ABSTRACT
We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Neutralization Tests , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , SARS-CoV-2/immunology , SARS-CoV-2/genetics , Humans , Antibodies, Neutralizing/immunology , Mice , COVID-19/immunology , COVID-19/virology , COVID-19/prevention & control , Antibodies, Viral/immunology , Neutralization Tests/methods , Mice, Transgenic , Coronavirus OC43, Human/immunology , Coronavirus OC43, Human/genetics , COVID-19 Vaccines/immunology , COVID-19 Vaccines/administration & dosage , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/immunology , Chlorocebus aethiops , Vero Cells , Macaca mulattaABSTRACT
The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.
Subject(s)
DNA, Viral , Oncogene Proteins, Viral , Papillomavirus Infections , Uterine Cervical Neoplasms , Virus Integration , Humans , Female , Uterine Cervical Neoplasms/virology , Uterine Cervical Neoplasms/genetics , Virus Integration/genetics , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/virology , Papillomavirus Infections/genetics , DNA, Viral/genetics , Human papillomavirus 16/genetics , Human papillomavirus 18/genetics , Cell Line, Tumor , Oncogenes/genetics , PolyadenylationABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is a lytic RNA-binding protein. We applied BCBL-1 cells in lytic KSHV infection and performed UV cross-linking immunoprecipitation (CLIP) followed by RNA-seq of the CLIPed RNA fragments (CLIP-seq). We identified ORF57-bound transcripts from 544 host protein-coding genes. By comparing with the RNA-seq profiles from BCBL-1 cells with latent and lytic KSHV infection and from HEK293T cells with and without ORF57 expression, we identified FOS and CITED2 RNAs being two common ORF57-specific RNA targets. FOS dimerizes with JUN as a transcription factor AP-1 involved in cell proliferation, differentiation, and transformation. Knockout of the ORF57 gene from the KSHV genome led BAC16-iSLK cells incapable of FOS expression in KSHV lytic infection. The dysfunctional KSHV genome in FOS expression could be rescued by Lenti-ORF57 virus infection. ORF57 protein does not regulate FOS translation but binds to the 13-nt RNA motif near the FOS RNA 5' end and prolongs FOS mRNA half-life 7.7 times longer than it is in the absence of ORF57. This binding of ORF57 to FOS RNA is competitive to the binding of a host nuclease AEN (also referred to as ISG20L1). KSHV infection inhibits the expression of AEN, but not exosomal RNA helicase MTR4. FOS expression mediated by ORF57 inhibits AEN transcription, but transactivates RGS2, a regulator of G-protein coupled receptors. FOS binds a conserved AP-1 site in the RGS2 promoter and enhances RGS2 expression to phosphorylate AKT. Altogether, we have discovered that KSHV ORF57 specifically binds and stabilizes FOS RNA to increase FOS expression, thereby disturbing host gene expression and inducing pathogenesis during KSHV lytic infection.
ABSTRACT
Background: Lynch syndrome (LS)-associated glioblastoma (GBM) is rare in clinical practice, and simultaneous occurrence with cutaneous porokeratosis is even rarer. In this study, we analyzed the clinicopathological and genetic characteristics of LS-associated GBMs and concurrent porokeratosis, as well as evaluated the tumor immune microenvironment (TIME) of LS-associated GBMs. Methods: Immunohistochemical staining was used to confirm the histopathological diagnosis, assess MMR and PD-1/PD-L1 status, and identify immune cell subsets. FISH was used to detect amplification of EGFR and PDGFRA, and deletion of 1p/19q and CDKN2A. Targeted NGS assay analyzed somatic variants, MSI, and TMB status, while whole-exome sequencing and Sanger sequencing were carried out to analyze the germline mutations. Results: In the LS family, three members (I:1, II:1 and II:4) were affected by GBM. GBMs with loss of MSH2 and MSH6 expression displayed giant and multinucleated bizarre cells, along with mutations in ARID1A, TP53, ATM, and NF1 genes. All GBMs had TMB-H but not MSI-H. CD8+ T cells and CD163+ macrophages were abundant in each GBM tissue. The primary and recurrent GBMs of II:1 showed mesenchymal characteristics with high PD-L1 expression. The family members harbored a novel heterozygous germline mutation in MSH2 and FDPS genes, confirming the diagnosis of LS and disseminated superficial actinic porokeratosis. Conclusion: LS-associated GBM exhibits heterogeneity in clinicopathologic and molecular genetic features, as well as a suppressive TIME. The presence of MMR deficiency and TMB-H may serve as predictive factors for the response to immune checkpoint inhibitor therapy in GBMs. The identification of LS-associated GBM can provide significant benefits to both patients and their family members, including accurate diagnosis, genetic counseling, and appropriate screening or surveillance protocols. Our study serves as a reminder to clinicians and pathologists to consider the possibility of concurrent genetic syndromes in individuals or families.
