Yaping Wang1,2†
Fang Chen1†
Wenjie Qu1,2Yingxin Gong1,2
Yan Wang1Limei Chen1Qi Zhou1Jiayin Mo1,2Hongwei Zhang1Lin Lin1
Tianyi Bi1,2
Xujie Wang3Jiashi Gu4Yanyun Li1,2*
Long Sui1,2*- 1Department of Gynecology and Obstetrics, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
- 2Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China
- 3Department of Obstetrics and Gynecology, Shanghai Changning Maternity and Infant Health Hospital, Shanghai, China
- 4Department of Obstetrics and Gynecology, Shanghai Pudong Hospital of Fudan University, Shanghai, China
Persistent infection with high-risk human papillomavirus (HR-HPV) is the main cause of cervical cancer. These chronic infections are characterized by high expression of the HPV E6 and E7 oncogenes and the absence of the L1 and L2 capsid proteins. The regulation of HPV gene expression plays a crucial role in both the viral life cycle and rare oncogenic events. Alternative splicing of HPV mRNA is a key mechanism in post-transcriptional regulation. Through alternative splicing, HPV mRNA is diversified into various splice isoforms with distinct coding potentials, encoding multiple proteins and influencing the expression of HPV genes. The spliced mRNAs derived from a donor splicing site within the E6 ORF and one of the different acceptor sites located in the early mRNA contain E6 truncated mRNAs, named E6*. E6* is one of the extensively studied splicing isoforms. However, the role of E6* proteins in cancer progression remains controversial. Here, we reviewed and compared the alternative splicing events occurring in the genomes of HR-HPV and LR-HPV. Recently, new HPV alternative splicing regulatory proteins have been continuously discovered, and we have updated the regulation of HPV alternative splicing. In addition, we summarized the functions of known splice isoforms from three aspects: anti-tumorigenic, tumorigenic, and other cancer-related functions, including not only E6*, but also E6^E7, E8^E2, and so on. Comprehending their contributions to cancer development enhances insights into the carcinogenic mechanisms of HPV and explores the potential utility of alternative splicing in the diagnosis and treatment of cervical cancer.
1 Introduction
Cervical cancer is the fourth most common cancer among women worldwide. In 2020, the disease resulted in over 300,000 deaths worldwide (Sung et al., 2021). HR-HPVs are the cause of the disease in most cases (Cohen et al., 2019). Hitherto, more than 400 HPV genotypes have been identified, and about 40 HPV types can infect the genital tract. Genital HPV can be divided into high-risk types (HR, which may cause invasive cancer) and low-risk types (LR, cause mainly genital warts) based on their oncogenic potential (McBride, 2022). Infection with HPV is usually transient and the majority of infections are cleared by the immune system (Stanley, 2012). In rare cases, infections with HR-HPVs may persist and lead to the development of cancer (Moscicki et al., 2012). Among approximately 15 HR-HPV types, which include HPV16, 18, 21, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82, HPV16 and HPV18 are responsible for more than 70% of global cervical cancers (Molina et al., 2024).
HPV is a non-enveloped DNA virus with a double-stranded genome containing around 8kb. The viral genome is separated by two polyadenylation (pA) signals, viral early (pAE) and viral late (pAL), into three parts: long control region (LCR), early region (E1, E2, E4, E5, E6, E7, E8) and late region (L1, L2) (Szymonowicz and Chen, 2020). The LCR contains most of the regulatory elements for viral DNA replication and transcription.
HPV infects undifferentiated basal cells through micro-wounds, activating the early promoter of episomal HPV DNA in the host nucleus to trigger transcription of early mRNA. The pre-mRNAs are polycistronic, undergo extensive alternative splicing, and are polyadenylated at the pAE site. This generates mature mRNA for early viral protein expression. As cells differentiate, the late promoter is activated and the viral life cycle enters a late stage. Similar to early gene expression, late mRNAs are generated through alternative splicing but polyadenylated at the pAL site (Burd, 2003; Doorbar et al., 2012). Therefore, the completion of the HPV life cycle and gene expression is inseparable from promoter switching, alternative splicing, and alternative polyadenylation sites. However, the small number of promoters somewhat limits the ability to fine-tune control of HPV gene expression at the transcriptional level (Bernard, 2013; Kajitani and Schwartz, 2020). In addition, the very compact genome and the absence of an internal ribosome entry site (IRES) in HPV RNA transcripts, which make all but the first ORF cannot be translated efficiently. So, HPV must evolve an efficient mode of RNA process, which can process polycistronic mRNAs for the expression of individual viral genes from the polycistronic RNA transcripts (Kozak, 1999). Besides, according to the translation scanning mechanism and 5’-cap dependent mechanism, the ribosome recognizes the strong Kozak start codon at the first ORF, initiating the translation of the first ORF. The ribosome falls off the mRNA after translation termination, so the strong Kozak start codon at the first ORF can efficiently block translation of downstream ORFs. Through splicing, the inhibition of the first start codon is removed and the downstream ORF is repositioned closer to the 5’ end of the mRNA, allowing downstream ORF expression (Kozak, 1992; Kozak, 2002; Zheng et al., 2004). The above two functions are mainly achieved by alternative splicing. So alternative splicing of HPV mRNAs following transcription is essential for the production of intact viral proteins. More importantly, relative levels of either early or late gene expression are regulated through alternative splicing, which affects the carcinogenic ability and infective ability of HPV. Therefore, even minor fluctuations in the efficacy of splicing could significantly affect the outcome of HPV infection (Johansson and Schwartz, 2013).
In recent years, the role of alternative splicing of tumor-associated viruses in the development of cancer has been increasingly emphasized, as it may offer potential targets for cancer therapy. Here, we will review and compare the alternative splicing events occurring in the genomes of HR-HPV and LR-HPV, with an update of the regulation of HPV RNA alternative splicing. In addition, many splice isoforms are produced during splicing, some of which with known coding functions. We will also summarize the function of these splice isoforms.
2 HPV alternative splicing in general
The process of removing introns from pre-mRNAs and connecting the remaining exons to produce mature mRNA is called splicing, while the different combinations of exons in the mRNA producing diversified mature mRNA are called alternative splicing (Bonnal et al., 2020). In HPVs, The mRNAs encoding E6 and E7 (Tang et al., 2006), E1 and E2 (Zheng et al., 2020a), as well as L1 and L2 mRNAs (Zhao et al., 2004; Dhanjal et al., 2015), are generated in a mutually exclusive manner from the same pre-mRNAs through alternative splicing. HPV mRNA splicing can be accomplished by an enzymatic machine termed the spliceosome inside the host cell nucleus (Will and Lührmann, 2011; Zhang et al., 2013; Graham and Faizo, 2017; Bowler and Oltean, 2019; Yang et al., 2019; Niño et al., 2022). The spliceosome is composed of 5 snRNPs (small nuclear ribonucleoproteins) (snRNPs: U1, U2, U4, U5, and U6 snRNPs) (Wahl et al., 2009; Hoskins et al., 2011). The spliceosome recognizes the junction between introns and exons by following the “GU-AG” rule and performs splicing (Faustino and Cooper, 2003; Hertel, 2008). Through alternative splicing, different mature mRNAs with different functions could be synthesized from a single gene, which increases the complexity of mRNA and the diversity of proteins (Zhang et al., 2021). The structure of spliced transcripts of different HPV types has been compiled by various laboratories (Van Doorslaer et al., 2013). HPVs’ transcription maps can be adopted from the PaVE (https://pave.niaid.nih.gov), these visions were updated in 2006 (Zheng and Baker, 2006). Later updates resulted in the current version of HPV16 (Chen et al., 2014; Yu et al., 2022) (Figure 1).
Figure 1. A schematic representation of the HPV16 genome and transcripts. The top part: linear genome, shows the eight open reading frames (ORF) (colored boxes), the three promoters (broken line arrow): p97, p670, pE8; and the early and late polyadenylation sites (thick black vertical lines): pAE and pAL; Black oval: 5’SS/splice-donor (SD). White oval: 3’SS/splice-acceptor (SA). Left lower part: early transcripts. Right lower part: late transcripts. Potential coding capacity is indicated to the right of each mRNA.
The process of alternative splicing is regulated by cis-acting elements and trans-acting factors. SR (serine and arginine-rich) proteins (Howard and Sanford, 2015) and hnRNPs (heterogeneous nuclear ribonucleoproteins) (Martinez-Contreras et al., 2007) are two essential auxiliary factors in enhancing or repressing splice site usage through the recognition of specific cis-acting RNA elements. In general, SR proteins play a positive role in splicing regulation and preferentially bind to exonic splicing enhancers (ESE) and intronic splicing enhancers (ISE). On the other hand, when hnRNPs bind to exonic splicing silencers (ESS) and intronic splicing silencers (ISS), it typically inhibits splicing (Busch and Hertel, 2012; Kędzierska and Piekiełko-Witkowska, 2017). Splicing efficiency determines the relative levels of viral proteins. Hence, tight regulation of splicing mechanisms must ensure adequate production of each HPV mRNA species and the optimal balance of viral proteins in HPV-infected and cancerous cells (Johansson and Schwartz, 2013).
