FIG. 1.

Structure of the MHV-68 rta gene. (A) Genomic location of ORF50. The nucleotide numbers are assigned on the basis of the MHV-68 sequence in the National Center for Biotechnology Information database (40). The open boxes represent ORFs predicted by computer analysis, and the numbers assigned to individual ORFs are based on homology with the corresponding ORFs of HVS and HHV-8 (40). The arrows in the open boxes indicate the orientation of ORFs. A major portion of the rta gene is carried by ORF50. Downstream of ORF50 is ORFM7 encoding gp150, which shares homology with EBV gp350 (34). (B) Sequencing analysis of the rta cDNA. RNA harvested from BHK-21 cells infected with MHV-68 at 2 PFU/cell was reverse transcribed with oligo(dT) and amplified, first by primers R3 and R4 and then by primers R3 and R6. The resultant product was cloned, and the positive clone was sequenced with the R7 primer. The splice donor and acceptor sites were mapped to nt 66795 and 67661, respectively. (C) Splicing of the rta gene. The splice donor (s.d.) and splice acceptor (s.a.) sites were localized to nt 66795 and 67661, respectively. An intron of 866 nt is removed, and two exons are joined together to generate the Rta ORF, with the translation initiation codon at nt 66760. Primers R3 and R4 were used for PCR amplification of the cDNA product and the genomic sequence spanning the rta gene. R3 and R6 were used to reamplify the cDNA PCR product of R3 and R4 for cloning. R7 was used to sequence the splicing junction. The probe shown in panel C was used for Northern analysis of the rta transcripts shown in Fig. 2A.