Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression

The Ll and L2 capsid genes of human papillomavirus type 31 (HPV-31) are exp ressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefo re pass through and ignore the early polyadenylation sequences to use the d ownstream late AAUAAA element located at the end of the Ll ORF. To identify sequences which modulate downstream capsid gene expression, a variety of s ubstitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter cons tructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor bi nding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-3 1 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 by of the L2 ORF wer e also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that co ntained a substitution in the AAUAAA sequence, an inserted strong CstF bind ing site, an inserted simian virus 40 (SV40) late poly(A) signal, or a subs titution of the 5'-most 800 nucleotides of the L2 ORF. Reductions in both t ransient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that al terations in the strength of the upstream poly(A) signal influence expressi on of viral replication factors. Similarly, disruption of the L2 ORF result ed in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AA UAAA sequence had increased levels of transient and stable replication. Qua ntitation of late transcripts following keratinocyte differentiation in met hylcellulose also showed a reduction in downstream capsid gene expression i n lines containing genomes with the strong CstF site or the late SV40 signa l mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.