E2 Open Reading Frame Research Articles

HnRNP H controls alternative splicing of human papillomavirus type 16 E1, E6, E7, and E6^E7 mRNAs via GGG motifs.

The mRNAs encoding the human papillomavirus type 16 (HPV16) E6 and E7 oncogene mRNAs are subjected to extensive alternative RNA splicing at multiple regulated splice sites. One of the most extensively used 5'-splice sites in the HPV16 genome is named SD880 and is located immediately downstream of the E7 open reading frame. Here, we show that a cluster of three GGG-motifs adjacent to HPV16 SD880 interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) H that cooperates with SD880 to stimulate splicing to the upstream HPV16 3'-splice site SA742. This splice site is located in the E7 coding region and is required for the production of the HPV16 226^742 mRNA that encodes the E6^E7 fusion protein. Enhancement of HPV16 E6^E7 mRNA production by hnRNP H occurred at the expense of the intron-retained E6 mRNAs and the spliced E7 mRNAs, demonstrating that hnRNP H controls the relative levels of E6, E7, and E6^E7 proteins. Unexpectedly, overexpression of hnRNP H also promoted retention of the downstream E1 encoding intron and enhanced E1 protein production. We concluded that hnRNP H plays an important role in the HPV16 gene expression program.IMPORTANCEHere, we show that hnRNP H binds to multiple GGG-motifs downstream of human papillomavirus type 16 (HPV16) splice site SD880 and acts in concert with SD880 to promote expression of the HPV16 E6^E7 mRNA. The E6^E7 protein has been shown previously to stabilize the HPV16 E6 and E7 oncoproteins and may as such contribute to the carcinogenic properties of HPV16. In its capacity of major regulator of HPV16 oncogene expression, hnRNP H may be exploited as a target for antiviral drugs to HPV16.

The level of expression of HPV16 early transcripts is not associated with the natural history of cervical lesions.

The natural history of cervical cancer is closely linked to that of high-risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high-grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening.

Novel lambdapapillomavirus in northern sea otters Enhydra lutris kenyoni, associated with oral hyperplastic nodules.

A novel papillomavirus (PV) associated with hyperplastic nodules scattered over the muco-cutaneous border of the oral cavity of a dead, wild, subadult northern sea otter Enhydra lutris kenyoni (NSO) in 2004 in Homer, Alaska, USA, was genetically characterized. Primers for the amplification of 2 large overlapping DNA fragments that contained the complete genome of the NSO PV were designed. Sanger methodology generated sequences from which new specific primers were designed for the primer-walking approach. The NSO PV genome consists of 8085 nucleotides and contains an early region composed of E6, E7, E1, and E2 open reading frames (ORFs), an E4 ORF (contained within E2) lacking an in-frame proximal ATG start codon, an unusually long (907 nucleotide) stretch lacking any ORFs, a late region that contains the capsid genes L2 and L1, and a non-coding regulatory region (ncRR). This NSO PV has been tentatively named Enhydra lutris kenyoni PV2 (ElkPV2). Pairwise and multiple sequence alignments of the complete L1 ORF nucleotides and concatenated E1-E2-L1 amino acid sequences showed that the NSO PV is a novel PV, phylogenetically most closely related to southern sea otter PV1. The carboxy end of the E6 oncoprotein does not contain the PDZ-binding motif with a strong correlation with oncogenicity, suggesting a low-risk PV, which is in agreement with histopathological findings. However, the ElkPV2 E7 oncoprotein does contain the retinoblastoma (pRb) binding domain LXCXE (LQCYE in ElkPV2), associated with oncogenicity in some high-risk PVs. Further studies on the prevalence and clinical significance of ElkPV2 infections in NSO are needed.

A Novel Role for the Adenovirus L4 Region 22K and 33K Proteins in Adeno-Associated Virus Production.

Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 open reading frame (ORF), and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for recombinant AAV (rAAV) production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for rAAV production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.