3 Alternative splicing within the HR-HPV E6 and E7 gene region
3.1 Splicing events within the HR-HPV E6 and E7 gene region
The E6 protein is a high-risk factor for HPV-infected cells to become cancerous, while the E7 protein is the major driver of cell proliferation in infected cells (Roman and Munger, 2013). The combined action of both may lead to malignant transformation of the cells. In HR-HPVs, E6 and E7 genes are transcribed as a single polycistronic E6/E7 pre-mRNA from a single early promoter, which undergoes splicing to produce several transcripts. The E6E7 polycistronic pre-mRNA contains at least one donor and one acceptor splicing site that can trigger the splicing process, inducing the expression of a variety of E6 spliced transcripts termed E6* (Ajiro and Zheng, 2014). There are three 5′SS in the E6 ORF (SD226,SD221,SD174) and three 3′SS either in the E6 or E7 ORFs (SA409、SA526、SA742) in HPV16. In particular, nt 226 5’SS and nt 409 3’SS are preferentially selected for splicing. The preference for nt 226 is due to the relatively higher base-pairing affinity between U1 snRNP and nt 226 compared to other 5’SS (Ajiro et al., 2012). For the selection of 3’SS, the functional branch point sequence (BPS) at nt 385, along with the splicing enhancer upstream of nt 409 and its regulatory protein TRAP150, facilitate the preferential selection of nt 409 (Ajiro et al., 2012; Jönsson et al., 2024). BPS, located 15~40 nucleotides upstream of the 3′ss, is recognized by U2 snRNA during pre-mRNA splicing to facilitate splicing process (Brant et al., 2019a). Additionally, the proximal rule, which dictates the choice of nt 226 and nt 409 to excise the minimal intron length, is energetically most favorable as it requires the least amount of splicing energy (Reed and Maniatis, 1986). E6∗I and E6∗II, produced by splicing at SD226^SA409 and SD226^SA526, respectively, are the two main E6 isoforms expressed in cervical cancer (Chen et al., 2014; Cerasuolo et al., 2017). The expression level of E6∗II is regularly higher than that of the unspliced E6 mRNA but lower than E6*I. More evidence supports that the E7 oncoprotein is mostly produced from the translation of E6*I mRNA, perhaps because of the shorter upstream E6*I ORF being less hindering for translation initiation at the E7 ATG (Sedman et al., 1991; Tang et al., 2006; Brant et al., 2019a). In addition, splicing at SD226^SA742 and SD226^SA3358 are used to produce E6^E7 and E6*III, respectively. In other HR-HPV types, multiple splice sites and transcripts in E6 and E7 gene regions were identified. However, compared with HPV16 and HPV18, the research about other HR-HPV types is still limited. Previous reviews have summarized the known splice sites and E6/E7 mRNA variants of HR-HPV (Olmedo-Nieva et al., 2018; Zheng et al., 2022).
3.2 Functions of HPV E6/E7 splicing isoforms
Considering the differences in HPV gene expression at different stages of the lesion, scientists are dedicated to investigate the relationship between alternative splicing and lesion occurrence, and to find potential diagnosis and treatment targets. The specific HPV16 E6-associated transcription patterns and dominant transcripts changed as low-grade squamous intraepithelial lesions progressed toward cancer (Lin et al., 2015). The detection rate of the E6* I transcripts increases with the progression of SIL grades, and a further increase is observed in cervical cancer tissue (Chen et al., 2014; McFarlane et al., 2015; Brant et al., 2019b; Baba et al., 2020). Although the E6*I protein has not been detected in infected cells in vivo, it has been found that ectopic expression of E6*I has been shown to reduce tumor formation in cervical cancer xenografts in nude mouse models, indicating that the E6*I protein is biologically active in vivo (Filippova et al., 2014). Therefore, we believe that summarizing the functions of the E6*I protein is still necessary. The difficulty in detecting the E6*I protein in vivo may be due to its extremely short half-life, as documented in previous studies (Filippova et al., 2009; Paget-Bailly et al., 2019). Subsequently, more precise methods are needed to detect the E6*I protein in infected cells in vivo (Chen et al., 2014; McFarlane et al., 2015; Brant et al., 2019b). E6*I is a multi-functional protein that has been extensively investigated and can to some extent mimic E6 activity to accelerate the degradation of some PDZ-containing proteins(such as Akt, Dlg, and MAGI-1) in the absence of E6, but its role in cancer development is still controversial (Pim et al., 2009). Scholars have explored the function of E6*I from many aspects such as the p53 signaling pathway, apoptosis, cell polarity, oxidative stress, inflammatory response, and tumor resistance (Pim and Banks, 1999; Filippova et al., 2009; Pim et al., 2009; Williams et al., 2014; Artaza-Irigaray et al., 2019; Paget-Bailly et al., 2019). The results show that E6*I not only has an anti-tumor function but is also involved in the development of HPV-related cancer in some instances (Williams et al., 2014; Muñoz-Bello et al., 2018; Olmedo-Nieva et al., 2018; Artaza-Irigaray et al., 2019; Paget-Bailly et al., 2019).
E6*I can play an anti-tumor role by inhibiting p53 degradation, which may be achieved by being independent of E6 or by interfering with the oncogenic activity of the E6. HPV18 E6 indirectly promotes the expression of p14ARF through p53 degradation, while the overexpression of E6*I only induces a slight increase of the p14ARF (Vazquez-Vega et al., 2013). This result suggests that independent of E6, E6*I may affect p53 levels to prevent p53 from regulating p14ARF. On the other hand, E6*I protein can interact with full-length E6 and E3 ubiquitin ligase E6-associated protein (E6-AP) to prevent E6-mediated p53 degradation (Pim and Banks, 1999; D’Costa et al., 2012; Filippova et al., 2014). Except for the p53 pathway, the co-expression of E6 and E6*I promotes TNF-induced apoptosis (Filippova et al., 2009). While HPV16 E6 can accelerate the degradation of caspase-8, the E6*I could stabilize it by binding to caspase-8 at different sites than E6 (Filippova et al., 2007; Tungteakkhun et al., 2010; Manzo-Merino et al., 2014), which may provide a molecular explanation for the different effects between E6 and E6*I. Interestingly, HPV18 E6 and E6*I induce caspase-8 activation and its nuclear translocation, but not apoptosis. Possibly, nuclear translocation is beneficial for executing the viral life cycle or maintaining cell proliferation (Manzo-Merino et al., 2014). The effects of E6*I vary differently among different HPV types, resulting in a more intricate functional network for E6*I.
It is also important to note that E6* isoforms may cooperate with E6 in malignant progression in a manner not yet described. First, in the context of HPV-driven carcinogenesis, it has been proposed that E6*I-induced oxidative stress could cause genome instability and thereby facilitate the integration of HPV genomes into the host cell genome (Williams et al., 2011; Williams et al., 2014; Letafati et al., 2024). In line with this hypothesis, the correlation between the severity of cervical lesions and increasing levels of spliced E6*I mRNA was detected (Williams et al., 2014; Paget-Bailly et al., 2019). In addition, abnormal activation of the Wnt cell signaling pathway has been reported in HPV-related tumors (Bello et al., 2015). It was found that E6* and E6 cooperate to up-regulate TCF-4 transcriptional activity to promote the expression of Wnt target genes. Proliferation enhanced by β-catenin was increased when E6 and E6*I were co-transfected (Muñoz-Bello et al., 2018). These findings support that E6 and E6* synergistically activate the Wnt signaling pathway, thereby promoting malignant progression. A recent study demonstrated that the co-expression of E6 and E6* I promotes greater IL-6 overexpression (Artaza-Irigaray et al., 2019). E6*I may help promote a pro-inflammatory and highly proliferative microenvironment and contribute to cervical tumorigenesis. Interestingly, the relationship between E6*I and drug resistance has also been studied. The increased HPV16 E6*I can facilitate the drug-resistant phenotype, such as doxorubicin and etoposide (Wanichwatanadecha et al., 2012). These findings could provide a new perspective on the treatment of drug-resistant cervical cancer.
By investigating E6*’s function, it has been observed that E6* exhibits a seemingly paradoxical role, potentially linked to the E6/E6* pattern, yet the exact nature of its function remains an unresolved and intricate issue.