ABSTRACT
Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.
Subject(s)
Processing Bodies , Stress Granules , Cytoplasmic Granules/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , RNA/metabolism , RNA Processing, Post-Transcriptional , Humans , Cell LineABSTRACT
Serine/arginine rich splicing factor 3 (SRSF3) is an important multi-functional splicing factor, and has attracted increasing attentions in the past thirty years. The importance of SRSF3 is evidenced by its impressively conserved protein sequences in all animals and alternative exon 4 which represents an autoregulatory mechanism to maintain its proper cellular expression level. New functions of SRSF3 have been continuously discovered recently, especially its oncogenic function. SRSF3 plays essential roles in many cellular processes by regulating almost all aspects of RNA biogenesis and processing of many target genes, and thus, contributes to tumorigenesis when overexpressed or disregulated. This review updates and highlights the gene, mRNA, and protein structure of SRSF3, the regulatory mechanisms of SRSF3 expression, and the characteristics of SRSF3 targets and binding sequences that contribute to SRSF3's diverse molecular and cellular functions in tumorigenesis and human diseases.
Subject(s)
Alternative Splicing , Carcinogenesis , Animals , Humans , Cell Line, Tumor , Exons , RNA Splicing Factors/metabolism , Carcinogenesis/genetics , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , Alternative Splicing/geneticsABSTRACT
SRSF3 (SRp20) is the smallest member of the serine/arginine (SR)-rich protein family. We found the annotated human SRSF3 and mouse Srsf3 RefSeq sequences are much larger than the detected SRSF3/Srsf3 RNA size by Northern blot. Mapping of RNA-seq reads from various human and mouse cell lines to the annotated SRSF3/Srsf3 gene illustrated only a partial coverage of its terminal exon 7. By 5' RACE and 3' RACE, we determined that SRSF3 gene spanning over 8422 bases and Srsf3 gene spanning over 9423 bases. SRSF3/Srsf3 gene has seven exons with exon 7 bearing two alternative polyadenylation signals (PAS). Through alternative PAS selection and exon 4 exclusion/inclusion by alternative RNA splicing, SRSF3/Srsf3 gene expresses four RNA isoforms. The major SRSF3 mRNA isoform with exon 4 exclusion by using a favorable distal PAS to encode a full-length protein is 1411 nt long (not annotated 4228 nt) and the same major mouse Srsf3 mRNA isoform is only 1295 nt (not annotated 2585 nt). The difference from the redefined RNA size of SRSF3/Srsf3 to the corresponding RefSeq sequence is at the 3' UTR region. Collectively, the redefined SRSF3/Srsf3 gene structure and expression will allow better understanding of SRSF3 functions and its regulations in health and diseases.