Combining oncolytic virus and radiation therapy for cancer management

Cancer has caused a tremendous burden in developing countries. Oncolytic virus (OV) therapy is an emerging modality with the potential to be a single or combination agent with radiation therapy (RT). Following entry of OV to the cell, OV will replicate and assemble before exiting from tumor cells. Construction of OV can be done by modifying the capsid, genome, and chemical material of viruses. Irradiation will induce double-strand breaks, and further integration of OV with DNA damage response pathway will interact with the MRE11-Rad50-Nbs1 complex to regulate the mobilization of E4 open reading frame 6, protein phosphatase 2A, poly(ADP-ribose) polymerase, apoptosis-inducing factor, and topoisomerase-IIβ-binding protein 1. Degradation of DNA-dependent protein kinase catalytic subunits via human simplex virus-1-infected cell polypeptide 0 will inhibit DNA repair. OV and RT have a synergistic interaction to cause viral oncolysis and upregulation of immune response. In the clinical setting, most studies have demonstrated that OV is a safe treatment with less toxicity. Moreover, OV + RT resulted in longer median survival (62.4 vs. 37.7 weeks) in malignant glioma.

The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression.

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF.

Cellular Functions of HPV16 E5 Oncoprotein during Oncogenic Transformation.

The human papillomavirus (HPV) is recognized as the main etiologic agent associated with cervical cancer. HPVs are epitheliotropic, and the ones that infect the mucous membranes are classified into low-risk (LR) and high-risk (HR) types. LR-HPVs produce benign lesions, whereas HR-HPVs produce lesions that may progress to cancer. HR-HPV types 16 and 18 are the most frequently found in cervical cancer worldwide. E6 and E7 are the major HPV oncogenic proteins, and they have been profusely studied. Moreover, it has been shown that the HPV16 E5 (16E5) oncoprotein generates transformation, although the molecular mechanisms through which it carries out its activity have not been well defined. In contrast to E6 and E7, the E5 open reading frame is lost during the integration of the episomal HPV DNA into the cellular genome. This suggests that E5 acts at the early stages of the transformation process. In this review, we focused on the biochemical characteristics and functions of the HPV E5 oncoprotein, mainly on its association with growth factor receptors and other cellular proteins. Knowledge of the HPV E5 biology is important to understand the role of this oncoprotein in maintaining the viral cycle through the modulation of proliferation, differentiation, and apoptosis, as well as the alteration of other processes, such as survival, adhesion, migration, and invasion during early carcinogenesis. Finally, we summarized recent research that uses the E5 oncoprotein as a therapeutic target, promising a novel approach to the treatment of cervical cancer in its early stages.

Genomic and phylogenetic characterization of ChPV2, a novel goat PV closely related to the Xi-PV1 species infecting bovines

BackgroundPapillomaviruses (PVs) infecting artiodactyls are very diverse, and only second in number to PVs infecting primates. PVs associated to lesions in economically important ruminant species have been isolated from cattle and sheep.MethodsPotential PV DNA from teat lesions of a Damascus goat was isolated, cloned and sequenced. The PV genome was analyzed using bioinformatics approaches to detect open reading frames and to predict potential features of encoded proteins as well as putative regulatory elements. Sequence comparison and phylogenetic analyses using the concatenated E1E2L2L1 nucleotide and amino acid alignments was used to reveal the relationship of the new PV to the known PV diversity and its closest relevants.ResultsWe isolated and characterized the full-genome of novel Capra hircus papillomavirus. We identified the E6, E7, E1, E2, L2, L1 open reading frames with protein coding potential and putative active elements in the ChPV2 proteins and putative regulatory genome elements. Sequence similarities of L1 and phylogenetic analyses using concatenated E1E2L2L1 nucleotide and amino acid alignments suggest the classification as a new PV type designated ChPV2 with a phylogenetic position within the XiPV genus, basal to the XiPV1 species. ChPV2 is not closely related to ChPV1, the other known goat PV isolated from healthy skin, although both of them belong confidently into a clade composed of PVs infecting cervids and bovids. Interestingly, ChPV2 contains an E6 open reading frame whereas all closely related PVs do notConclusionChPV2 is a novel goat PV closely related to the Xi-PV1 species infecting bovines. Phylogenetic relationships and genome architecture of ChPV2 and closely related PV types suggest at least two independent E6 losses within the XiPV clade.