Studies on other E6 splice isoforms are limited. Regarding the differences in E6*II expression levels in the different lesion grades, conclusions were inconsistent among different studies. Some studies have found an increase of E6*II in high-grade lesions (Cricca et al., 2009; Pastuszak-Lewandoska et al., 2014), while a study has found a decrease of E6*II in high-grade lesions (McNicol et al., 1995). A study has proposed that the expression level of E6*II gene might be used as an indicator of cervical cancer severity (Pastuszak-Lewandoska et al., 2014) It should be noted that the study had a small sample size, and the results still need to be examined in larger patient cohorts. The E6*II protein was also shown to accelerate the degradation of p53 and had the opposite effect on cisplatin-induced apoptosis compared to E6*I (del Moral-Hernández et al., 2010; Vaisman et al., 2018). Consequently, it is necessary to investigate whether there is a relationship between its role in promoting apoptosis and the function of p53 degradation. Another isoform, HPV16 E6^E7, which can stabilize E6 and E7 oncoproteins via HSP90 and GRP78 (Ajiro and Zheng, 2015). E6^E7 is expressed at low levels, yet it may be a potent protein that can function well at a very low level. The functions of E6 isoforms are summarized in Table 1.
Table 1. Summary of the functions of splice isoforms.
3.3 Regulation of alternative splicing within the E6 and E7 region
The expression levels of E6, E7, and E6* proteins significantly influence the process of viral carcinogenesis, and the expression of these proteins is affected by splicing efficiency. Therefore, it is important to explore the regulatory mechanism of alternative splicing. E7 is derived from transcripts spliced within E6 ORF, and the production of E6 requires unspliced transcripts. Since E6 and E7 are both oncogenes, the regulation of the expression level of both genes must be stringent and maintained. The imbalance of E6 and E7 was found in infected cells showing senescence or apoptosis. A previous study suggested that splicing in the HPV16 E6 region is regulated by hnRNP A1 through the epidermal growth factor (EGF) pathway. It was shown that activation of the Erk1/2-kinase pathway promoted the retention of the E6 intron and the production of E6 mRNAs (Rosenberger et al., 2010). Further studies showed that hnRNP A1 and hnRNP D can bind to the splicing enhancer located in the E7-coding region of HPV16. This binding negatively regulates the splicing of 226^409, thereby promoting the production of E6 mRNAs and inhibiting the production of E7 mRNAs (Zheng et al., 2020b; Cui et al., 2022).The same conclusion was also obtained in HPV18 (233^416) (Ajiro et al., 2016). Even if hnRNP A1 is required to maintain the level of intron-retained E6 mRNA, the results from another study argues that there must be another role to control the action of hnRNP A1 and promote SA409 splicing to produce the appropriate amount of E7 (Jönsson et al., 2024). The research identified a novel splicing enhancer in the E6-coding region, located 35 nucleotides downstream of SA409. This enhancer interacts with TRAP150 to promote the splicing between SD226 and SA409, therefore ending up with more isoform of E6*I/E7 mRNA (Jönsson et al., 2024). In addition, hnRNP A2 interacts with the same splicing silencer as hnRNP A1 does to inhibit SA409 but is different from hnRNP A1 in that it redirects splicing to the downstream 3′SS (SA742) in HPV16 (Zheng et al., 2020b). Structural differences between the two proteins may explain their different effects on splicing (Zheng et al., 2020b). Figure 2A summarizes the identified splicing regulation within the E6 and E7 region. Current research has only identified some of the regulators, which are generally considered to have a singular function, that is, to either promote or inhibit splicing events. However, the true purpose of alternative splicing is to maintain a relative balance in the expression levels of E6 and E7 through these splicing events. To achieve this balance, there may be upstream regulatory mechanisms that modulate the activity intensity of factors that promote or inhibit splicing, but our understanding of these regulatory mechanisms is still quite limited at present. Exploring the upstream pathways of these regulatory factors is expected to resolve this problem.
Figure 2. Schematic presentation of identified splicing factors involved in the splicing regulation of the HPV16 genome. (A) E6/E7 region. (B) E1/E2 region. (C) L1/L2 region. Small red squares on gene regions represent splicing silencers (SS) and small green squares represent splicing enhancers (SE); inhibitive splicing factors are shown in red, enhanced splicing factors are shown in green; and the sequence of the corresponding element below the cis-regulatory elements. Black oval: 5’SS/splice-donor (SD). White oval: 3’SS/splice-acceptor (SA).
4 Alternative splicing within the HR-HPV E1 and E2 gene region
4.1 Splicing events within the E1 and E2 gene region
E1 and E2 proteins play a crucial role in the initiation and regulation of HPV replication. In addition, the E2 protein is the negative transcription regulator of E6 and E7. Integration disrupts the E2 gene, resulting in increased expression of the E6 and E7 oncoproteins and cell transformation (Bhattacharjee et al., 2022). The generation of E2-coding mRNA involves splicing, specifically a splicing event that removes the E1-coding region (Kajitani and Schwartz, 2022). Similar to E6 and E7, the generation of E1 and E2 are mutually exclusive. In the HPV16 E1 and E2 gene region, there are three donor splicing sites (SD880, SD1302 and SD3632) and three acceptor splicing sites (SA2582, SA2709 and SA3358) (Van Doorslaer et al., 2013). SD880 is the most commonly used 5′ splice site (5′SS), which is used to generate E1^E4 mRNAs (880^3358) (Doorbar et al., 1990) and E2 mRNAs (880^2582, 880^2709) (Zheng et al., 2020a), respectively. After integration, a splicing event between SD880 with a nearby acceptor splicing site in the human genome leads to the generation of the fusion transcripts, which are used to produce E6*I and E7 (Brant et al., 2019b; Liu et al., 2023).
SA3358 in HPV16, recognized as the most commonly used 3′ splice site (3′SS), is efficiently utilized during both the early and late stages of the HPV16 life cycle. This site is used to generate HPV16 early mRNAs that encode the E6 and its splice variants E6*I, E6*II, and E6*III, as well as the E7 and E5, all of which are polyadenylated at the pAE. In the late stages of the viral life cycle, the majority of late pre-mRNAs are spliced from SD880 to SA3358 and polyadenylated at the pAE site to generate E1^E4 transcripts, a small fraction is polyadenylated at the pAL site to produce L2 transcripts or spliced from SD880 to SA3358 and then from SD3632 to SA5639, where they are polyadenylated at the pAL site to produce L1 transcripts (Doorbar et al., 1990; Somberg and Schwartz, 2010). E1^E4 has been demonstrated to induce G2/M cell cycle arrest, aid virus replication, and facilitate virus release (Wilson et al., 2007; McIntosh et al., 2010; Biryukov et al., 2017; Egawa et al., 2017). Thus, the E1^E4 protein functions as biomarkers indicative for active virus infection and the associated disease severity (Doorbar, 2013). In addition to these functions, E1^E4 has been shown to exert an inhibitory effect on the phosphorylation of SRPK1, a kinase involved in the regulation of SR protein functions (Prescott et al., 2014). Therefore, E1^E4 may be involved in the regulation of alternative splicing. SA3358 may be required for the production of E6, E6*, E7, E5, as well as E1^E4 and late proteins, while E1 and E2 expression is negatively affected by the efficient use of SA3358 (Li et al., 2013a). Other sites are used at relatively low frequencies. SD1302 is mainly used to produce E8^E2 mRNA (SD1302^SA3358), the precursor mRNA of which is generated from a separate promoter within the E1 gene (Stubenrauch et al., 2000; Lace et al., 2008). E8^E2 has been found to bind to viral genomes and represses viral transcription and genome replication (Dreer et al., 2016). Shortly after the virus infection, the level of E8^E2 determines whether the infection becomes latent or productive and during the productive phase, E8^E2 levels determine how much virus is produced (Dreer et al., 2016). Thus, E8^E2 is closely related to infection outcome. SD3362 is specifically used to produce late mRNA and will be discussed in the later section. Another two acceptor splicing sites, SA2582 and SA2709, are utilized at comparatively low frequencies. SA2709 is closer to the E2 ATG than SA2582, allowing SA2709 the preferred choice for producing transcripts encoding E2 compared to the suboptimal SA2582 (Zheng et al., 2020a).