ABSTRACT
Persistent high-risk human papillomavirus (HR-HPV) infections cause cervical cancer and a fraction of head and neck cancer. To investigate whether HR-HPV infection might be also involved in the development of gastric cancer (GC), we developed a platform utilizing a rolling circle amplification (RCA)-based nested L1 polymerase chain reaction with Sanger sequencing to genotype the HPV DNA in cancer tissues of 361 GC and 89 oropharyngeal squamous cell carcinomas (OPSCC). HPV transcriptional activity was determined by E6/E7 mRNA expression and a 3' rapid amplification of cDNA ends was performed to identify HPV integration and expression of virus-host fusion transcripts. Ten of 361 GC, 2 of 89 OPSCC, and 1 of 22 normal adjacent tissues were HPV L1 DNA-positive. Five of the 10 HPV-positive GC were genotyped as HPV16 by sequencing and 1 of 2 GC with RCA/nested HPV16 E6/E7 DNA detection exhibited HPV16 E6/E7 mRNA. Two OPSCC displayed HPV16 L1 DNA and E6/E7 mRNA, of which 1 OPSCC tissue showed virus-host RNA fusion transcripts from an intron region of KIAA0825 gene. Together, our data reveal viral oncogene expression and/or integration in GC and OPSCC and a possible etiology role of HPV infections in gastric carcinogenesis.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Oncogene Proteins, Viral , Papillomavirus Infections , Stomach Neoplasms , Humans , Squamous Cell Carcinoma of Head and Neck , Human papillomavirus 16/genetics , Papillomavirus Infections/complications , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Human Papillomavirus Viruses , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Stomach Neoplasms/genetics , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins/genetics , RNA, Viral/genetics , RNA, Viral/analysis , Oncogenes , RNA, Messenger/genetics , DNA, Viral/genetics , DNA, Viral/analysisABSTRACT
Approximately 5% of all human cancers are attributable to human papillomavirus (HPV) infections. HPV-associated diseases and cancers remain a substantial public health and economic burden worldwide despite the availability of prophylactic HPV vaccines. Current diagnosis and treatments for HPV-associated diseases and cancers are predominantly based on cell/tissue morphological examination and/or testing for the presence of high-risk HPV types. There is a lack of robust targets/markers to improve the accuracy of diagnosis and treatments. Several naturally occurring animal papillomavirus models have been established as surrogates to study HPV pathogenesis. Among them, the Cottontail rabbit papillomavirus (CRPV) model has become known as the gold standard. This model has played a pivotal role in the successful development of vaccines now available to prevent HPV infections. Over the past eighty years, the CRPV model has been widely applied to study HPV carcinogenesis. Taking advantage of a large panel of functional mutant CRPV genomes with distinct, reproducible, and predictable phenotypes, we have gained a deeper understanding of viral-host interaction during tumor progression. In recent years, the application of genome-wide RNA-seq analysis to the CRPV model has allowed us to learn and validate changes that parallel those reported in HPV-associated cancers. In addition, we have established a selection of gene-modified rabbit lines to facilitate mechanistic studies and the development of novel therapeutic strategies. In the current review, we summarize some significant findings that have advanced our understanding of HPV pathogenesis and highlight the implication of the development of novel gene-modified rabbits to future mechanistic studies.
Subject(s)
Cottontail rabbit papillomavirus , Neoplasms , Papillomavirus Infections , Papillomavirus Vaccines , Animals , Cottontail rabbit papillomavirus/genetics , Humans , Papillomaviridae/genetics , RabbitsABSTRACT
In the published review [...].
ABSTRACT
RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. No circRNA was detectable from the PAN region. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with eight exons and seven introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF57 inhibits splicing of both K15 introns 1 and 2. ORF70/K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70/K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70/K3 locus, we further confirmed ORF57 regulation of ORF70/K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70/K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.
