Michael Janda

In vitro transcripts from mixtures of appropriate brome mosaic virus (BMV) cDNA clones are infectious when inoculated onto barley plants. Infectivity depends on in vitro transcription and on the presence of transcripts from clones of all three BMV genetic components. Infectivity is destroyed by RNase after transcription, but it is insensitive to RNase before or to DNase after transcription. Virion RNAs from plants infected with cDNA transcripts hybridize to BMV-specific probes and coelectrophorese with virion RNAs propagated from conventional inoculum. Direct RNA sequencing shows that a deletion in the noncoding region of one infectious BMV clone is preserved in viral RNA from plants systemically infected with transcript mixtures representing that clone.

The nucleotide sequence of 7200 bases of encephalomyocarditis (EMC) viral RNA, including the complete polyprotein-coding region, was determined. The polyprotein is encoded within a unique translational reading frame, 6870 bases in length. Protein synthesis begins with the sequence Met-Ala-Thr, and ends with the sequence Leu-Phe-Trp, 126 bases from the 3' end of the RNA. Viral capsid and noncapsid proteins were aligned with the deduced amino acid sequence of the polyprotein. The proteolytic processing map follows the standard 4-3-4 picornaviral pattern except for a short leader peptide (8 kd), which precedes the capsid proteins. Identification of the proteolytic cleavage sites showed that EMC viral protease, p22, has cleavage specificity for gln-gly or gln-ser sequences with adjacent proline residues. The cleavage specificity of the host-coded protease(s) includes both tyr-pro and gln-gly sequences.

Brome mosaic virus (BMV) is a plant virus whose genome consists of three RNA components. A previously described viral complementary DNA expression system has been used to express both wild-type and altered genomic RNA's in barley protoplasts. Variants of BMV RNA3 were constructed in which the coat gene had been removed or replaced with sequences encoding chloramphenicol acetyltransferase (CAT). CAT sequences were also inserted near the 5' end of the intact coat gene. When inoculated on protoplasts together with transcripts of BMV RNA's 1 and 2, all of these RNA3 derivatives were replicated and produced subgenomic RNA's analogous to the normal subgenomic coat protein messenger RNA. RNA3 derivatives in which the CAT coding sequences were oriented with the same polarity as viral genes produced significant CAT activity in protoplasts. CAT expression was improved by inserting the CAT gene in frame with the upstream coat protein initiation codon, and exceeded expression in plant cells transformed with Ti plasmid-based vectors.