4.2 Regulation of alternative splicing within the E1 and E2 region
As the most commonly used site, the regulation of SA3358 splicing has been extensively studied. There are many identified ESE sequences downstream of SA3358 that can directly interact with RNA-binding proteins. SRSF1 and SRSF3 are two splicing positive regulatory factors that have been extensively studied, And their transcriptional activity is regulated by the level of E2 protein (Johansson and Schwartz, 2013). In undifferentiated cells, low to medium levels of E2 protein result in enhanced transcription of SRSF1 and SRSF3. The high levels of SRSF1 and SRSF3 binding to corresponding ESE sequences promote the splicing of the SA3358 and concurrently inhibit the production of L1 mRNAs. However, this effect is significantly reduced when E2 level is high (Mole et al., 2009; Klymenko et al., 2016). Given that the E2 is typically peak in the late stage, it can be inferred that moderate levels of SRSF1 and SRSF3 in terminally differentiated cells lead to the production of L1 mRNAs (Rush et al., 2005; Jia et al., 2009; Somberg and Schwartz, 2010; Ajiro et al., 2016; Klymenko et al., 2016). E2 has the greatest transactivation effect on the expression of SRSF3, so differentially expressed SRSF3 controls the papillomavirus early-to-late switch (Graham, 2016). In contrast, SRSF9 and hnRNP G have been identified as inhibitors of splicing at SA3358. Specifically, SRSF9 inhibits splicing at SA3358 while redirecting splicing to SA5639 (Somberg et al., 2011). The binding of hnRNP G to the ESE downstream of SA3358 may inhibit exon inclusion between SA3358 and SD3632 (Yu et al., 2018). Most of the mRNAs encoding L1 protein contained the sequence between SA3358 and SD3632. Therefore, the role of hnRNP G may be to prevent the premature expression of late genes. In summary, SA3358 is effectively utilized throughout the early and late phases, with its regulation being influenced by varying concentrations of E2 and splicing regulatory factors, making the regulation at this site intricate.
Although SD880 is frequently utilized, there has been little research on its regulatory mechanism. It has been shown in RNA-mediated protein pull-down assays that interactions of splicing components(U1snRNP component U1-70K) with SD880 are under the control of the Akt kinase (Kajitani et al., 2017). Further investigation is warranted to elucidate the cis-acting elements and trans-regulatory factors at this site. Furthermore, the nucleotide around this site is recurrently present at the boundaries between HPV/human sequences after HPV integration (Brant et al., 2019b; Liu et al., 2023). It is an interesting question to ask if there are regulatory elements around the site and whether integration can affect the splicing of this site. Another site, SA2709, the splicing of which is regulated by hnRNP G and hnRNP D. hnRNP G binds to a splicing enhancer sequence in the E1 region, and this binding promotes the splicing of SA2709, leading to an increase in E2 mRNA production in HPV16 (Hao et al., 2022). In addition, hnRNP G is also involved in the regulation of splicing in the E6/E7-coding region (Hao et al., 2022). The timing of hnRNP G to perform these two different functions may depend on the state of cell differentiation. However, hnRNP D inhibits the splicing of 880^2709 and promotes the generation of E1 mRNAs (Cui et al., 2022). The contrasting regulatory roles of hnRNP D and hnRNP G in the production of E1 and E2 indicate the necessity of investigating the upstream regulatory pathway of both hnRNP D and hnRNP G proteins. This exploration is crucial to elucidate how the effects of them are properly controlled to produce appropriate levels of E1 and E2. The regulatory mechanism of SD1302 and SA2582 has not yet been explored. The regulation of these splicing factors is schematic in Figure 2B.
5 Late splice sites for the production of proteins L1 and L2
5.1 Splicing events between late splice sites
L1 and L2 are viral capsid proteins whose expression is suppressed at the early stage of infection and are expressed in highly differentiated epithelial cells during the late stage to assemble virions. The absence of L1 and L2 capsid proteins allows the virus to evade the immune system and persist, so suppression of L1 and L2 gene expression is a prerequisite for cancer progression and maintenance (Bodily and Meyers, 2005; Chow et al., 2010). The production of L1 and L2 proteins requires corresponding transcripts produced by alternative splicing of late pre-mRNAs. In addition, alternative splicing may be involved in the inhibition of these two protein expressions at the early stage of infection. SD3632 and SA5639 of HPV16 are dedicated to the generation of HPV16 L1 mRNAs. They are conditionally inactivated during the early stage of the HPV16 life cycle and activated during the late stage (Kajitani and Schwartz, 2020). It is worth noting that the intronic sequence between SD3632 and SA5639 encodes L2. The full activation of these two sites inhibits L2 mRNA production. As a result, the utilization of these two sites is strictly regulated to produce L1 and L2 at the late stage.
5.2 Regulation of alternative splicing at late splice sites
At the early stage of the life cycle, the utilization of SD3632 and SA5639 is suppressed (Johansson and Schwartz, 2013; Salma et al., 2016). For one thing, SD3632 lies between SA3358 and pAE, so SD3632 silence is crucial for early mRNA expression. In addition, regulatory elements upstream of SD3632 and downstream of SA5639 inhibit the activity of two sites. Also, many regulatory proteins that interact with these elements to regulate splicing at these sites have been identified. hnRNP D, hnRNP DL, hnRNP AB, and hnRNP A2/B1 are identified as suppressors of SD3632, which interact with splicing silencer elements located upstream of SD3632 (Li et al., 2013b; Li et al., 2013c; Dhanjal et al., 2015). hnRNP A1 interacts with a splicing silencer located downstream of SA5639 in the L1-coding region, leading to the inhibition of SA5639 splicing (Zhao et al., 2004). The phosphorylated hnRNP L binds to cis-elements around SD3362, SA5639, and pAE, resulting in the repression of splicing at both sites and promoting polyadenylation at pAE. Phosphorylation of hnRNP L is mediated by Akt kinase and the inhibition of Akt kinase will lead to dephosphorylate and induce the expression of viral late genes (Kajitani et al., 2017). Therefore, it can be speculated that Akt-related pathways play an important role in the regulation of late gene splicing.
At the late stage of the virus life cycle, repressive factors are relieved, and certain RNA-binding proteins bind to the early U-rich region (eUTR) upstream of the pAE, which inhibits the activity of pAE and activates these two late sites, SD3632 and SA5639, for the expression of late proteins. In addition to these regulatory factors, DNA damage response (DDR) is also involved in the regulation of late gene splicing (Nilsson et al., 2018b). hnRNP C was recruited to the HPV16 DNA by the DDR factors, and this recruitment increased the chances of hnRNP C binding to newly synthesized mRNAs and polyadenylation factors, thereby inhibiting pAE and activating SD3632 splicing (Nilsson et al., 2018a). Strongly related to the DDR factor BCLAF1, TRAP150 responds to DNA damage by recruiting U2AF65 and enhances the late mRNA splicing, which leads to late gene expression (Nilsson et al., 2018a). As mentioned earlier, TRAP150 also enhances the splicing of E6*I. Higher staining of TRAP150 was observed in the basal and middle cell keratinocyte layers than in the upper differentiated cells (www.proteinatlas.org). Similar to many SR proteins, TRAP150’s function may be related to its expression level.
Importantly, SD3632 and SA5639 cannot be activated completely considering the necessity of L2 mRNA production, since the intronic sequence between SD3632 and SA5639 encodes L2 (Kajitani and Schwartz, 2020; Kajitani and Schwartz, 2022). The regulatory mechanisms that control the activation of the two sites to produce L1 and L2 proteins simultaneously are not well understood. Additionally, the utilization of SD3358 has been found to exert an inhibitory effect on both SD3632 and SD5639 (Li et al., 2013a). However, during the late stage of the viral life cycle, the concurrent activation of all three sites is essential (Figure 1). These enigmatic regulatory factors constitute a complex regulatory network of late gene expression. The control of late gene expression is important for the establishment of the persistence of HPV infection. Therefore, it is meaningful to identify the factors that control the expression of late genes in HPV. The regulation of these splicing factors is schematic in Figures 2B, C.
6 Splicing within LR-HPV
Compared to HR-HPVs, research on alternative splicing of LR-HPVs is very limited. The main focus was on HPV11 and HPV6, and transcription from cell lines and benign condyloma acuminatum was mapped. It is widely known the E6 transcript from LR-HPVs does not have an E6 intron and thus does not undergo RNA splicing in the E6 coding region (Mesplède et al., 2012). The transcription of the E6 mRNAs and E7 mRNAs of LR-HPV were started from two different early promoters. Notably, the splicing of the E6 intron might serve as a key event for the expression of biologically active E1 protein (Remm et al., 1999; Hubert and Laimins, 2002). However, the E6 transcripts from LR-HPVs do not undergo splicing in the E6 coding region, effective expression of E1 protein may be achieved through the use of other regulatory factors within the E6 and E7 regions (Isok-Paas et al., 2015). In the E1 and E2 gene region, three splicing donor sites SD847, SD1272, SD1459 and three splicing acceptor sites SA2622, SA3325, SA3593 have been identified, corresponding to SD880, SD1302 and SA2582, SA2709, SA3358 in HPV16, respectively. Similar to HPV16, splicing at 847^3325 is used to produce E1^E4 transcripts and 1272^3325 to E8^E2 transcripts. HPV11 E8^E2 proteins also inhibit viral DNA replication (Isok-Paas et al., 2015). SD3593 and SA5771 are used for HPV11 late protein production. In addition, the transcription pattern of HPV6 identified in HPV6-positive condyloma acuminatum samples was similar to that of HPV16 in HPV16-positive CIN2 (Chen et al., 2014). Therefore, LR-HPVs and HR-HPVs appear to have similar splicing patterns except for splicing differences in the E6E7 region. The unique splicing in the E6E7 region of HR-HPVs and enriched E6* expression in HR-HPV-related cervical cancer indicate that splicing in this region may be one of the mechanisms of HPV carcinogenesis.
