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

Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) data supporting ISF 3' UTR pape

Noncoding RNA of Zika Virus Affects Interplay between Wnt-Signaling and Pro-Apoptotic Pathways in the Developing Brain Tissue

Zika virus (ZIKV) has a unique ability among flaviviruses to cross the placental barrier and infect the fetal brain causing severe abnormalities of neurodevelopment known collectively as congenital Zika syndrome. In our recent study, we demonstrated that the viral noncoding RNA (subgenomic flaviviral RNA, sfRNA) of the Zika virus induces apoptosis of neural progenitors and is required for ZIKV pathogenesis in the developing brain. Herein, we expanded on our initial findings and identified biological processes and signaling pathways affected by the production of ZIKV sfRNA in the developing brain tissue. We employed 3D brain organoids generated from induced human pluripotent stem cells (ihPSC) as an ex vivo model of viral infection in the developing brain and utilized wild type (WT) ZIKV (producing sfRNA) and mutant ZIKV (deficient in the production of sfRNA). Global transcriptome profiling by RNA-Seq revealed that the production of sfRNA affects the expression of >1000 genes. We uncovered that in addition to the activation of pro-apoptotic pathways, organoids infected with sfRNA-producing WT, but not sfRNA-deficient mutant ZIKV, which exhibited a strong down-regulation of genes involved in signaling pathways that control neuron differentiation and brain development, indicating the requirement of sfRNA for the suppression of neurodevelopment associated with the ZIKV infection. Using gene set enrichment analysis and gene network reconstruction, we demonstrated that the effect of sfRNA on pathways that control brain development occurs via crosstalk between Wnt-signaling and proapoptotic pathways

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

The Flavivirus Non-Structural Protein 5 (NS5): Structure, Functions, and Targeting for Development of Vaccines and Therapeutics

Flaviviruses, including dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), yellow fever (YFV), and tick-borne encephalitis (TBEV) viruses, pose a significant global emerging threat. With their potential to cause widespread outbreaks and severe health complications, the development of effective vaccines and antiviral therapeutics is imperative. The flaviviral non-structural protein 5 (NS5) is a highly conserved and multifunctional protein that is crucial for viral replication, and the NS5 protein of many flaviviruses has been shown to be a potent inhibitor of interferon (IFN) signalling. In this review, we discuss the functions of NS5, diverse NS5-mediated strategies adopted by flaviviruses to evade the host antiviral response, and how NS5 can be a target for the development of vaccines and antiviral therapeutics

Archivo adicional 4: La infección por el virus del Nilo Occidental y el tratamiento con interferón alfa alteran el espectro y los niveles de los ARN codificantes y no codificantes del huésped secretados en vesículas extracelulares

Table S1. EV miRNAs significantly (FDR1) altered in response to WNV infection comparing to mock EVs. Table S2. EV miRNAs significantly (FDR1) altered in response to IFN-alpha treatment comparing to mock EVs. Table S3. EV sncRNAs significantly (FDR1) altered in response to WNV infection comparing to mock EVs. Table S4. EV sncRNAs significantly (FDR1) altered in response to IFN-alpha treatment comparing to mock EVs. Table S5. EV mRNAs significantly (FDR1) altered in response to WNV infection comparing to mock EVs. Table S6. EV mRNAs significantly (FDR1) altered in response to IFN-alpha treatment comparing to mock EVs. Table S7. GO terms associated with mRNAs exhibiting decreased levels in EVs secreted by WNV infected A549 cells. Table S8. GO terms associated with mRNAs exhibiting decreased levels in EVs secreted by IFN-alpha treated A549 cell. Table S9. PCR primers used in the study. (XLSX 349 kb)Tabla S1. Los miRNAs de las VE se alteraron significativamente (FDR1) en respuesta a la infección por el VNO en comparación con las VE de prueba. Tabla S2. MiRNAs de EV significativamente (FDR1) alterados en respuesta al tratamiento con IFN-alfa en comparación con EVs de prueba. Tabla S3. Los sncRNAs de las VE alterados significativamente (FDR1) en respuesta a la infección por el VNO en comparación con las VE de prueba. Tabla S4. Los sncRNAs de las VE alterados significativamente (FDR1) en respuesta al tratamiento con IFN-alfa en comparación con las VE de prueba. Tabla S5. ARNm de las VE significativamente (FDR1) alterados en respuesta a la infección por el VNO en comparación con las VE de prueba. Tabla S6. ARNm de EV significativamente (FDR1) alterados en respuesta al tratamiento con IFN-alfa en comparación con EVs de prueba. Tabla S7. Términos GO asociados con los mRNAs que muestran niveles reducidos en las EVs secretadas por las células A549 infectadas por el VNO. Tabla S8. Términos GO asociados con los ARNm que muestran niveles reducidos en las VE secretadas por las células A549 tratadas con IFN-alfa. Tabla S9. Cebadores de PCR utilizados en el estudio. (XLSX 349 kb)Fil: Slonchak, Andrii. University of Queensland; Australia.Fil: Clarke, Brian. Universidad de Arizona; Estados Unidos.Fil: Mackenzie, Jason. University of Queensland; Australia.Fil: Amarilla, Leonardo D. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.Fil: Amarilla, Leonardo D. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto Multidisciplinario de Biología Vegetal; Argentina.Fil: Setoh, Yin. University of Queensland; Australia.Fil: Khromykh, Alexander. University of Queensland; Australia

