Experiments with virus genomes containing an HPV31 E2 transactivation mutant, E2:IL-73, confirmed that a wild-type E2 activation domain is required to achieve high levels of SF2/ASF in virus-infected cells
Experiments with virus genomes containing an HPV31 E2 transactivation mutant, E2:IL-73, confirmed that a wild-type E2 activation domain is required to achieve high levels of SF2/ASF in virus-infected cells. vitro. This is the first time that HPV16 E2 has been shown to regulate cellular gene expression and the first report of viral regulation of expression of an Flurizan RNA processing factor. Such E2-mediated control during differentiation of infected epithelial cells may facilitate late capsid protein expression and completion of the virus life cycle. Human papillomavirus type 16 (HPV16) infects cervical epithelial cells, causing mainly benign lesions (cervical dysplasia). However, in some rare cases upon persistent infection, lesions can progress to cervical cancer (66). The life cycle of this 7.9-kb double-stranded DNA virus is highly dependent upon the differentiation status of the epithelial tissue it infects. Of particular importance is restriction of expression of the highly immunogenic capsid proteins L1 and L2 to the most differentiated cells of the structure, where immune surveillance is low. However, while L1 and L2 RNAs have been detected in less-differentiated epithelial cells (5,60), fully processed messenger RNAs (mRNAs) are found only in differentiated epithelial cells (42), and capsid protein expression is restricted to the granular layer cells (48). Thus, expression of the virus capsid proteins is regulated at least partly at posttranscriptional levels (20).cis-Acting RNA regulatory motifs have been identified in the HPV16 genome that may regulate capsid protein expression at one or more posttranscriptional levels (10,11,12,29,45,50,55,65). The elements are proposed to act via interactions with cellular RNA splicing factors, including U1 snRNP, hnRNP A1, SF2/ASF, PTB hnRNP C1/C2, and CUG-BP1 (9,10,12,19,30,41,56,64). Such RNA-protein interactions Flurizan are proposed to be essential in regulating virus TNFRSF11A late gene expression, for example by regulating alternative Flurizan splicing and polyadenylation of virus late mRNAs (20). There are two key protein families involved in regulation of alternative splicing in human cells. These are the SR (serine/arginine-rich) and hnRNP protein families. SF2/ASF is the key SR splicing protein (7), and hnRNP A1 is the main antagonistic counterpart of SF2/ASF (15). SF2/ASF has roles in constitutive splicing by bringing U1 snRNP to the 5 splice site and by bridging spliceosome complexes across Flurizan exons and introns. This leads to efficient recognition of intron/exon junctions and determination of RNA splicing patterns. In relation to this, SF2/ASF has extremely well-documented roles in alternative splicing by binding intronic and exonic sequence enhancers to modulate the efficiency of recognition of upstream and downstream splice sites by the splicing machinery. The binding pattern of SR and hnRNP proteins has been termed the splicing code, as it determines which mRNA isoforms and therefore proteins are ultimately expressed from a gene. Surprisingly, our recent studies indicated that 13 different late mRNAs encode the virus capsid proteins (42) in differentiated W12 cells, cervical epithelial cells that contain nuclear episomal copies of the HPV16 genome (57). These mRNAs that are the products of extensive alternative splicing of late pre-mRNAs also encode E6/E7 isoforms, E1^E4 and E5. Overlapping mRNAs may be required to ensure that each open reading frame is first in one of the mRNAs to ensure efficient translation of each protein (31). Previously, comparing undifferentiated and differentiated cells, we showed that abundance of SF2/ASF increased approximately four- to eightfold in differentiated W12 cells (41) and in cervical tissue isolated from patients with HPV16-positive, low-grade cervical lesions (unpublished data). In contrast, SF2/ASF levels diminished in uninfected differentiated epithelial cells such as HaCaT, a spontaneously immortalized epithelial cell line, and normal human keratinocytes (41). This indicated that HPV16 infection upregulated SF2/ASF expression in a differentiation stage-specific manner. Furthermore, SF2/ASF was found to be upregulated in U2OS cells stably expressing HPV16 E2, the virus transcription factor, suggesting it could transcriptionally regulate SF2/ASF (41). E2 is a typical transcription factor with a DNA binding and a transactivation domain separated by a flexible hinge.