The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt 282 to 2143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 39-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for its growth. The UL37 promoter drives production of an unspliced and several alternatively spliced RNAs. The UL37 exon 1 (UL37x1) unspliced RNA is abundant from IE to late times of HCMV infection, whereas the UL37 spliced RNAs are markedly less abundant. Production of the UL37x1 unspliced RNA requires polyadenylation (PA) at nucleotide 50998, which lies within intron 1, upstream of the UL37 exon 2 (UL37x2) acceptor. The physical proximity of its cis elements suggests steric hindrance between PA and splicing machineries for UL37 pre-mRNA. To test this possibility, we generated site-specific mutants in Target 1 PA and RNA splicing cis elements and compared the PA and splicing efficiencies of mutant RNAs with those of wild-type RNA. The mutually exclusive processing events of UL37x1 PA and UL37x1-UL37x2 splicing have been accurately recapitulated in transfected permissive human fibroblasts (HFFs) expressing a Target 1 minigene RNA, which contains the required splicing and PA cis elements. Two mutants in the invariant PA signal dramatically decreased UL37x1 PA as expected and, concomitantly, increased the efficiency of UL37x1-UL37x2 RNA splicing. Consistent with these results, changes to consensus UL37x1 donor and UL37x2 acceptor sites increased the efficiency of UL37x1-UL37x2 RNA splicing but decreased the efficiency of UL37x1 PA. Moreover, HCMV infection of HFFs increased the abundance of the PA cleavage stimulatory factor CstF-64, the potent splicing suppressor PTB, and the hypophosphorylated form of the splicing factor SF2 at 4 h postinfection. Induction of these factors further favors production of the UL37x1 unspliced RNA over that of the spliced RNAs. Taken together, these results suggest that there is a convergence in UL37 RNA regulation by cis elements and cellular proteins which favors production of the UL37x1 unspliced RNA during HCMV infection at the posttranscriptional level.