The nature of naturally occurring mutations in the hemagglutinin gene of vaccinia virus and the sequence of immediately adjacent genes

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Abstract
The expression of the vaccinia IHD-J hemagglutinin (HA) gene is regulated by two promoters, an early/late and a second distinct late promoter. The first promoter results in transcripts that begin early and are synthesized throughout the infection. All transcripts from this promoter initiate at the same 5′ site. A second promoter is only active late in infection and initiates transcription some distance upstream of the first promoter. We have previously shown that the transcriptional start site controlled by this second, “late only” promoter lies within the coding sequence of an upstream reading frame (p 16-ORF), whereas the termination of early transcription of the HA gene utilizes a transcription termination signal (TTTTTNT) located just beyond the coding region of an immediately downstream reading frame (p 17-ORF). In order to assist our understanding of HA gene expression, we report here the sequence of these two ORFs adjacent to the HA gene. The HA-ORF itself consists of 945 bp, whereas the upstream p 16-ORF consists of 429 bp and the downstream p 17-ORF of 453 bp, sufficient to encode polypeptides of 16 and 17 kD, respectively. While many strains of vaccinia are HA+, rabbitpox virus and the variant of vaccinia IHD-J, designated IHD-W, are HA-. We report and compare here the HA gene sequences of wild-type rabbit poxvirus, two spontaneous HA+ revertants of rabbit poxvirus, and the HA- vaccinia strain IHD-W to that of the previously sequenced (1) prototype HA+ IHD-J strain of vaccinia. All differences were found to occur within the HA open reading frame. Vaccinia virus IHD-W contains a two nucleotide (CA) insertion, leading to the creation of a premature translation termination signal. The HA gene of rabbitpox virus contains several base changes, two deletions of three and six bases, and a single adenine nucleotide deletion within a seven-base adenine repeat, creating a nonfunctional frameshift. Reversion of the HA- rabbitpox virus to HA+ variants is accompanied on both occasions by reinsertion of the single deleted adenine nucleotide.