Subgenomic RNAs with nucleotide sequences derived from RNAs 1 and 2 of cucumber mosaic virus can act as messenger RNAsin Vitro

Gordon, Karl H.J.; Symons, Robert H.

Abstract Encapsidated RNAs of cucumber mosaic virus (CMV) were analyzed by hybridization to specific probes after gel electrophoresis. [32P]-complementary DNA (cDNA) probes were prepared by transcription of genomic RNA 1 and RNA 2 nucleotide sequences that had been cloned in a bacteriophage M13 vector. Probes that correspond to unique sequences near the 3′ ends of RNA 1 and RNA 2 revealed over 20 smaller RNAs. The subgenomic RNAs derived from each genomic RNA were analyzed more definitively by hybrid selection from total encapsidated RNA, using minus DNA clones derived from sequences in either RNA 1 or RNA 2, and a cDNA probe for the 3′ sequence conserved among all the genomic RNAs. Different patterns of over 20 minor RNA species, which were 3′-coterminal with RNAs 1 and 2, were detected, and they were reproducible irrespective of the host, cucumber orNicotiana clevelandii, from which the virus was isolated. The same RNA patterns were found in RNA extracted from the particulate fraction of CMV-infected cucumber orN. clevelandii. In order to determine whether the subgenomic RNAs could function as messenger RNAs, hybrid-selected RNAs were tested byin vitro translation, using the rabbit reticulocyte lysate. The subgenomic RNAs from RNA 1 produced over 10 major polypeptides from Mr 27,000 to Mr 90,000 all of which could be translated from a few RNA species over about 2,300 nucleotides long. The 3′-coterminal subgenomic RNAs derived from RNA 2 gave less than 10 products from Mr , 17,000 toMr 85,000. The smallest product (Mr 17,000) was produced by an RNA about 880 nucleotides long, whereas longer RNAs from 1400 to 2500 nucleotides were efficient mRNAs for polypeptides from Mr 30,000 up to the largest translation products consistent with the size of the RNA.