Genomic length cDNAs of papaya mosaic virus (PMV) RNA were generated utilizing 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 plasmid pUC18 and infectious RNA transcripts were synthesized from a bacteriophage T7 RNA polymerase promoter incorporated into the 5′ specific primer. The infectivity of transcripts was 16% that of native PMV RNA. Increasing the poly(A) tail length from A to A produced a 43% increase in infectivity. Transcripts synthesized with or without an mGpppG cap structure were biologically active although uncapped transcripts were much less infectious. The addition of up to 2434 non-viral nucleotides at the 3′ end of transcripts decreased but did not abolish infectivity. Insertions of two amino acid residues within the polymerase coding region inactivated viral transcripts. A single amino acid deletion within the capsid protein (CP) produced local lesions of a reduced size as compared to native PMV RNA. Viral particles could not be observed in crude extracts from lesions produced by this deletion mutant suggesting that it exists as a naked RNA species within the host. 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.


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