Subgenomic flaviviral RNAs: what do we know after the first decade of research

The common feature of flaviviral infection is the accumulation of abundant virus-derived noncoding RNA, named flaviviral subgenomic RNA (sfRNA) in infected cells. This RNA represents a product of incomplete degradation of viral genomic RNA by the cellular 5′-3′ exoribonuclease XRN1 that stalls at the conserved highly structured elements in the 3′ untranslated region (UTR). This mechanism of sfRNA generation was discovered a decade ago and since then sfRNA has been a focus of intense research. The ability of flaviviruses to produce sfRNA was shown to be evolutionary conserved in all members of Flavivirus genus. Mutations in the 3′UTR that affect production of sfRNAs and their interactions with host factors showed that sfRNAs are responsible for viral pathogenicity, host adaptation, and emergence of new pathogenic strains. RNA structural elements required for XRN1 stalling have been elucidated and the role of sfRNAs in inhibiting host antiviral responses in arthropod and vertebrate hosts has been demonstrated. Some molecular mechanisms determining these properties of sfRNA have been recently characterized, while other aspects of sfRNA functions remain an open avenue for future research. In this review we summarise the current state of knowledge on the mechanisms of generation and functional roles of sfRNAs in the life cycle of flaviviruses and highlight the gaps in our knowledge to be addressed in the future

Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells

Aim. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and post-translational. We concentrated our attention on the transcriptional level of regulation. Methods. Transient transfection of Me45 melanoma cells with constructs containing the luciferase gene under the control of complete and truncated GSTP1 promoter was utilized to identify a role of different promoter regions in regulation of the gene transcription in Me45 cells. To identify the transcription factors, interacting with the GSTP1 promoter sites, the competitive EMSA and super shift assay were applied. Results. GSTP1 transcription in Me45 cells is positively regulated by binding NF-kB to the cognate site and ERb in complex with unknown protein to the ARE site; the complex of ERb with c-Fos negatively regulates the gene expression via CRE site. The interaction of c-Fos/ERb with GSTP1 CRE site and indirect interaction of ERb with GSTP1 ARE were identified. Conclusions. The positive regulation of the human GSTP1 gene in Me45 melanoma cells is exerted via NF-kB and ARE sites and the negative one via CRE site of the promoter. ERb is indirectly involved in the regulation of GSTP1 transcription. It is bound via c-Fos with CRE site and via unknown protein with ARE site

Injection site vaccinology of a recombinant vaccinia-based vector reveals diverse innate immune signatures

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design.Jessamine E. Hazlewood, Troy Dumenil, Thuy T. Le, Andrii Slonchak, Stephen H. Kazakoff, Ann-Marie Patch ... et al

Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

Contains fulltext : 171518.PDF (publisher's version ) (Open Access)In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species

Structure and functions of glutathione S-transferase Pl-1

Glutathione S-transferase Pl-1 (GSTP1-1) is a multifunctional enzyme which protects the cell from the influence of genotoxic factors and apop-tosis. Contemporary knowledge about the GSTP1 molecule structure and the enzyme functions are summarized in this review. Also PI isoform is compared to other glutathione S-transferases and structure-function relationships are emphasized. Novel functions of GSTP1 in cell signaling modulation, NO storage and metabolism are highlighted in this paper

Functional non-coding RNAs derived from the flavivirus 3' untranslated region

Flaviviruses are single-stranded positive sense RNA enveloped viruses. The flavivirus genus includes important human pathogens such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and Murray Valley encephalitis virus (MVEV). In addition to the viral proteins and viral genomic RNA, flaviviruses produce at least two functional non-coding RNAs derived from the 3′ untranslated region (3′UTR), the subgenomic flavivirus RNA (sfRNA) and a putative WNV miRNA (KUN-miR-1). In this review we summarize published data from studies with WNV, YFV, DENV, JEV, and MVEV on sfRNA production following incomplete degradation of the viral genomic RNA by the cellular 5′–3′ exoribonuclease 1 (XRN1), RNA structural elements involved in stalling XRN1 to generate sfRNA, and functions of sfRNA in modulating cellular mRNA decay and RNAi pathways as well as in modulating anti-viral type I interferon response. In addition, we also summarize data on the mechanisms of biogenesis of 3′UTR-derived KUN-miR-1 and its function in WNV replication in mosquito host, along with recent findings on a discovery of a second potential flaviviral miRNA vsRNA5, derived from the 3′UTR of DENV. This review thus summarizes the known mechanisms of generation and the functions of flaviviral 3′UTR-derived non-coding RNAs