Characteristic of slow growth in cell culture of adenovirus type 54 causing nationwide outbreak epidemic keratoconjunctivitis in Japan

To characterize the virological features of adenovirus type 54 (Ad54) causing nationwide outbreak of severe epidemic keratoconjunctivitis (EKC) in Japan, we comparatively analysed the viral propagation phenotype of Ad54 and other Ads: type 37 (Ad37), 64 (Ad64), and 5 (Ad5), in A549 cells quantitatively. Laboratory investigation. We compared the growth rate of Ads using copy numbers and cytopathic effect observation during propagation in A549 cell lines. Expressions of mRNA of E1 gene were also calculated and compared. Phylogenetic analysis of the region, including putative promoter of E1 gene and E1 open reading frame (ORF), were performed. Increases in viral loads, growth rate, and viral propagation were slower for Ad54 than for other Ads. The expression level of the E1 gene per infected cell was lower for Ad54 than for other Ad types on post-infection day 1. Phylogenetic analysis of the E1 gene putative promoter and ORF revealed Ad54 was the closest to Ad type 8. The propagation of Ad54 in A549 is slow compared with Ad37, Ad64 and Ad5. This slow propagation could have been caused by slow genomic replication resulting from delayed viral entry or E1 transcription initiation. The EKC caused by Ad54 needs more attention because the slow propagation of Ad54 may contribute to prolonged disease duration.

HPV transcription in skin tumors.

BackgroundAlthough more than 95% of viral sequences found in skin tumors typically belong to human papillomaviruses (HPVs), HPV transcription has so far not been detected. As current technology allows very deep transcriptome sequencing, we examined skin tumors and precursor lesions for HPV transcription.MethodFresh frozen biopsies from 12 skin specimens (11/12 were positive for HPV DNA) were subjected to total RNA sequencing. The cervical cancer cell line CaSki was included as a positive control for HPV transcription.ResultsHPV RNA was detected and confirmed in 1/12 skin lesions at a median depth of 66 million reads per sample. One specimen was positive for HPV 110 transcripts mapping to E6, E7, E2/E4 and L2 open reading frames, as well as to a spliced E1^E4 transcript.ConclusionIn conclusion, the study revealed that a minority of skin lesions contains HPV transcripts and that HPV DNA detection does not predict HPV transcriptional activity.

Genome Plasticity in Papillomaviruses and De Novo Emergence of E5 Oncogenes.

The clinical presentations of papillomavirus (PV) infections come in many different flavors. While most PVs are part of a healthy skin microbiota and are not associated to physical lesions, other PVs cause benign lesions, and only a handful of PVs are associated to malignant transformations linked to the specific activities of the E5, E6, and E7 oncogenes. The functions and origin of E5 remain to be elucidated. These E5 open reading frames (ORFs) are present in the genomes of a few polyphyletic PV lineages, located between the early and the late viral gene cassettes. We have computationally assessed whether these E5 ORFs have a common origin and whether they display the properties of a genuine gene. Our results suggest that during the evolution of Papillomaviridae, at least four events lead to the presence of a long noncoding DNA stretch between the E2 and the L2 genes. In three of these events, the novel regions evolved coding capacity, becoming the extant E5 ORFs. We then focused on the evolution of the E5 genes in AlphaPVs infecting primates. The sharp match between the type of E5 protein encoded in AlphaPVs and the infection phenotype (cutaneous warts, genital warts, or anogenital cancers) supports the role of E5 in the differential oncogenic potential of these PVs. In our analyses, the best-supported scenario is that the five types of extant E5 proteins within the AlphaPV genomes may not have a common ancestor. However, the chemical similarities between E5s regarding amino acid composition prevent us from confidently rejecting the model of a common origin. Our evolutionary interpretation is that an originally noncoding region entered the genome of the ancestral AlphaPVs. This genetic novelty allowed to explore novel transcription potential, triggering an adaptive radiation that yielded three main viral lineages encoding for different E5 proteins, displaying distinct infection phenotypes. Overall, our results provide an evolutionary scenario for the de novo emergence of viral genes and illustrate the impact of such genotypic novelty in the phenotypic diversity of the viral infections.