7 Perspectives for further research on HPV alternative splicing
In the regulation of HPV alternative splicing, in addition to the regulatory proteins in the host cells, the viral proteins are also involved in this process. However, limited knowledge is known for the regulation of the viral proteins expression, the further understanding of which will be crucial for constructing a clearer virus-host interaction network. Current research on HPV RNA splicing is primarily focused on exploring single promoting and inhibiting mechanisms. However, the relative levels of viral proteins are essential for HPV carcinogenesis and the completion of the viral life cycle. Despite the unclear mechanisms that maintain this balance, disrupting the balance could be a promising therapeutic strategy for treating cervical lesions. Furthermore, SR proteins and hnRNPs, involved in the regulation of alternative splicing, can be used as therapeutic targets. Several splicing factor inhibitors are being tested in pre-clinical and clinical trials in other cancers. In addition, the relationship between HPV alternative splicing and tumor immunity has been noted (Li et al., 2018; Jiang et al., 2022), but more in-depth studies are still needed. Exploring the relationship between HPV RNA alternative splicing and tumor immunity will be helpful to improve the implementation of immunotherapy. At last, current research mainly focuses on HPV16 and 18, and in some areas, some non-16/18 types are more prevalent, such as HPV31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, and other probable HR-HPVs. It would be interesting to study these types of alternative splicing. Because different types have different oncogenic potentials, and the reasons for this are not entirely clear. Understanding the alternative splicing characteristics of these types can provide part of the explanation for this issue.
Author contributions
YPW: Writing – original draft, Writing – review & editing. FC: Writing – original draft, Writing – review & editing. WQ: Writing – review & editing. YG: Writing – review & editing. YW: Writing – review & editing. LC: Writing – review & editing. QZ: Writing – review & editing. JM: Writing – review & editing. HZ: Writing – review & editing. LL: Writing – review & editing. TB: Writing – review & editing. XW: Writing – review & editing. JG: Writing – review & editing. YL: Writing – review & editing. LS: Writing – review & editing.
Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by the National Natural Science Foundation of China (Grant No.82272970) and the Science and Technology Commission of Shanghai Municipality (No.22ZR1408800; No.21Y11906500).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
Ajiro, M., Jia, R., Zhang, L., Liu, X., Zheng, Z. M. (2012). Intron definition and a branch site adenosine at nt 385 control RNA splicing of HPV16 E6*I and E7 expression. PloS One 7, e46412. doi: 10.1371/journal.pone.0046412
Ajiro, M., Tang, S., Doorbar, J., Zheng, Z. M. (2016). Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements. J. Virol. 90, 9138–9152. doi: 10.1128/JVI.00965-16
Ajiro, M., Zheng, Z.-M. (2014). Oncogenes and RNA splicing of human tumor viruses. Emerging Microbes infections 3, e63–e63. doi: 10.1038/emi.2014.62
Ajiro, M., Zheng, Z. M. (2015). E6^E7, a novel splice isoform protein of human papillomavirus 16, stabilizes viral E6 and E7 oncoproteins via HSP90 and GRP78. mBio 6, e02068–e02014. doi: 10.1128/mBio.02068-14
Artaza-Irigaray, C., Molina-Pineda, A., Aguilar-Lemarroy, A., Ortiz-Lazareno, P., Limón-Toledo, L. P., Pereira-Suárez, A. L., et al. (2019). E6/E7 and E6(*) from HPV16 and HPV18 upregulate IL-6 expression independently of p53 in keratinocytes. Front. Immunol. 10, 1676. doi: 10.3389/fimmu.2019.01676
Baba, S., Taguchi, A., Kawata, A., Hara, K., Eguchi, S., Mori, M., et al. (2020). Differential expression of human papillomavirus 16-, 18-, 52-, and 58-derived transcripts in cervical intraepithelial neoplasia. Virol. J. 17, 32. doi: 10.1186/s12985-020-01306-0
Bello, J. O., Nieva, L. O., Paredes, A. C., Gonzalez, A. M., Zavaleta, L. R., Lizano, M. (2015). Regulation of the wnt/β-catenin signaling pathway by human papillomavirus E6 and E7 oncoproteins. Viruses 7, 4734–4755. doi: 10.3390/v7082842
Bernard, H. U. (2013). Regulatory elements in the viral genome. Virology 445, 197–204. doi: 10.1016/j.virol.2013.04.035
Bhattacharjee, R., Das, S. S., Biswal, S. S., Nath, A., Das, D., Basu, A., et al. (2022). Mechanistic role of HPV-associated early proteins in cervical cancer: Molecular pathways and targeted therapeutic strategies. Crit. Rev. Oncol. Hematol. 174, 103675. doi: 10.1016/j.critrevonc.2022.103675
Biryukov, J., Myers, J. C., McLaughlin-Drubin, M. E., Griffin, H. M., Milici, J., Doorbar, J., et al. (2017). Mutations in HPV18 E1^E4 impact virus capsid assembly, infectivity competence, and maturation. Viruses 9, 385. doi: 10.3390/v9120385
Bodily, J. M., Meyers, C. (2005). Genetic analysis of the human papillomavirus type 31 differentiation-dependent late promoter. J. Virol. 79, 3309–3321. doi: 10.1128/JVI.79.6.3309-3321.2005
Bonnal, S. C., López-Oreja, I., Valcárcel, J. (2020). Roles and mechanisms of alternative splicing in cancer - implications for care. Nat. Rev. Clin. Oncol. 17, 457–474. doi: 10.1038/s41571-020-0350-x
Bowler, E., Oltean, S. (2019). Alternative splicing in angiogenesis. Int. J. Mol. Sci. 20, 2067. doi: 10.3390/ijms20092067
Brant, A. C., Majerciak, V., Moreira, M. A. M., Zheng, Z. M. (2019a). HPV18 utilizes two alternative branch sites for E6*I splicing to produce E7 protein. Virol. Sin. 34, 211–221. doi: 10.1007/s12250-019-00098-0
Brant, A. C., Menezes, A. N., Felix, S. P., de Almeida, L. M., Sammeth, M., Moreira, M. A. M. (2019b). Characterization of HPV integration, viral gene expression and E6E7 alternative transcripts by RNA-Seq: A descriptive study in invasive cervical cancer. Genomics 111, 1853–1861. doi: 10.1016/j.ygeno.2018.12.008
Burd, E. M. (2003). Human papillomavirus and cervical cancer. Clin. Microbiol. Rev. 16, 1–17. doi: 10.1128/CMR.16.1.1-17.2003
Busch, A., Hertel, K. J. (2012). Evolution of SR protein and hnRNP splicing regulatory factors. Wiley Interdiscip Rev. RNA 3, 1–12. doi: 10.1002/wrna.v3.1
Cerasuolo, A., Annunziata, C., Tortora, M., Starita, N., Stellato, G., Greggi, S., et al. (2017). Comparative analysis of HPV16 gene expression profiles in cervical and in oropharyngeal squamous cell carcinoma. Oncotarget 8, 34070–34081. doi: 10.18632/oncotarget.15977
Chen, J., Xue, Y., Poidinger, M., Lim, T., Chew, S. H., Pang, C. L., et al. (2014). Mapping of HPV transcripts in four human cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic progression. Virology 462-463, 14–24. doi: 10.1016/j.virol.2014.05.026
Chow, L. T., Broker, T. R., Steinberg, B. M. (2010). The natural history of human papillomavirus infections of the mucosal epithelia. Apmis 118, 422–449. doi: 10.1111/j.1600-0463.2010.02625.x
Cohen, P. A., Jhingran, A., Oaknin, A., Denny, L. (2019). Cervical cancer. Lancet 393, 169–182. doi: 10.1016/S0140-6736(18)32470-X
Cricca, M., Venturoli, S., Leo, E., Costa, S., Musiani, M., Zerbini, M. (2009). Molecular analysis of HPV 16 E6I/E6II spliced mRNAs and correlation with the viral physical state and the grade of the cervical lesion. J. Med. Virol. 81, 1276–1282. doi: 10.1002/jmv.21496
