degree than the parent virus. Restriction of specific inf ectivity and virus growth of the 5' NTR mutants correlated with reduced amo unts of accumulated viral RNAs.