Role of Viral Ribonucleoproteins in Human Papillomavirus Type 16 Gene Expression

Abstract

Human papillomaviruses (HPVs) depend on the cellular RNA-processing machineries including alternative RNA splicing and polyadenylation to coordinate HPV gene expression. HPV RNA processing is controlled by cis-regulatory RNA elements and trans-regulatory factors since the HPV splice sites are suboptimal. The definition of HPV exons and introns may differ between individual HPV mRNA species and is complicated by the fact that many HPV protein-coding sequences overlap. The formation of HPV ribonucleoproteins consisting of HPV pre-mRNAs and multiple cellular RNA-binding proteins may result in the different outcomes of HPV gene expression, which contributes to the HPV life cycle progression and HPV-associated cancer development. In this review, we summarize the regulation of HPV16 gene expression at the level of RNA processing with focus on the interactions between HPV16 pre-mRNAs and cellular RNA-binding factors.

Keywords: SR proteins; hnRNP; human papillomavirus (HPV); papillomavirus; polyadenylation; splicing.

Figure 8

Proteins that regulate HPV RNA processing are controlled by extracellular stimuli and cellular responses. HPV activates the DNA damage response (DDR), the Akt signaling pathway and the ERK signaling pathways upon infection. These activations may affect HPV RNA processing in different ways. The DDR is activated by HPV E1 and E7 by an unknown mechanism to replicate the HPV DNA in differentiating cells [28]. DDR activation also affects HPV mRNA processing by (1) dissociating Fip1 and CPSF30 from HPV16 early UTR, (2) inducing hnRNP C binding to the HPV16 early UTR, thereby alleviating suppression of HPV16 late splice site SD3632 and reducing the activity of HPV16 pAE. As a consequence, the activation of the DDR induces HPV16 late gene expression [73]. The activity of Akt is restricted to epithelial basal cells and Akt is inactivated during epithelial cell differentiation [199] or HPV E7 expression [202], but is often overexpressed in cervical cancer [200]. Inactivation of Akt reduces hnRNP L phosphorylation, which alleviates the silencing of HPV16 late splice sites SD3632 and SA5639. Thus, inhibition of the Akt kinase activates HPV late gene expression at the level of RNA processing [48]. The ERK signaling pathway is activated by HPV E6 [203], and ERK activity affects HPV RNA splicing by controlling E6 exon inclusion via ERK-responding splicing factors Sam68, Brm, and hnRNP A1 [204].

Figure 1

Schematic representation of human papillomavirus type 16 (HPV16) genome. The HPV16 genome is a circular double-stranded DNA molecule of eight kilobases. Blue cylinders represent HPV16 early genes (E1, E2, E4, E5, E6, and E7), and yellow cylinders represent HPV16 late genes (L1 and L2). HPV16 early and late promoters P97 and P670, respectively, and HPV16 early and late polyadenylation signals pAE and pAL, respectively, are indicated. The long control region (LCR) situated between the L1 stop codon and the E6 start codon contains the origin of DNA replication and the HPV16 early promoter/enhancer p97.

Figure 2

HPV16 genome and transcript map. (A) Schematic representation of the HPV16 genome, here depicted as a linearized molecule. Cylinders represent HPV16 early (E1, E2, E4, E5, E6, and E7) and late genes (L1 and L2). HPV16 early and late promoters P97 and P670, respectively, and HPV16 early and late polyadenylation signals pAE and pAL, respectively, are indicated. HPV16 5′-splice sites/splice donors (SD) and 3′-splice sites/splice acceptors (SA) are shown. The long control region situated between the L1 stop codon and the E6 start codon contains the origin of DNA replication and the HPV16 early promoter/enhancer P97. (B) A subset of HPV16 mRNAs initiated at the HPV16 early promoter P97 (middle part, blue) or at the HPV16 late promoter P670 (lower part, yellow) are displayed. The transcript map is adapted from the Papillomavirus Episteme website (PaVE) (https://pave.niaid.nih.gov), except the full E1 mRNA (species C), which has not been identified in HPV16-infected cells. Potential coding capacity is indicated to the right of each mRNA.