Discovery, characterisation and genomic variation of six novel Gammapapillomavirus types from penile swabs in South Africa

Six novel human papillomaviruses from penile swabs were characterised. Multiple full genome clones for each novel type were generated, and complete genome sizes were: HPV211 (7253bp), HPV212 (7208bp), HPV213 (7096bp), HPV214 (7357), HPV215 (7186bp) and HPV216 (7233bp). Phylogenetically the novel papillomaviruses all clustered with Gammapapillomaviruses: HPV211 is most closely related to HPV168 (72% identity in the L1 nucleotide sequence) of the Gamma-8 species, HPV212 is most closely related to HPV144 (82.9%) of the Gamma-17 species, HPV213 is most closely related to HPV153 (71.8%) of the Gamma-13 species, HPV214 is most closely related to HPV103 (75.3%) of the Gamma-6 species, HPV215 and HPV216 are most closely related to HPV129 (76.8% and 79.2% respectively) of the Gamma-9 species. The novel HPV types demonstrated the classical genomic organisation of Gammapapillomavirusess, with seven open reading frames (ORFs) encoding five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) proteins. Typical of Gammapapillomavirusess the novel types all lacked the E5 ORF and HPV214 also lacked the E6 ORF. HPV212 had nine unique variants, HPV213 had five and HPV215 had four variants. Conserved domains observed among the novel types are the Zinc finger Binding Domain and PDZ domains. A retinoblastoma binding domain (pRB) binding domain in E7 protein was additionally identified in HPV214. This study expands the knowledge of the rapidly growing Gammapapillomavirus genus.

The role of HPV16 regulatory region methylation in viral oncogenes E6 and E7 eхpression in primary cervical cancer lesions

Background.Overexpression of the human papillomavirus oncogenes E6 and E7 is a major factor in initiation and progression of HPV-induced tumors. Inactivation of negative regulatory function of E2 protein – the main viral transcription and replication regulator – is considered to be an important mechanism leading to the viral oncogenes overexpression. It is known that the loss of E2 functions occurs due to disruption of E2 open reading frame during the viral DNA integration into a cell genome in a part of HPV-positive tumors. An alternative mechanism of E2 function blocking in tumors retained its expression can be methylation of the HPV regulatory region, since it is known that E2 is incapable to bind its methylated binding sites.The study objectiveis to analyze methylation of the HPV16 regulatory region and expression of the viral E6 and E7 oncogenes in E2 expressing or non-expressing clinical samples of cervical cancer.Results.It has been demonstrated that the level of the HPV16 URR methylation in E2-expressing lesions is significantly higher than that in non-expressing cervical cancer lesions. Demethylation of the HPV16 promoter in cervical cell line Caski is followed by decrease of the viral E6 и E7 oncogenes mRNA levels, supporting the hypothesis that methylation is necessary for effective E6 and E7 transcription and indicates on restitution of E2 regulatory function in E2-expressing cervical cancer cells.Conclusion.These data suggest that methylation of E2 binding sites in HPV16 regulatory region blocking E2 protein binding represents an important mechanism ensuring high level of the viral E6 and E7 oncogenes expression.

Phylogeny and Classification of Human Papillomavirus (HPV)16 and HPV18 Variants Based on E6 and L1 genes in Tunisian Women with Cervical Lesions

Background:High-risk human papillomavirus (HPV) types are the main etiological factors for cervical cancer. HPV16 and HPV18 are generally the most common forms associated with development of high-grade cervical lesions. This study was undertaken to identify intratypic variants of HPV16 and HPV18 among women with cervical lesions in Tunisia.Materials and Methods:DNA was extracted from cervical samples collected from 49 women. using a PureLinkTM Genomic DNA mini Kit (Invitrogen). E6 and L1 open reading frames (ORF) were amplified by PCR and viral DNA amplicons were subjected to automated sequencing using Big Dye Terminators technology (Applied Biosystems). The obtained sequences were analyzed using an appropriate software program to allow phylogenetic trees to be generated.Results:HPV16 and HPV18 were detected in 15 and 5 cases, respectively. HPV16 E6 sequences clustered with the European German lineage (A2) whereas one isolate diverged differently in the L1 region and clustered with the African sub-lineage (B1). HPV 18 E6 sequences clustered with the European sub-lineage (A1) but L1 sequences clustered as a new clade which diverged from A1-A5.Conclusions:Our results suggest that the distribution of HPV16 and HPV18 sequences in women with cervical lesions in Tunisia is mainly related to European epidemiological conditions and point to the presence of recombinant HPV forms.

