INFECTIOUS RNA TRANSCRIPTS DERIVED FROM CLONED CDNA OF PAPAYA MOSAIC-VIRUS - EFFECT OF MUTATIONS TO THE CAPSID AND POLYMERASE PROTEINS

Genomic length cDNAs of papaya mosaic virus (PMV) RNA were generated u tilizing reverse transcriptase (RNase H-) for first strand synthesis, Sequenase for second strand synthesis and primers specific for the 5' and 3' termini of the viral genome. These cDNAs were cloned into plasm id pUC18 and infectious RNA transcripts were synthesized in vitro from a bacteriophage T7 RNA polymerase promoter incorporated into the 5' s pecific primer. The infectivity of transcripts was 16 % that of native PMV RNA. Increasing the poly(A) tail length from A24 to A71 produced a 43% increase in infectivity. Transcripts synthesized with or without an m7GpppG cap structure were biologically active although uncapped t ranscripts were much less infectious. The addition of up to 2434 non-v iral nucleotides at the 3' end of transcripts decreased but did not ab olish infectivity. Insertions of two amino acid residues within the po lymerase coding region inactivated viral transcripts. A single amino a cid deletion within the capsid protein (CP) produced local lesions of a reduced size as compared to native PMV RNA. Viral particles could no t be observed in crude extracts from lesions produced by this deletion mutant suggesting that it exists as a naked RNA species within the ho st. Mutations to the CP suggest that it is required not only for viral assembly but also for some other unidentified function(s) during the replication cycle.