Archivo adicional 4: La infección por el virus del Nilo Occidental y el tratamiento con interferón alfa alteran el espectro y los niveles de los ARN codificantes y no codificantes del huésped secretados en vesículas extracelulares

Table S1. EV miRNAs significantly (FDR1) altered in response to WNV infection comparing to mock EVs. Table S2. EV miRNAs significantly (FDR1) altered in response to IFN-alpha treatment comparing to mock EVs. Table S3. EV sncRNAs significantly (FDR1) altered in response to WNV infection comparing to mock EVs. Table S4. EV sncRNAs significantly (FDR1) altered in response to IFN-alpha treatment comparing to mock EVs. Table S5. EV mRNAs significantly (FDR1) altered in response to WNV infection comparing to mock EVs. Table S6. EV mRNAs significantly (FDR1) altered in response to IFN-alpha treatment comparing to mock EVs. Table S7. GO terms associated with mRNAs exhibiting decreased levels in EVs secreted by WNV infected A549 cells. Table S8. GO terms associated with mRNAs exhibiting decreased levels in EVs secreted by IFN-alpha treated A549 cell. Table S9. PCR primers used in the study. (XLSX 349 kb)Tabla S1. Los miRNAs de las VE se alteraron significativamente (FDR1) en respuesta a la infección por el VNO en comparación con las VE de prueba. Tabla S2. MiRNAs de EV significativamente (FDR1) alterados en respuesta al tratamiento con IFN-alfa en comparación con EVs de prueba. Tabla S3. Los sncRNAs de las VE alterados significativamente (FDR1) en respuesta a la infección por el VNO en comparación con las VE de prueba. Tabla S4. Los sncRNAs de las VE alterados significativamente (FDR1) en respuesta al tratamiento con IFN-alfa en comparación con las VE de prueba. Tabla S5. ARNm de las VE significativamente (FDR1) alterados en respuesta a la infección por el VNO en comparación con las VE de prueba. Tabla S6. ARNm de EV significativamente (FDR1) alterados en respuesta al tratamiento con IFN-alfa en comparación con EVs de prueba. Tabla S7. Términos GO asociados con los mRNAs que muestran niveles reducidos en las EVs secretadas por las células A549 infectadas por el VNO. Tabla S8. Términos GO asociados con los ARNm que muestran niveles reducidos en las VE secretadas por las células A549 tratadas con IFN-alfa. Tabla S9. Cebadores de PCR utilizados en el estudio. (XLSX 349 kb)Fil: Slonchak, Andrii. University of Queensland; Australia.Fil: Clarke, Brian. Universidad de Arizona; Estados Unidos.Fil: Mackenzie, Jason. University of Queensland; Australia.Fil: Amarilla, Leonardo D. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.Fil: Amarilla, Leonardo D. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto Multidisciplinario de Biología Vegetal; Argentina.Fil: Setoh, Yin. University of Queensland; Australia.Fil: Khromykh, Alexander. University of Queensland; Australia

West Nile virus infection and interferon alpha treatment alter the spectrum and the levels of coding and noncoding host RNAs secreted in extracellular vesicles

Extracellular vesicles (EVs) are small membrane vesicles secreted by the cells that mediate intercellular transfer of molecules and contribute to transduction of various signals. Viral infection and action of pro-inflammatory cytokines has been shown to alter molecular composition of EV content. Transfer of antiviral proteins by EVs is thought to contribute to the development of inflammation and antiviral state. Altered incorporation of selected host RNAs into EVs in response to infection has also been demonstrated for several viruses, but not for WNV. Considering the medical significance of flaviviruses and the importance of deeper knowledge about the mechanisms of flavivirus-host interactions we assessed the ability of West Nile virus (WNV) and type I interferon (IFN), the main cytokine regulating antiviral response to WNV, to alter the composition of EV RNA cargo.We employed next generation sequencing to perform transcriptome-wide profiling of RNA cargo in EVs produced by cells infected with WNV or exposed to IFN-alpha. RNA profile of EVs secreted by uninfected cells was also determined and used as a reference. We found that WNV infection significantly changed the levels of certain host microRNAs (miRNAs), small noncoding RNAs (sncRNAs) and mRNAs incorporated into EVs. Treatment with IFN-alpha also altered miRNA and mRNA profiles in EV but had less profound effect on sncRNAs. Functional classification of RNAs differentially incorporated into EVs upon infection and in response to IFN-alpha treatment demonstrated association of enriched in EVs mRNAs and miRNAs with viral processes and pro-inflammatory pathways. Further analysis revealed that WNV infection and IFN-alpha treatment changed the levels of common and unique mRNAs and miRNAs in EVs and that IFN-dependent and IFN-independent processes are involved in regulation of RNA sorting into EVs during infection.WNV infection and IFN-alpha treatment alter the spectrum and the levels of mRNAs, miRNAs and sncRNAs in EVs. Differentially incorporated mRNAs and miRNAs in EVs produced in response to WNV infection and to IFN-alpha treatment are associated with viral processes and host response to infection. WNV infection affects composition of RNA cargo in EVs via IFN-dependent and IFN-independent mechanisms