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

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

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

Helicase Domain of West Nile Virus NS3 Protein Plays a Role in Inhibition of Type I Interferon Signalling

West Nile virus (WNV) is a neurotropic flavivirus that can cause encephalitis in mammalian and avian hosts. In America, the virulent WNV strain (NY99) is causing yearly outbreaks of encephalitis in humans and horses, while in Australia the less virulent Kunjin strain of WNV strain has not been associated with significant disease outbreaks until a recent 2011 large outbreak in horses (but not in humans) caused by NSW2011 strain. Using chimeric viruses between NY99 and NSW2011 strains we previously identified a role for the non-structural proteins of NY99 strain and especially the NS3 protein, in enhanced virus replication in type I interferon response-competent cells and increased virulence in mice. To further define the role of NY99 NS3 protein in inhibition of type I interferon response, we have generated and characterised additional chimeric viruses containing the protease or the helicase domains of NY99 NS3 on the background of the NSW2011 strain. The results identified the role for the helicase but not the protease domain of NS3 protein in the inhibition of type I interferon signalling and showed that helicase domain of the more virulent NY99 strain performs this function more efficiently than helicase domain of the less virulent NSW2011 strain. Further analysis with individual amino acid mutants identified two amino acid residues in the helicase domain primarily responsible for this difference. Using chimeric replicons, we also showed that the inhibition of type I interferon (IFN) signalling was independent of other known functions of NS3 in RNA replication and assembly of virus particles

Full genome sequence of Rocio virus reveal substantial variations from the prototype Rocio virus SPH 34675 sequence

Rocio virus (ROCV) is an arbovirus belonging to the genus Flavivirus, family Flaviviridae. We present an updated sequence of ROCV strain SPH 34675 (GenBank: AY632542.4), the only available full genome sequence prior to this study. Using next-generation sequencing of the entire genome, we reveal substantial sequence variation from the prototype sequence, with 30 nucleotide differences amounting to 14 amino acid changes, as well as significant changes to predicted 3’UTR RNA structures. Our results present an updated and corrected sequence of a potential emerging human-virulent flavivirus uniquely indigenous to Brazil (GenBank: MF461639)

Sequencing of historical isolates, k‐mer mining and high serological cross‐reactivity with Ross River virus argue against the presence of Getah virus in Australia

Getah virus (GETV) is a mosquito‐transmitted alphavirus primarily associated with disease in horses and pigs in Asia. GETV was also reported to have been isolated from mosquitoes in Australia in 1961; however, retrieval and sequencing of the original isolates (N544 and N554), illustrated that these viruses were virtually identical to the 1955 GETVMM2021 isolate from Malaysia. K‐mer mining of the >40,000 terabases of sequence data in the Sequence Read Archive followed by BLASTn confirmation identified multiple GETV sequences in biosamples from Asia (often as contaminants), but not in biosamples from Australia. In contrast, sequence reads aligning to the Australian Ross River virus (RRV) were readily identified in Australian biosamples. To explore the serological relationship between GETV and other alphaviruses, an adult wild‐type mouse model of GETV was established. High levels of cross‐reactivity and cross‐protection were evident for convalescent sera from mice infected with GETV or RRV, highlighting the difficulties associated with the interpretation of early serosurveys reporting GETV antibodies in Australian cattle and pigs. The evidence that GETV circulates in Australia is thus not compelling