Cui, X., Hao, C., Gong, L., Kajitani, N., Schwartz, S. (2022). HnRNP D activates production of HPV16 E1 and E6 mRNAs by promoting intron retention. Nucleic Acids Res. 50, 2782–2806. doi: 10.1093/nar/gkac132
D’Costa, Z. J., Jolly, C., Androphy, E. J., Mercer, A., Matthews, C. M., Hibma, M. H. (2012). Transcriptional repression of E-cadherin by human papillomavirus type 16 E6. PloS One 7, e48954. doi: 10.1371/journal.pone.0048954
del Moral-Hernández, O., López-Urrutia, E., Bonilla-Moreno, R., Martínez-Salazar, M., Arechaga-Ocampo, E., Berumen, J., et al. (2010). The HPV-16 E7 oncoprotein is expressed mainly from the unspliced E6/E7 transcript in cervical carcinoma C33-A cells. Arch. Virol. 155, 1959–1970. doi: 10.1007/s00705-010-0787-9
Dhanjal, S., Kajitani, N., Glahder, J., Mossberg, A. K., Johansson, C., Schwartz, S. (2015). Heterogeneous nuclear ribonucleoprotein C proteins interact with the human papillomavirus type 16 (HPV16) early 3’-untranslated region and alleviate suppression of HPV16 late L1 mRNA splicing. J. Biol. Chem. 290, 13354–13371. doi: 10.1074/jbc.M115.638098
Doorbar, J. (2013). The E4 protein; structure, function and patterns of expression. Virology 445, 80–98. doi: 10.1016/j.virol.2013.07.008
Doorbar, J., Parton, A., Hartley, K., Banks, L., Crook, T., Stanley, M., et al. (1990). Detection of novel splicing patterns in a HPV16-containing keratinocyte cell line. Virology 178, 254–262. doi: 10.1016/0042-6822(90)90401-C
Doorbar, J., Quint, W., Banks, L., Bravo, I. G., Stoler, M., Broker, T. R., et al. (2012). The biology and life-cycle of human papillomaviruses. Vaccine 30 Suppl 5, F55–F70. doi: 10.1016/j.vaccine.2012.06.083
Dreer, M., Fertey, J., van de Poel, S., Straub, E., Madlung, J., Macek, B., et al. (2016). Interaction of NCOR/SMRT repressor complexes with papillomavirus E8^E2C proteins inhibits viral replication. PloS Pathog. 12, e1005556. doi: 10.1371/journal.ppat.1005556
Egawa, N., Wang, Q., Griffin, H. M., Murakami, I., Jackson, D., Mahmood, R., et al. (2017). HPV16 and 18 genome amplification show different E4-dependence, with 16E4 enhancing E1 nuclear accumulation and replicative efficiency via its cell cycle arrest and kinase activation functions. PloS Pathog. 13, e1006282. doi: 10.1371/journal.ppat.1006282
Faustino, N. A., Cooper, T. A. (2003). Pre-mRNA splicing and human disease. Genes Dev. 17, 419–437. doi: 10.1101/gad.1048803
Filippova, M., Evans, W., Aragon, R., Filippov, V., Williams, V. M., Hong, L., et al. (2014). The small splice variant of HPV16 E6, E6, reduces tumor formation in cervical carcinoma xenografts. Virology 450-451, 153–164. doi: 10.1016/j.virol.2013.12.011
Filippova, M., Filippov, V. A., Kagoda, M., Garnett, T., Fodor, N., Duerksen-Hughes, P. J. (2009). Complexes of human papillomavirus type 16 E6 proteins form pseudo-death-inducing signaling complex structures during tumor necrosis factor-mediated apoptosis. J. Virol. 83, 210–227. doi: 10.1128/JVI.01365-08
Filippova, M., Johnson, M. M., Bautista, M., Filippov, V., Fodor, N., Tungteakkhun, S. S., et al. (2007). The large and small isoforms of human papillomavirus type 16 E6 bind to and differentially affect procaspase 8 stability and activity. J. Virol. 81, 4116–4129. doi: 10.1128/JVI.01924-06
Graham, S. V. (2016). Human papillomavirus E2 protein: linking replication, transcription, and RNA processing. J. Virol. 90, 8384–8388. doi: 10.1128/JVI.00502-16
Graham, S. V., Faizo, A. A. A. (2017). Control of human papillomavirus gene expression by alternative splicing. Virus Res. 231, 83–95. doi: 10.1016/j.virusres.2016.11.016
Hao, C., Zheng, Y., Jonsson, J., Cui, X., Yu, H., Wu, C., et al. (2022). hnRNP G/RBMX enhances HPV16 E2 mRNA splicing through a novel splicing enhancer and inhibits production of spliced E7 oncogene mRNAs. Nucleic Acids Res. 50, 3867–3891. doi: 10.1093/nar/gkac213
Hertel, K. J. (2008). Combinatorial control of exon recognition. J. Biol. Chem. 283, 1211–1215. doi: 10.1074/jbc.R700035200
Hoskins, A. A., Friedman, L. J., Gallagher, S. S., Crawford, D. J., Anderson, E. G., Wombacher, R., et al. (2011). Ordered and dynamic assembly of single spliceosomes. Science 331, 1289–1295. doi: 10.1126/science.1198830
Howard, J. M., Sanford, J. R. (2015). The RNAissance family: SR proteins as multifaceted regulators of gene expression. Wiley Interdiscip Rev. RNA 6, 93–110. doi: 10.1002/wrna.2015.6.issue-1
Hubert, W. G., Laimins, L. A. (2002). Human papillomavirus type 31 replication modes during the early phases of the viral life cycle depend on transcriptional and posttranscriptional regulation of E1 and E2 expression. J. Virol. 76, 2263–2273. doi: 10.1128/jvi.76.5.2263-2273.2002
Isok-Paas, H., Männik, A., Ustav, E., Ustav, M. (2015). The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome. Virol. J. 12, 59. doi: 10.1186/s12985-015-0292-6
Jia, R., Liu, X., Tao, M., Kruhlak, M., Guo, M., Meyers, C., et al. (2009). Control of the papillomavirus early-to-late switch by differentially expressed SRp20. J. Virol. 83, 167–180. doi: 10.1128/JVI.01719-08
Jiang, S., Zhou, Y., Ye, M., Li, X., Zhang, L., Yang, Y. (2022). Construction of an immune-related ceRNA network in cervical cancer based on HPV E6 splicing. Front. Oncol. 12, 979884. doi: 10.3389/fonc.2022.979884
Johansson, C., Schwartz, S. (2013). Regulation of human papillomavirus gene expression by splicing and polyadenylation. Nat. Rev. Microbiol. 11, 239–251. doi: 10.1038/nrmicro2984
Jönsson, J., Wang, L., Kajitani, N., Schwartz, S. (2024). A novel HPV16 splicing enhancer critical for viral oncogene expression and cell immortalization. Nucleic Acids Res. 52, 316–336. doi: 10.1093/nar/gkad1099
Kędzierska, H., Piekiełko-Witkowska, A. (2017). Splicing factors of SR and hnRNP families as regulators of apoptosis in cancer. Cancer Lett. 396, 53–65. doi: 10.1016/j.canlet.2017.03.013
Kajitani, N., Glahder, J., Wu, C., Yu, H., Nilsson, K., Schwartz, S. (2017). HnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner. Nucleic Acids Res. 45, 9654–9678. doi: 10.1093/nar/gkx606
Kajitani, N., Schwartz, S. (2020). Role of viral ribonucleoproteins in human papillomavirus type 16 gene expression. Viruses 12, 1110. doi: 10.3390/v12101110
Kajitani, N., Schwartz, S. (2022). The role of RNA-binding proteins in the processing of mRNAs produced by carcinogenic papillomaviruses. Semin. Cancer Biol. 86, 482-496. doi: 10.1016/j.semcancer.2022.02.014
Klymenko, T., Hernandez-Lopez, H., MacDonald, A. I., Bodily, J. M., Graham, S. V. (2016). Human papillomavirus E2 regulates SRSF3 (SRp20) to promote capsid protein expression in infected differentiated keratinocytes. J. Virol. 90, 5047–5058. doi: 10.1128/JVI.03073-15
Kozak, M. (1992). Regulation of translation in eukaryotic systems. Annu. Rev. Cell Biol. 8, 197–225. doi: 10.1146/annurev.cb.08.110192.001213
Kozak, M. (1999). Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187–208. doi: 10.1016/S0378-1119(99)00210-3
Kozak, M. (2002). Pushing the limits of the scanning mechanism for initiation of translation. Gene 299, 1–34. doi: 10.1016/S0378-1119(02)01056-9
Kuehner, F., Stubenrauch, F. (2022). Functions of papillomavirus E8^E2 proteins in tissue culture and in vivo. Viruses 14, 953. doi: 10.3390/v14050953
Lace, M. J., Anson, J. R., Thomas, G. S., Turek, L. P., Haugen, T. H. (2008). The E8–E2 gene product of human papillomavirus type 16 represses early transcription and replication but is dispensable for viral plasmid persistence in keratinocytes. J. Virol. 82, 10841–10853. doi: 10.1128/JVI.01481-08