Transcription regulation in differential expression of the human GSTP1 gene in breast and choriocarcinoma cells

Glutathione S-transferase P1 is a major phase II detoxification enzyme in most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. GSTP1 gene transcription is regulated by promoter methylation and by transcription factors. To elucidate the mechanisms responsible for the different levels of GSTP1 expression observed in Hbl-100 and BeWo cells we utilized truncated promoter constructs to compare the functional role of different promoter elements. We also identified transcription factors binding the responsive elements by electrophoretic mobility shift assay. The applied approaches provided the evidence that binding of transcription factors to ARE, CRE and NF-kappaB sites are responsible for the cell specific levels of GSTP1 expression in Hbl-100 and BeWo cells. It was also indicated that partial promoter methylation occurs in BeWo cells

Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells

Aim. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and post-translational. We concentrated our attention on the transcriptional level of regulation. Methods. Transient transfection of Me45 melanoma cells with constructs containing the luciferase gene under the control of complete and truncated GSTP1 promoter was utilized to identify a role of different promoter regions in regulation of the gene transcription in Me45 cells. To identify the transcription factors, interacting with the GSTP1 promoter sites, the competitive EMSA and super shift assay were applied. Results. GSTP1 transcription in Me45 cells is positively regulated by binding NF-κB to the cognate site and ERβin complex with unknown protein to the ARE site; the complex of ERβ with c-Fos negatively regulates the gene expression via CRE site. The interaction of c-Fos/ERβ with GSTP1 CRE site and indirect interaction of ERβ with GSTP1 ARE were identified. Conclusions. The positive regulation of the human GSTP1 gene in Me45 melanoma cells is exerted via NF-κB and ARE sites and the negative one via CRE site of the promoter. ERis indirectly involved in the regulation of GSTP1 transcription. It is bound via c-Fos with CRE site and via unknown protein with ARE site

Human miRNA miR-532-5p exhibits antiviral activity against West Nile virus via suppression of host genes SESTD1 and TAB3 required for virus replication

West Nile virus (WNV) is a mosquito-transmitted flavivirus that naturally circulates between mosquitos and birds but can also infect humans, causing severe neurological disease. The early host response to WNV infection in vertebrates primarily relies on the type I interferon pathway; however, recent studies suggest that microRNAs (miRNAs) may also play a notable role. In this study, we assessed the role of host miRNAs in response to WNV infection in human cells. We employed small RNA sequencing (RNA-seq) analysis to determine changes in the expression of host miRNAs in HEK293 cells infected with an Australian strain of WNV, Kunjin (WNVKUN), and identified a number of host miRNAs differentially expressed in response to infection. Three of these miRNAs were confirmed to be significantly upregulated in infected cells by quantitative reverse transcription (qRT)-PCR and Northern blot analyses, and one of them, miR-532-5p, exhibited a significant antiviral effect against WNVKUN infection. We have demonstrated that miR-532-5p targets and downregulates expression of the host genes SESTD1 and TAB3 in human cells. Small interfering RNA (siRNA) depletion studies showed that both SESTD1 and TAB3 were required for efficient WNVKUN replication. We also demonstrated upregulation of mir-532-5p expression and a corresponding decrease in the expression of its targets, SESTD1 and TAB3, in the brains of WNVKUN-infected mice. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent a host antiviral response aimed at limiting WNVKUN infection and highlight the important role of miRNAs in controlling RNA virus infections in mammalian hosts