Figure 3

Consequences of RNA-binding proteins on the processing of mRNAs. (Left panel) pre-mRNAs are subject to alternative splicing and/or alternative polyadenylation controlled by interactions of the pre-mRNAs with cellular RBPs. These interactions result in the selection of appropriate splice sites or polyadenylation sites. The interactions between pre-mRNAs and RBPs can be modulated by (1) cotranscriptional regulation by altered by Pol II elongation kinetics or by interactions between Pol II and RBPs, (2) change of protein function by posttranslational modification (PTM), which is regulated by extracellular signaling and/or, for example, cellular responses to HPV infection, (3) multivalent effects and/or competition between RBPs that bind to the same or adjacent cis-acting regulatory RNA elements or hot spots for RNA-binding proteins, (4) m6A modifications on RNAs that affect recruitment of m6A readers that control RNA processing. (Right panel). Some of the RBPs may stick to the mRNAs after RNA splicing and polyadenylation. These RBPs may affect additional RNA processing steps subsequent to splicing, such as nuclear RNA export and RNA degradation.

Figure 4

Regulation of HPV16 pre-mRNA splicing and polyadenylation during the HPV16 early life cycle. HPV16 early pre-mRNA splicing and polyadenylation is regulated in a multi-stepwise fashion. Although not yet shown for HPV16, RNA polymerase II (Pol II) kinetics may affect HPV16 splice site selection. Pol II kinetics can be regulated by epigenetic modifications on DNA and/or PTMs of the Pol II C-terminal domain (CTD). In addition, Pol II interacts with various RBPs that may influence RNA splicing. There are multiple cis-acting regulatory RNA elements on HPV16 early mRNAs, indicated here as green cylinders (splicing enhancers) and red cylinders (splicing silencers), that interact with RNA-binding proteins (RBPs) that either activate (green spheres) or suppress (red spheres) splicing or polyadenylation. Proteins that control HPV16 splice sites SA409, SA3358, SD3632 or the HPV16 early and late polyadenylation signals pAE and pAL, respectively, have been identified. Proteins controlling pAE and pAL bind to the early or late untranslated regions, eUTR and lUTR, respectively. Red arrows indicate suppression, and green arrows indicate activation. Proteins that have been identified and shown to control HPV16 splice sites or polyadenylation signals are listed in boxes. See text for more details.

Figure 5

Control of HPV16 and HPV18 E6/E7 mRNA splicing by splicing silencers located in the E7-coding region. Schematic representation of the HPV E6- and E7-coding regions as blue cylinders. The location of a splicing silencer element in the E7-coding region of HPV16 or HPV18 is shown as a red cylinder. The sequence of each splicing silencer element is indicated. The HPV16 splicing silencer interacts with hnRNP A1 and hnRNP A2 to inhibit HPV16 3′-splice site SA409. Inhibition of HPV16 SA409 by hnRNP A1 results in the production of unspliced E6-encoding mRNAs (red arrow), whereas inhibition of HPV16 SA409 by hnRNP A2 results in redirection of splicing to the downstream HPV16 3′-splice site SA742 (green arrow). In HPV18, binding of hnRNP A1 to the splicing silencer results in inhibition of SA416 and production of unspliced E6-encoding mRNAs (red arrow).

Figure 6

Hot spots for RNA-binding proteins (RBPs) at HPV16 splice sites SA3358, SD3632, and SA5639. Extensive mapping of protein interactions with HPV16 RNAs revealed the existence of hot spots for RNA-binding proteins (RBPs) on HPV16 mRNAs. These “RBP hot spots” were centered around HPV16 splice sites SA3358, SD3632, and SAA5639. Quantitations of the RNA–protein interactions and determination of relative binding occupancy of various RBPs (hnRNP A1, PTB, SRSF1, hnRNP L, Tra2B, and hnRNP D) at these hot spots are shown. The results shown here are adapted from [48].

Figure 7

Regulation of HPV16 pre-mRNA splicing and polyadenylation during the HPV16 late life cycle. There are multiple cis-acting regulatory RNA elements on HPV16 late mRNAs, indicated here as green cylinders (splicing enhancers) and red cylinders (splicing silencers), that interact with RNA-binding proteins (RBPs) that either activate (green spheres) or suppress (red spheres) splicing or polyadenylation. Proteins that control HPV16 splice sites SA3358, SD3632, and SA5639 or the HPV16 early and late polyadenylation signals pAE and pAL, respectively, have been identified. Proteins controlling pAE and pAL bind to the early or late untranslated regions, eUTR and lUTR, respectively. Red arrows indicate suppression, and green arrows indicate activation. Dotted lines represent events that must be reversed during switch from early to late gene expression. Proteins that have been identified and shown to control HPV16 splice sites or polyadenylation signals are listed in boxes. See text for more details.