Mutational analysis of the pseudoknot region in the 3' noncoding region of tobacco mosaic virus RNA

Abstract

The approximately 200-nucleotide-long 3'-terminal noncoding region of tobacco mosaic virus (TMV) RNA contains a tRNA-like structure and, in its immediate upstream region, three consecutive pseudoknots, each of which is composed of two double-helical segments. To elucidate the biological functions of the pseudoknot region, we constructed several deletion mutant TMV-L (a tomato strain) RNAs by using an in vitro transcription system and tested their ability to multiply in both tobacco plants and protoplasts. When deletions were introduced just downstream of the termination codon of the coat protein gene in the 5'-to-3' direction progressively, five of six double-helical segments were dispensable for viral multiplication, indicating that the pseudoknot structures are not essential for multiplication. However, extension of the deletion into the central pseudoknot region resulted in reduction in viral multiplication, accompanied by loss of development of mosaic symptoms on systemic tobacco plants. Cessation of multiplication was observed when the sequence involved in formation of double-helical segment I just upstream of the tRNA-like structure was deleted irrespective of the start point and extent of deletion. Point mutations that destabilized double-helical segment I resulted in a loss or great reduction of viral multiplication, whereas the double mutants in which the double helix was restored by additional compensating base substitutions restored multiplication to nearly the wild-type level. Thus, double-helical segment I just upstream of the tRNA-like structure is a structural feature essential for viral multiplication.