Transcriptional and translational analysis of the vaccinia virus late gene L65

Among the products of vaccinia virus genes which are expressed late in infection is a major polypeptide (Mr, 65,000) designated L65. Pulse-chase analyses indicated that L65 is not subject to posttranslational cleavage as is the core polypeptide p4b which migrates to a similar position in sodium dodecyl sulfate-polyacrylamide gels. A polypeptide of 65,000 molecular weight produced in reticulocyte lysates programmed with viral mRNA isolated late in infection was identified as L65 by peptide mapping. L65 mRNA was purified by hybridization selection to restriction fragments of the viral genome and translated in vitro. This allowed the gene encoding L65 to be mapped to the rightmost 4.5 kilobase pairs of the HindIII D fragment. Transcriptional mapping of this region of the genome detected a late mRNA which was initiated at 450 base pairs to the right of the HindIII D-A junction, was transcribed in the leftward direction, and was terminated in the nondescript manner typical of vaccinia virus late mRNAs.