Letafati, A., Taghiabadi, Z., Zafarian, N., Tajdini, R., Mondeali, M., Aboofazeli, A., et al. (2024). Emerging paradigms: unmasking the role of oxidative stress in HPV-induced carcinogenesis. Infect. Agent Cancer 19, 30. doi: 10.1186/s13027-024-00581-8
Li, Y., Cai, Q., Lin, L., Xu, C. (2018). MiR-875 and miR-3144 switch the human papillomavirus 16 E6/E6* mRNA ratio through the EGFR pathway and a direct targeting effect. Gene 679, 389–397. doi: 10.1016/j.gene.2018.09.015
Li, X., Johansson, C., Cardoso Palacios, C., Mossberg, A., Dhanjal, S., Bergvall, M., et al. (2013a). Eight nucleotide substitutions inhibit splicing to HPV-16 3’-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes. PloS One 8, e72776. doi: 10.1371/journal.pone.0072776
Li, X., Johansson, C., Glahder, J., Mossberg, A.-K., Schwartz, S. (2013b). Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs. Nucleic Acids Res. 41, 10488–10508. doi: 10.1093/nar/gkt803
Li, X., Johansson, C., Glahder, J., Mossberg, A. K., Schwartz, S. (2013c). Suppression of HPV-16 late L1 5’-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs. Nucleic Acids Res. 41, 10488–10508. doi: 10.1093/nar/gkt803
Lin, K., Lu, X., Chen, J., Zou, R., Zhang, L., Xue, X. (2015). E6-associated transcription patterns in human papilloma virus 16-positive cervical tissues. Oncol. Lett. 9, 478–482. doi: 10.3892/ol.2014.2698
Liu, M., Han, Z., Zhi, Y., Ruan, Y., Cao, G., Wang, G., et al. (2023). Long-read sequencing reveals oncogenic mechanism of HPV-human fusion transcripts in cervical cancer. Transl. Res. 253, 80–94. doi: 10.1016/j.trsl.2022.09.004
Manzo-Merino, J., Massimi, P., Lizano, M., Banks, L. (2014). The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8. Virology 450-451, 146–152. doi: 10.1016/j.virol.2013.12.013
Martinez-Contreras, R., Cloutier, P., Shkreta, L., Fisette, J. F., Revil, T., Chabot, B. (2007). hnRNP proteins and splicing control. Adv. Exp. Med. Biol. 623, 123–147. doi: 10.1007/978-0-387-77374-2_8
Martinez-Zapien, D., Ruiz, F. X., Poirson, J., Mitschler, A., Ramirez, J., Forster, A., et al. (2016). Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53. Nature 529, 541–545. doi: 10.1038/nature16481
McBride, A. A. (2022). Human papillomaviruses: diversity, infection and host interactions. Nat. Rev. Microbiol. 20, 95–108. doi: 10.1038/s41579-021-00617-5
McFarlane, M., MacDonald, A. I., Stevenson, A., Graham, S. V. (2015). Human papillomavirus 16 oncoprotein expression is controlled by the cellular splicing factor SRSF2 (SC35). J. Virol. 89, 5276–5287. doi: 10.1128/JVI.03434-14
McIntosh, P. B., Laskey, P., Sullivan, K., Davy, C., Wang, Q., Jackson, D. J., et al. (2010). E1–E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium. J. Cell Sci. 123, 2810–2822. doi: 10.1242/jcs.061978
McNicol, P., Guijon, F., Wayne, S., Hidajat, R., Paraskevas, M. (1995). Expression of human papillomavirus type 16 E6-E7 open reading frame varies quantitatively in biopsy tissue from different grades of cervical intraepithelial neoplasia. J. Clin. Microbiol. 33, 1169–1173. doi: 10.1128/jcm.33.5.1169-1173.1995
Mesplède, T., Gagnon, D., Bergeron-Labrecque, F., Azar, I., Sénéchal, H., Coutlée, F., et al. (2012). p53 degradation activity, expression, and subcellular localization of E6 proteins from 29 human papillomavirus genotypes. J. Virol. 86, 94–107. doi: 10.1128/JVI.00751-11
Mole, S., Milligan, S. G., Graham, S. V. (2009). Human papillomavirus type 16 E2 protein transcriptionally activates the promoter of a key cellular splicing factor, SF2/ASF. J. Virol. 83, 357–367. doi: 10.1128/JVI.01414-08
Molina, M. A., Steenbergen, R. D. M., Pumpe, A., Kenyon, A. N., Melchers, W. J. G. (2024). HPV integration and cervical cancer: a failed evolutionary viral trait. Trends Mol. Med. 30, 890-902. doi: 10.1016/j.molmed.2024.05.009
Moscicki, A. B., Schiffman, M., Burchell, A., Albero, G., Giuliano, A. R., Goodman, M. T., et al. (2012). Updating the natural history of human papillomavirus and anogenital cancers. Vaccine 30 Suppl 5, F24–F33. doi: 10.1016/j.vaccine.2012.05.089
Muñoz-Bello, J. O., Olmedo-Nieva, L., Castro-Muñoz, L. J., Manzo-Merino, J., Contreras-Paredes, A., González-Espinosa, C., et al. (2018). HPV-18 E6 oncoprotein and its spliced isoform E6*I regulate the wnt/β-catenin cell signaling pathway through the TCF-4 transcriptional factor. Int. J. Mol. Sci. 19, 3153. doi: 10.3390/ijms19103153
Nilsson, K., Wu, C., Kajitani, N., Yu, H., Tsimtsirakis, E., Gong, L., et al. (2018a). The DNA damage response activates HPV16 late gene expression at the level of RNA processing. Nucleic Acids Res. 46, 5029–5049. doi: 10.1093/nar/gky227
Nilsson, K., Wu, C., Schwartz, S. (2018b). Role of the DNA damage response in human papillomavirus RNA splicing and polyadenylation. Int. J. Mol. Sci. 19, 1735. doi: 10.3390/ijms19061735
Niño, C. A., Scotto di Perrotolo, R., Polo, S. (2022). Recurrent spliceosome mutations in cancer: mechanisms and consequences of aberrant splice site selection. Cancers (Basel) 14, 281. doi: 10.3390/cancers14020281
Olmedo-Nieva, L., Munoz-Bello, J. O., Contreras-Paredes, A., Lizano, M. (2018). The role of E6 spliced isoforms (E6*) in human papillomavirus-induced carcinogenesis. Viruses 10, 45. doi: 10.3390/v10010045
Paget-Bailly, P., Meznad, K., Bruyère, D., Perrard, J., Herfs, M., Jung, A. C., et al. (2019). Comparative RNA sequencing reveals that HPV16 E6 abrogates the effect of E6*I on ROS metabolism. Sci. Rep. 9, 5938. doi: 10.1038/s41598-019-42393-6
Pastuszak-Lewandoska, D., Bartosinska-Dyc, A., Migdalska-Sek, M., Czarnecka, K. H., Nawrot, E., Domanska, D., et al. (2014). HPV16 E6*II gene expression in intraepithelial cervical lesions as an indicator of neoplastic grade: a pilot study. Med. Oncol. 31, 842. doi: 10.1007/s12032-014-0842-6
Pim, D., Banks, L. (1999). HPV-18 E6*I protein modulates the E6-directed degradation of p53 by binding to full-length HPV-18 E6. Oncogene 18, 7403–7408. doi: 10.1038/sj.onc.1203134
Pim, D., Tomaic, V., Banks, L. (2009). The human papillomavirus (HPV) E6* proteins from high-risk, mucosal HPVs can direct degradation of cellular proteins in the absence of full-length E6 protein. J. Virol. 83, 9863–9874. doi: 10.1128/JVI.00539-09
Prescott, E. L., Brimacombe, C. L., Hartley, M., Bell, I., Graham, S., Roberts, S. (2014). Human papillomavirus type 1 E1^E4 protein is a potent inhibitor of the serine-arginine (SR) protein kinase SRPK1 and inhibits phosphorylation of host SR proteins and of the viral transcription and replication regulator E2. J. Virol. 88, 12599–12611. doi: 10.1128/JVI.02029-14
Reed, R., Maniatis, T. (1986). A role for exon sequences and splice-site proximity in splice-site selection. Cell 46, 681–690. doi: 10.1016/0092-8674(86)90343-0
Remm, M., Remm, A., Ustav, M. (1999). Human papillomavirus type 18 E1 protein is translated from polycistronic mRNA by a discontinuous scanning mechanism. J. Virol. 73, 3062–3070. doi: 10.1128/JVI.73.4.3062-3070.1999
Roman, A., Munger, K. (2013). The papillomavirus E7 proteins. Virology 445, 138–168. doi: 10.1016/j.virol.2013.04.013
Rosenberger, S., De-Castro Arce, J., Langbein, L., Steenbergen, R. D., Rösl, F. (2010). Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation. Proc. Natl. Acad. Sci. U.S.A. 107, 7006–7011. doi: 10.1073/pnas.1002620107