Subject(s)
Herpesvirus 8, Human , Repressor Proteins/genetics , Trans-Activators/genetics , DNA Replication , DNA, Viral/metabolism , Gene Expression Regulation, Viral , Genome, Viral , Herpesvirus 8, Human/physiology , RNA Splicing/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Virus Replication/geneticsABSTRACT
Glucagon-like peptide-1 (GLP-1) is easily degraded by dipeptidyl peptidase-4 (DPP-4) in the human body, limiting its therapeutic effect on type II diabetes. Therefore, improving GLP-1 receptor agonist (GLP-1RA) stability is a major obstacle for drug development. We analyzed human GLP-1, DPP-4, and GLP-1 receptor structures and designed three GLP-1RAs, which were introduced into fusion protein fragments and changed in the overall conformation. This modification effectively prevented GLP-1RAs from entering the DPP-4 active center without affecting GLP-1RAs' ability to bind to GLP-1R, the new GLP-1RA hypoglycemic effect lasting for >24 h. Through molecular modeling, molecular dynamics calculation, and simulation, possible tertiary structure models of GLP-1RAs were obtained; molecular docking with DPP-4 and GLP-1R showed access to the fusion protein. The overall conformational change of GLP-1RAs prevented DPP-4 binding, without affecting GLP-1RAs' affinity to GLP-1R. This study provides important drug design ideas for GLP-1RA development and a new example for application of structural biology-based protein design in drug development.
ABSTRACT
Human papillomaviruses (HPV) are a group of small non-enveloped DNA viruses whose infection causes benign tumors or cancers. HPV16 and HPV18, the two most common high-risk HPVs, are responsible for ~70% of all HPV-related cervical cancers and head and neck cancers. The expression of the HPV genome is highly dependent on cell differentiation and is strictly regulated at the transcriptional and post-transcriptional levels. Both HPV early and late transcripts differentially expressed in the infected cells are intron-containing bicistronic or polycistronic RNAs bearing more than one open reading frame (ORF), because of usage of alternative viral promoters and two alternative viral RNA polyadenylation signals. Papillomaviruses proficiently engage alternative RNA splicing to express individual ORFs from the bicistronic or polycistronic RNA transcripts. In this review, we discuss the genome structures and the updated transcription maps of HPV16 and HPV18, and the latest research advances in understanding RNA cis-elements, intron branch point sequences, and RNA-binding proteins in the regulation of viral RNA processing. Moreover, we briefly discuss the epigenetic modifications, including DNA methylation and possible APOBEC-mediated genome editing in HPV infections and carcinogenesis.
Subject(s)
Genome, Viral , Human papillomavirus 16 , Human papillomavirus 18 , Oncogene Proteins, Viral , Alternative Splicing , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Human papillomavirus 18/genetics , Human papillomavirus 18/metabolism , Humans , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Papillomavirus Infections/genetics , RNA Processing, Post-Transcriptional , RNA, Viral/genetics , RNA, Viral/metabolismABSTRACT
Kaposi sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with CHD4 and ADNP proteins, components of the cellular ChAHP complex. The CHD4 and ADNP proteins occupy the 5'-region of the highly inducible lncRNAs and terminal repeats of the KSHV genome together with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation sequesters CHD4 from the KSHV genome, which is also accompanied by detachment of KSHV episomes from host chromosome docking sites. We propose a model in which robust KSHV lncRNA expression determines the latency-lytic decision by regulating LANA/CHD4 binding to KSHV episomes.
