Jiandong Shi

Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches

BackgroundIt is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described.MethodsHepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs.ResultsUsing human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p.ConclusionThese results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.

Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection

MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host‐virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome‐wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/ China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep‐sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav‐miR‐1‐5p and hav‐miR‐2‐5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV‐derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre‐miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post‐transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA‐like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host‐RNA virus interactions mediated by RNA virus‐derived miRNAs.—Shi, J., Sun, J., Wang, B., Wu, M., Zhang, J., Duan, Z., Wang, H., Hu, N., Hu, Y., Novel microRNA‐like viral small regulatory RNAs arising during human hepatitis A virus infection. FASEB J. 28, 4381–4393 (2014). www.fasebj.org

Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases.

Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases.

Gene expression profiles identify both MyD88-independent and MyD88-dependent pathways involved in the maturation of dendritic cells mediated by heparan sulfate: a novel adjuvant.

The traditional vaccine adjuvant research is mainly based on the trial and error method, and the mechanisms underlying the immune system stimulation remaining largely unknown. We previously demonstrated that heparan sulfate (HS), a TLR-4 ligand and endogenous danger signal, effectively enhanced humoral and cellular immune responses in mice immunized by HBsAg. This study aimed to evaluate whether HS induces better humoral immune responses against inactivated Hepatitis A or Rabies Vaccines, respectively, compared with traditional adjuvants (e.g. Alum and complete Freunds adjuvant). In order to investigate the molecular mechanisms of its adjuvanticity, the gene expression pattern of peripheral blood monocytes derived DCs (dendritic cells) stimulated with HS was analyzed at different times points. Total RNA was hybridized to Agilent SurePrint G3 Human Gene Expression 8 × 60 K one-color oligo-microarray. Through intersection analysis of the microarray results, we found that the Toll-like receptor signaling pathway was significantly activated, and NF-kB, TRAF3 and IRF7 were activated as early as 12 h, and MyD88 was activated at 48 h post-stimulation. Furthermore, the expression of the surface marker CD83 and the co-stimulatory molecules CD80 and CD86 was up-regulated as early as 24 h. Therefore, we speculated that HS-induced human monocyte-derived DC maturation may occur through both MyD88-independent and dependent pathways, but primarily through the former (TRIF pathway). These data provide an important basis for understanding the mechanisms underlying HS enhancement of the immune response.

Small-Activating RNA Can Change Nucleosome Positioning in Human Fibroblasts.

RNA activation (RNAa) is a mechanism of positive gene expression regulation mediated by small-activating RNAs (saRNAs), which target gene promoters and have been used as tools to manipulate gene expression. Studies have shown that RNAa is associated with epigenetic modifications at promoter regions; however, it is unclear whether these modifications are the cause or a consequence of RNAa. In this study, we examined changes in nucleosome repositioning and the involvement of RNA polymerase II (RNAPII) in this process. We screened saRNAs for OCT4 (POU5F1), SOX2, and NANOG, and identified several novel saRNAs. We found that nucleosome positioning was altered after saRNA treatment and that the formation of nucleosome-depleted regions (NDRs) contributed to RNAa at sites of RNAPII binding, such as the TATA box, CpG islands (CGIs), proximal enhancers, and proximal promoters. Moreover, RNAPII appeared to be bound specifically to NDRs. These results suggested that changes in nucleosome positions resulted from RNAa. We thus propose a hypothesis that targeting promoter regions using exogenous saRNAs can induce the formation of NDRs, exposing regulatory binding sites to recruit RNAPII, a key component of preinitiation complex, and leading to increased initiation of transcription.

Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97–99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

Attenuation of antigenic immunogenicity by kynurenine, a novel suppressive adjuvant

A novel therapeutic strategy is required for autoimmune diseases characterized by the production of autoantibody, because current clinical strategies have limitations. Vaccination against autoimmune diseases is a feasible strategy because vaccines induce immune response memory and the antigen specificity. However, no suitable adjuvant is available to direct the immune response toward tolerance or suppression. In the current study, we evaluated whether kynurenine (Kyn) could serve as a novel suppressive adjuvant to decrease the humoral immune responses against hepatitis A virus (HAV) in the ICR mouse model in vivo and lipopolysaccharide (LPS) in B cells in vitro. The underlying mechanisms of Kyn-mediated suppression of LPS-induced IgM responses were explored. The results showed that Kyn significantly decreased HAV immunogenicity when co-administered with HAV, and that Kyn (100 μM/1000 μM) impaired IgM generation compared with that induced by LPS alone. We also demonstrated that microRNA30b (miR30b) played a critical role in the process of Kyn-mediated suppression of IgM responses induced by LPS, and that Bach2, a transcriptional repressor of B cell terminal differentiation, was a novel target of miR30b. These findings suggest that Kyn can serve as a novel and effective suppressive adjuvant for vaccines.