Rush, M., Zhao, X., Schwartz, S. (2005). A splicing enhancer in the E4 coding region of human papillomavirus type 16 is required for early mRNA splicing and polyadenylation as well as inhibition of premature late gene expression. J. Virol. 79, 12002–12015. doi: 10.1128/JVI.79.18.12002-12015.2005
Salma, U., Xue, M., Ali Sheikh, M. S., Guan, X., Xu, B., Zhang, A., et al. (2016). Role of transforming growth factor-β1 and smads signaling pathway in intrauterine adhesion. Mediators Inflammation 2016, 4158287. doi: 10.1155/2016/4158287
Sedman, S. A., Barbosa, M. S., Vass, W. C., Hubbert, N. L., Haas, J. A., Lowy, D. R., et al. (1991). The full-length E6 protein of human papillomavirus type 16 has transforming and trans-activating activities and cooperates with E7 to immortalize keratinocytes in culture. J. Virol. 65, 4860–4866. doi: 10.1128/jvi.65.9.4860-4866.1991
Somberg, M., Li, X., Johansson, C., Orru, B., Chang, R., Rush, M., et al. (2011). Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism. J. Gen. Virol. 92, 2411–2421. doi: 10.1099/vir.0.033183-0
Somberg, M., Schwartz, S. (2010). Multiple ASF/SF2 sites in the human papillomavirus type 16 (HPV-16) E4-coding region promote splicing to the most commonly used 3’-splice site on the HPV-16 genome. J. Virol. 84, 8219–8230. doi: 10.1128/JVI.00462-10
Stanley, M. A. (2012). Epithelial cell responses to infection with human papillomavirus. Clin. Microbiol. Rev. 25, 215–222. doi: 10.1128/CMR.05028-11
Stubenrauch, F., Hummel, M., Iftner, T., Laimins, L. A. (2000). The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes. J. Virol. 74, 1178–1186. doi: 10.1128/JVI.74.3.1178-1186.2000
Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., et al. (2021). Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 71, 209–249. doi: 10.3322/caac.21660
Szymonowicz, K. A., Chen, J. (2020). Biological and clinical aspects of HPV-related cancers. Cancer Biol. Med. 17, 864–878. doi: 10.20892/j.issn.2095-3941.2020.0370
Tang, S., Tao, M., McCoy, J. P., Jr., Zheng, Z. M. (2006). The E7 oncoprotein is translated from spliced E6*I transcripts in high-risk human papillomavirus type 16- or type 18-positive cervical cancer cell lines via translation reinitiation. J. Virol. 80, 4249–4263. doi: 10.1128/JVI.80.9.4249-4263.2006
Tungteakkhun, S. S., Filippova, M., Fodor, N., Duerksen-Hughes, P. J. (2010). The full-length isoform of human papillomavirus 16 E6 and its splice variant E6* bind to different sites on the procaspase 8 death effector domain. J. Virol. 84, 1453–1463. doi: 10.1128/JVI.01331-09
Vaisman, C. E., Del Moral-Hernandez, O., Moreno-Campuzano, S., Aréchaga-Ocampo, E., Bonilla-Moreno, R., Garcia-Aguiar, I., et al. (2018). C33-A cells transfected with E6*I or E6*II the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis. Virus Res. 247, 94–101. doi: 10.1016/j.virusres.2018.02.009
Van Doorslaer, K., Tan, Q., Xirasagar, S., Bandaru, S., Gopalan, V., Mohamoud, Y., et al. (2013). The Papillomavirus Episteme: A central resource for papillomavirus sequence data and analysis. Nucleic Acids Res. 41, D571–D578. doi: 10.1093/nar/gks984
Vazquez-Vega, S., Sanchez-Suarez, L. P., Andrade-Cruz, R., Castellanos-Juarez, E., Contreras-Paredes, A., Lizano-Soberon, M., et al. (2013). Regulation of p14ARF expression by HPV-18 E6 variants. J. Med. Virol. 85, 1215–1221. doi: 10.1002/jmv.23568
Wahl, M. C., Will, C. L., Lührmann, R. (2009). The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701–718. doi: 10.1016/j.cell.2009.02.009
Wanichwatanadecha, P., Sirisrimangkorn, S., Kaewprag, J., Ponglikitmongkol, M. (2012). Transactivation activity of human papillomavirus type 16 E6*I on aldo-keto reductase genes enhances chemoresistance in cervical cancer cells. J. Gen. Virol. 93, 1081–1092. doi: 10.1099/vir.0.038265-0
Will, C. L., Lührmann, R. (2011). Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 3, a003707. doi: 10.1101/cshperspect.a003707
Williams, V. M., Filippova, M., Filippov, V., Payne, K. J., Duerksen-Hughes, P. (2014). Human papillomavirus type 16 E6* induces oxidative stress and DNA damage. J. Virol. 88, 6751–6761. doi: 10.1128/JVI.03355-13
Williams, V. M., Filippova, M., Soto, U., Duerksen-Hughes, P. J. (2011). HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress. Future Virol. 6, 45–57. doi: 10.2217/fvl.10.73
Wilson, R., Ryan, G. B., Knight, G. L., Laimins, L. A., Roberts, S. (2007). The full-length E1E4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression. Virology 362, 453–460. doi: 10.1016/j.virol.2007.01.005
Yang, Q., Zhao, J., Zhang, W., Chen, D., Wang, Y. (2019). Aberrant alternative splicing in breast cancer. J. Mol. Cell Biol. 11, 920–929. doi: 10.1093/jmcb/mjz033
Yu, H., Gong, L., Wu, C., Nilsson, K., Li-Wang, X., Schwartz, S. (2018). hnRNP G prevents inclusion on the HPV16 L1 mRNAs of the central exon between splice sites SA3358 and SD3632. J. Gen. Virol. 99, 328–343. doi: 10.1099/jgv.0.001019
Yu, L., Majerciak, V., Zheng, Z. M. (2022). HPV16 and HPV18 genome structure, expression, and post-transcriptional regulation. Int. J. Mol. Sci. 23, 7903. doi: 10.3390/ijms23094943
Zhang, L., Li, X., Zhao, R. (2013). Structural analyses of the pre-mRNA splicing machinery. Protein Sci. 22, 677–692. doi: 10.1002/pro.v22.6
Zhang, Y., Qian, J., Gu, C., Yang, Y. (2021). Alternative splicing and cancer: a systematic review. Signal Transduct Target Ther. 6, 78. doi: 10.1038/s41392-021-00486-7
Zhao, X., Rush, M., Schwartz, S. (2004). Identification of an hnRNP A1-dependent splicing silencer in the human papillomavirus type 16 L1 coding region that prevents premature expression of the late L1 gene. J. Virol. 78, 10888–10905. doi: 10.1128/JVI.78.20.10888-10905.2004
Zheng, Z. M., Baker, C. C. (2006). Papillomavirus genome structure, expression, and post-transcriptional regulation. Front. Biosci. 11, 2286–2302. doi: 10.2741/1971
Zheng, Y., Cui, X., Nilsson, K., Yu, H., Gong, L., Wu, C., et al. (2020a). Efficient production of HPV16 E2 protein from HPV16 late mRNAs spliced from SD880 to SA2709. Virus Res. 285, 198004. doi: 10.1016/j.virusres.2020.198004
Zheng, Y., Jönsson, J., Hao, C., Shoja Chaghervand, S., Cui, X., Kajitani, N., et al. (2020b). Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNP A1) and hnRNP A2 Inhibit Splicing to Human Papillomavirus 16 Splice Site SA409 through a UAG-Containing Sequence in the E7 Coding Region. J. Virol. 94, e01509-20. doi: 10.1128/JVI.01509-20
Zheng, Y., Li, X., Jiao, Y., Wu, C. (2022). High-risk human papillomavirus oncogenic E6/E7 mRNAs splicing regulation. Front. Cell Infect. Microbiol. 12, 929666. doi: 10.3389/fcimb.2022.929666
Keywords: HPV, alternative splicing, splice sites, RBPs, splicing isoforms
Citation: Wang Y, Chen F, Qu W, Gong Y, Wang Y, Chen L, Zhou Q, Mo J, Zhang H, Lin L, Bi T, Wang X, Gu J, Li Y and Sui L (2024) Alternative splicing in the genome of HPV and its regulation. Front. Cell. Infect. Microbiol. 14:1443868. doi: 10.3389/fcimb.2024.1443868
Received: 04 June 2024; Accepted: 30 September 2024;
Published: 22 October 2024.
Edited by:
Filipe Colaco Mariz, Deutsches Krebsforschungszentrum (DKFZ), GermanyReviewed by:
Hengyi Xu, The University of Texas at Austin, United StatesStefan Schwartz, Uppsala University, Sweden
Copyright © 2024 Wang, Chen, Qu, Gong, Wang, Chen, Zhou, Mo, Zhang, Lin, Bi, Wang, Gu, Li and Sui. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yanyun Li, bGl5YW55dW5AZnVkYW4uZWR1LmNu; Long Sui, c3VpbG9uZ0BmdWRhbi5lZHUuY24=
†These authors have contributed equally to this work