Subject(s)
Herpesvirus 8, Human , RNA, Long Noncoding , Sarcoma, Kaposi , Antigens, Viral/genetics , Antigens, Viral/metabolism , Chromosomes/metabolism , Herpesvirus 8, Human/genetics , Humans , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Plasmids , RNA, Long Noncoding/genetics , Tumor Microenvironment , Virus Latency/geneticsABSTRACT
Human papillomavirus type 16 (HPV16) E7 oncoprotein plays an essential role in cervical carcinogenesis and is encoded predominantly by an E6*I mRNA through alternative RNA splicing of a P97 promoter-transcribed bicistronic E6E7 pre-mRNA. Recently, an HPV16 circular RNA, circE7, was detected in two HPV16-positive cervical cancer cell lines, CaSki and SiHa. It was generated through back-splicing of the E6E7 pre-mRNA. The reported findings showed that, because viral E6*I RNA was nuclear, E7 was mainly translated from the cytoplasmic circE7, and knockdown of circE7 in CaSki cells led to reduction of E7 oncoprotein, cell proliferation, and xenograft tumor formation. We have reanalyzed the published data, conducted detailed experiments, and found that the circE7 in CaSki cells is only 0.4 copies per cell, which is â¼1,640-fold lower than E6*I RNA and also barely detectable from two W12 subclone cell lines, 20861 (integrated HPV16) and 20863 (extrachromosomal HPV16) cells derived from a low-grade cervical lesion. We also determined HPV16 E6*I and E6*II RNAs in CaSki cells are mainly cytoplasmic in cell fractionation analyses, as reported in other studies. We further demonstrated that the claimed circE7 functions in the published report have resulted from off-target effects on E6*I RNA by the circE7 small interfering RNAs used in the reported study. IMPORTANCE RNA back-splicing is a rare splicing event accounting for <1% of canonical RNA splicing and, thus, is thought to have little or no biological significance. Recently, circular RNAs (circRNAs) from RNA back-splicing have been found widely in cells and tissues and may have a role in modulating RNA transcription, splicing, and interference and antiviral innate immunity. A recent report claimed that the predominant HPV16 E6*I RNA was nuclear and unable to encode E7. Rather, a viral circE7 was responsible for translating the oncoprotein E7 in CaSki cells, a cervical cancer cell line. However, we found that both HPV16 E6*I and circE7 RNAs in CaSki cells are primarily cytoplasmic and that the copy number of viral E6*I RNA is 656 copies per cell, whereas the viral circE7 is only 0.4 copies per cell. Most importantly, we found that the claimed circE7 function resulted from off-target effect on viral E6*I RNA by the small interfering RNA (siRNA) si-circE7 designed to knock down the back-spliced circE7 RNA.
Subject(s)
Oncogene Proteins, Viral , Uterine Cervical Neoplasms , Female , Humans , RNA, Circular/metabolism , Oncogene Proteins, Viral/genetics , Human papillomavirus 16/genetics , Human Papillomavirus Viruses , Uterine Cervical Neoplasms/genetics , RNA Precursors/metabolism , Repressor Proteins/genetics , Papillomavirus E7 Proteins/genetics , RNA, Small Interfering/genetics , Cell Line, TumorABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding posttranscriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57 cross-linking immunoprecipitation (CLIP) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIP RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host noncoding and protein-coding mRNAs. We found that ORF57 binds and regulates expression of a subset of host long noncoding RNAs (lncRNAs), including LINC00324, LINC00355, and LINC00839, which are involved in cell growth. ORF57 binds small nucleolar RNAs (snoRNAs) responsible for 18S and 28S rRNA modifications but does not interact with fibrillarin or NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57 RNA immunoprecipitation (RIP)-snoRNA array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57 RIP and Northern blot analyses using a 32P-labeled oligonucleotide probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligonucleotides from the rRNA regions that ORF57 binds for oligonucleotide pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and that its association with polysomes increases PABPC1 binding to polysomes but prevents Ago2 association with polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. IMPORTANCE As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus-infected cells. In this study, ORF57 was found to interact with many host noncoding RNAs, including lncRNAs, snoRNAs, and rRNAs, to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the RNA motifs identified by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins and with rRNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4 but does not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC1 but prevents Ago2 association with polysomes. Data provide compelling evidence on how ORF57 in KSHV-infected cells might regulate protein synthesis by blocking Ago2's hostile activities on translation.
Subject(s)
Herpesviridae Infections/genetics , Herpesviridae Infections/virology , Herpesvirus 8, Human/physiology , Host-Pathogen Interactions/genetics , Polyribosomes/metabolism , RNA, Untranslated/genetics , Viral Regulatory and Accessory Proteins/metabolism , Gene Expression Regulation , Gene Expression Regulation, Viral , Genome-Wide Association Study , Herpesviridae Infections/metabolism , High-Throughput Nucleotide Sequencing , Humans , Nucleic Acid Conformation , Protein Binding , RNA-Binding Proteins/metabolism , Virus ReplicationABSTRACT
High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6-interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. IMPLICATIONS: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.