Deep RNA Sequencing Reveals a Repertoire of Human Fibroblast Circular RNAs Associated with Cellular Responses to Herpes Simplex Virus 1 Infection

Background/Aims: Circular RNAs (circRNAs), a new class of regulators of gene expression, are involved in diverse physiological and pathogenic processes. However, their role in cellular responses to virus infection is yet unclear. Methods: A human lung fibroblast cell line was infected or mock infected by herpes simplex virus 1 (HSV-1). Deep RNA sequencing was used to profile the global changes in circRNAs, genes, and miRNAs following HSV-1 infection. Altered circRNAs, genes, and miRNAs were validated using quantitative reverse transcription polymerase chain reaction (RT-qPCR). An integration analysis of circRNAs, genes, and miRNAs was applied to investigate the putative function of the dysregulated circRNAs. Results: A total of 536 circRNAs, 3,885 genes, and 207 miRNAs were significantly dysregulated after HSV-1 infection. An integration analysis of circRNAs, genes, and miRNAs revealed the alleged involvement of dysregulated circRNAs in cellular responses to HSV-1 infection via the circRNA-miRNA-gene regulatory axis. These genes regulated by circRNAs were enriched to NOD-like receptor/JAK-STAT signaling pathway, and pathways of apoptosis, cell cycle progression, and cell death, all of which may be implicated in the viral pathogenesis and cellular immunity. Conclusions: These data present a comprehensive view for circRNAs induced by HSV-1 and their interplay with miRNAs and genes during HSV-1 infection, thus offering new insights into the mechanisms of interactions between HSV-1 and the host.

Molecular characterization of an imported dengue virus serotype 4 isolate from Thailand

The epidemic of dengue virus infections has spread markedly in Yunnan province of China in recent years due to an increase in the number of imported dengue cases. To the best of our knowledge, the present study is the first to report a whole genome sequence and molecular characterization of an imported DENV-4 isolate from Thailand. The current strain, 2013JH285, has an RNA genome of 10,772 nucleotides that shares 99.0% nucleotide and 99.7% amino acid sequence identity with the 2013 Thailand strain CTI2-13. Phylogenetic analysis of the whole genome sequence revealed that the 2013JH285 strain belongs to genotype I of DENV-4. Recombination analysis suggested that the 2013JH285 strain originated from inter-genotypic recombination of DENV-4 strains. The new complete DENV-4 genome sequence reported here might contribute to further understanding of the molecular epidemiology and disease surveillance of DENV-4 in China.

Unique expression signatures of circular RNAs in response to DNA tumor virus SV40 infection

Circular RNAs (circRNAs), identified as a class of widely expressed endogenous regulatory RNAs, are involved in diverse physiological and pathological processes. However, their role in viral pathogenesis and cellular antiviral response remains unexplored. In this study, a potent DNA tumor virus, simian virus 40 (SV40), was used as a model to investigate the viral influences on cellular circRNA transcriptome. Using RNA-seq, 15,241 putative circRNAs were identified de novo from 5,057 parental genes in monkey kidney–derived Vero cells. The expression of selected circRNAs was confirmed by reverse transcription-polymerase chain reaction and Sanger sequencing. Further analysis showed that most circRNAs comprised multiple exons, and most parental genes produced multiple circRNA isoforms. A total of 134 significantly dysregulated circRNAs, including 103 upregulated and 31 downregulated circRNAs, were found after SV40 infection. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that various cancer-related pathways, including p53 and Wnt pathway, could be affected by SV40 infection via the alteration of the circRNA hosting genes. Moreover, diverse cellular immune pathways, including Toll-like receptor pathway and Janus kinase–signal transducer and activator of transcription pathway, could also be affected by SV40 infection. An integrated circRNA-miRNA-gene analysis suggested the putative function of circRNAs as cellular and viral miRNA decoys to indirectly regulate the gene expression during SV40 infection. This study presented the first comprehensive expression and functional profile of circRNAs in response to SV40 infection, thus providing new insights into the mechanisms of viral pathogenesis and cellular immune response.