Splicing and Polyadenylation of Human Papillomavirus Type 16 mRNAs

Abstract

The human papillomavirus type 16 (HPV16) life cycle can be divided into an early stage in which the HPV16 genomic DNA is replicated, and a late stage in which the HPV16 structural proteins are synthesized and virions are produced. A strong coupling between the viral life cycle and the differentiation state of the infected cell is highly characteristic of all HPVs. The switch from the HPV16 early gene expression program to the late requires a promoter switch, a polyadenylation signal switch and a shift in alternative splicing. A number of cis-acting RNA elements on the HPV16 mRNAs and cellular and viral factors interacting with these elements are involved in the control of HPV16 gene expression. This review summarizes our knowledge of HPV16 cis-acting RNA elements and cellular and viral trans-acting factors that regulate HPV16 gene expression at the level of splicing and polyadenylation.

Keywords: HPV16; SR-protein; hnRNP; human papillomavirus; polyadenylation; splicing.

Figure 1

Schematic drawing of the human papillomavirus type 16 (HPV16) genome [3]. Boxes indicate open reading frames, filled triangles represent 5′-splice sites and open triangles represent 3′-splice sites. The early promoter p97 and the late promoter p670 are indicated, early and late polyadenylation signals pAE and pAL are indicated. A selection of alternatively spliced HPV16 mRNAs are shown below the HPV16 genome. L1 and L1i represent two alternatively spliced L1 mRNAs.

Figure 2

The HPV16 E2 proteins can be produced from two mRNAs generated either from the HPV16 early promoter p97, or the late promoter p670 [3]. These mRNAs are indicated above the schematic drawing of the HPV16 genome. The E2 protein affects HPV16 gene expression by inhibiting transcription from the HPV16 early promoter p97, which inhibits E6 and E7 expression [17], and by inhibiting the HPV16 early polyadenylation signal pAE, which promotes HPV16 late gene expression by inducing read-through into the HPV16 late region [22]. E2 could potentially also indirectly affect HPV16 RNA processing by activating expression of genes encoding serine/arginine rich (SR) proteins [23,24]. A direct induction of HPV16 late gene expression by E2 occurs when HPV16 affects the conformation of the polyadenylation complex that forms on pAE, thereby inhibiting pAE [22]. E2 therefore could function as a switch from HPV16 early gene expression to HPV16 late gene expression.

Figure 3

Schematic drawing of the middle and late regions of the HPV16 genome. Upper panel: splicing to HPV16 3′-splice site SA3358 followed by polyadenylation at the HPV early polyadenylation signal pAE at the early stage of the HPV16 infection. The suppressed HPV16 late 5′-splice site SD3632 is encircled [6]; Lower panel: at the late stage of the HPV16 infection the three late mRNAs L2, L1 and E4 co-exist. This is the result of partial inhibition of pAE, which causes read-through into the late region and polyadenylation at pAL to produce the L2 mRNAs. Activation of the two exclusively late splice sites SD3632 and SA5639 results in production of L1 mRNAs, while a fraction of the mRNAs that are polyadenylated at pAE produces E4 mRNAs. The E4 protein is produced at both early and late stages of the HPV16 life cycle. The regulated HPV16 late 5′-splice site SD3632 and the early polyA signal pAE are encircled in blue and HPV16 late 3′-splice site SA5639 in yellow.

Figure 4

Schematic drawing of the middle region of the HPV16 genome ranging from the HPV16 3′-splice site SA3358 to the early polyadenylation signal pAE. The suppressed HPV16 late 5′-splice site SD3632 is indicated [41]. The blow up shows the splicing enhancer that is required for splicing to SA3358 [34,35,36]. Binding sites for the splicing factors serine/argine rich splicing factors 1 and 3 (SRSF1 and SRSF3) are indicated in red [6,7,9,35,36,37]. Question marks indicate unidentified cellular proteins that bind to the splicing enhancer.

Figure 5

Schematic drawing of the middle region of the HPV16 genome with the HPV16 3′-splice site SA3358 used by both early and late mRNAs, the exclusively late 5′-splice site SD3632 and the early polyadenylation signal pAE. The blow up shows the suppressed HPV16 late 5′-splice site SD3632 with upstream splicing silencer elements [34] consisting of two AUAGUA-motifs that bind heterogenous nuclear ribonuclear protein D (hnRNP D) and two ACAC-motifs that interact with a yet unknown cellular factor [6,9,41]. Question mark indicates unidentified cellular proteins that bind to the splicing enhancer. Binding sited for hnRNP D and the intronic region of HPV16 late 5′-splice site SD3632 are in boldface, and entire SD3632 is underlined.

Figure 6

Schematic drawing of the middle region of the HPV16 genome. HPV16 3′-splice site SA3358, used by both early and late mRNAs is indicated. The exclusively late 5′-splice site SD3632 with hnRNP D binding to upstream splicing silencer elements to suppress SD3632 [41]. heterogenous nuclear ribonuclear protein C (hnRNP C), HuR, polypyrimidine tract binding protein (PTB) and Fip1 binding to the HPV16 early untranslated region (UTR) are indicated [6,9,22,43,48,51]. hnRNP H binds to sequences downstream of the HPV16 early polyadenylation signal pAE to promote polyadenylation at pAE [6,9,46,47]. Double arrow represents the interactions between hnRNP C that binds to the HPV16 early UTR, and hnRNP D that binds to the splicing silencers upstream of SD3632 [9,48]. The interaction of hnRNP C with hnRNP D activates HPV16 late 5′-splice site SD3632 and induces splicing from SD3632 to SA5639 and thereby activates production of the spliced, major late L1 mRNAs [9,48].

Figure 7

Schematic drawing of the late region of the HPV16 genome with the HPV16 3′-splice site SA5639 and the late polyadenylation signal pAL and the late untranslated region (UTR). The blow up shows the splicing silencer elements downstream of the suppressed HPV16 late 3′-splice site SA5639 [6,59,60]. These RNA elements consist of purine-rich sequences that bind hnRNP A1 [6,9,58,59,60]. Question marks indicate unidentified cellular proteins that bind to the splicing silencer. In addition to the splicing silencer elements downstream of SA5639, the HPV16 late mRNAs encode a negative regulatory element in the HPV16 late untranslated region [62]. The late UTR of many HPVs has an inhibitory function [63]. These elements consist of 5′-splice site-like sequences that bind to U1 small nuclear ribonucleoprotein (snRNP) to inhibit HPV16 late polyadenylation [64,65], and GU-rich elements that bind CUG-triplet repeat RNA binding protein (CUG-BP) [66], and other proteins [62]. Binding sites for hnRNP A1, U1 snRNP and CUG-BP are in boldface.