HPV16 E5 is produced from an HPV16 early mRNA spliced from SD226 to SA3358

The HPV16 E5 open reading frame (ORF) is present on the majority of all alternatively spliced HPV16 mRNAs, but it is currently unknown how well it is translated into E5 protein. To identify HPV16 mRNAs that are efficiently translated into E5, we have generated cDNA plasmids expressing individual, alternatively spliced HPV16 mRNAs with the potential to produce E5. By replacing the E5 ORF with sLuc, we could quantitate sLuc and determine how well each cDNA was translated. Our results showed that the upstream E1 and E7 AUGs inhibited translation of the E5 ORF and revealed that only one HPV16 mRNA produced high levels of E5. This was an HPV16 early mRNA spliced from SD226 to SA3358. These results were confirmed in the context of the entire HPV16 genome. Taken together, our results indicate that E5 is expressed early in the HPV16 replication cycle since it is translated efficiently only by one early mRNA.

5azadC treatment upregulates miR-375 level and represses HPV16 E6 expression.

High-risk human papillomaviruses are the etiological agents of cervical cancer and HPV16 is the most oncogenic genotype. Immortalization and transformation of infected cells requires the overexpression of the two viral oncoproteins E6 and E7 following HPV DNA integration into the host cell genome. Integration often leads to the loss of the E2 open reading frame and the corresponding protein can no longer act as a transcriptional repressor on p97 promoter. Recently, it has been proposed that long control region methylation also contributes to the regulation of E6/E7 expression.To determine which epigenetic mechanism is involved in HPV16 early gene regulation, 5-aza-2′-deoxycytidine was used to demethylate Ca Ski and SiHa cell DNA. Decreased expression of E6 mRNA and protein levels was observed in both cell lines in an E2-independent manner. E6 repression was accompanied by neither a modification of the main cellular transcription factor expression involved in long control region regulation, nor by a modification of the E6 mRNA splicing pattern. In contrast, a pronounced upregulation of miR-375, known to destabilize HPV16 early viral mRNA, was observed. Finally, the use of miR-375 inhibitor definitively proved the involvement of miR-375 in E6 repression. These results highlight that cellular DNA methylation modulates HPV16 early gene expression and support a role for epigenetic events in high-risk HPV associated-carcinogenesis.

Determination of the Physical Status (Episomal/Integral) of HPV by qPCR in Esophageal Squamous Cell Carcinoma

Background: In cervical cancer, the carcinogenic mechanism of human papillomavirus (HPV) occurs through the integration of viral DNA into the host genome. This process initiates with a disruption in the E2 open reading frame (ORF) of the viral genome. Disruption of E2 ORF results in an increased expression of the viral oncoproteins, E6 and E7, by removal of E2 suppression effect on their promoters. E6 and E7 interfere with the normal cell cycle by degrading the p53 and pRb tumor suppressor proteins, respectively. Objectives: The objective of this study was to determine the physical status (episomal/integral) of HPV genome in esophageal squamous cell carcinoma (ESCC). Materials and Methods: The rate of copy numbers of E2 and E6 genes in HPV-18 and HPV-16 positive samples were analyzed by quantitative polymerase chain reaction (qPCR) in order to assess the physical status (episomal/integral) of HPV. DNA extracts from HeLa cell line were used as the positive control. Results: The E2 gene was detected in 1 sample, co-infected with HPV-16 and HPV-18. While, E6 gene was detected in all 11 HPV positive samples. The qPCR analysis showed the presence of integrated form of viral DNA in all HPV positive samples and only 1 mixed episomal-integrated form was detected. Conclusion: The presence of integrated forms of high risk HPV-16 and HPV-18 genomes might reflect a crucial process towards malignant transformation of ESCC.

Molecular archeological evidence in support of the repeated loss of a papillomavirus gene.

It is becoming clear that, in addition to gene gain, the loss of genes may be an important evolutionary mechanism for many organisms. However, gene loss is often associated with an increased mutation rate, thus quickly erasing evidence from the genome. The analysis of evolutionarily related sequences can provide empirical evidence for gene loss events. This paper analyzes the sequences of over 300 genetically distinct papillomaviruses and provides evidence for a role of gene loss during the evolution of certain papillomavirus genomes. Phylogenetic analysis suggests that the viral E6 gene was lost at least twice. Despite belonging to distant papillomaviral genera, these viruses lacking a canonical E6 protein may potentially encode a highly hydrophobic protein from an overlapping open reading frame, which we designate E10. Evolutionary pressure working on this alternative frame, may explain why, despite having lost the E6 open reading frame between 20 and 60 million years ago, evidence of an E6-like protein is conserved.

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.

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences.

Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.