ABSTRACT: Dengue and Zika are closely related members of the Flaviviridae family of positive, single-stranded RNA viruses and are of global clinical importance. These viruses utilize an 11kb RNA genome for translation and replication, and much remains to be learnt about how the entire genome folds to enable virus function. Here, we performed high throughput RNA secondary structure and pair-wise interaction mapping on four dengue serotypes and four Zika strains within their virus particles. We identified structures that are associated with translation pausing, and are evolutionary conserved by integrating synonymous mutation rates into our analysis. Genome-wide interaction mapping revealed alternative structures, as well as extensive long-range RNA interactions – including the known circularization signals– within the virus particles. Many of these long-range interactions are conserved across the viruses and/or clustered into “hubs” that are shown to be functionally important. This comprehensive structural resource of dengue and Zika viruses reveals that viral genome organization is much more complex than previously appreciated and deepens our understanding of the molecular basis for viral pathogenesis.
ORGANISM(S): dengue virus type 3 dengue virus type 4 dengue virus type 2 Homo sapiens Zika virus dengue virus type 1
Project description:Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. Live attenuated vaccines have been successfully used to combat infection by flaviviruses, such as yellow fever and Japanese encephalitis viruses. A Zika virus harboring combined mutations in the envelope protein glycosylation site and in the nonstructural 4B protein amino acid 36 (ZE4B-36) was generated and assessed for stability, attenuation, and protection against infection. To determine the genetic stability of its RNA genome, ZE4B-36 was serially passaged in vitro in Vero cells. Virus harvested from passages (P)1 to P6 was subjected to next generation sequencing and downstream analysis to determine its nucleotide sequence variability. Specifically, single nucleotide variant analysis showed that the ZE4B-36 genome decreased its genetic diversity and resulted in a more stable nucleotide sequence. Thus, in addition to showing attenuation and protection, ZE4B-36 is a stable live attenuated virus that possesses characteristics important for a vaccine to combat Zika disease.
Project description:The modification N6-methyladenosine (m6A) affects rates of translation and degradation of mRNA transcripts. We analyzed m6A across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV) using MeRIP-seq. We used the uninfected replicates, among which we would expect little biological variation in methylation, as negative controls to validate statistical methods for the detection of m6A changes in MeRIP-seq data. Applying validated statistical methods, we found that innate immune response to Flaviviridae viruses alters m6A modification of specific cellular transcripts compared to uninfected controls. Finally, we find that these changes in m6A can in turn affect splicing or translation of genes relevant to infection.
Project description:Zika virus (ZIKV) and dengue virus (DENV) are members of the Flaviviridae family of RNA viruses and cause severe disease in humans. ZIKV and DENV share over 90% of their genome sequences, however the clinical features of Zika and dengue infections are very different reflecting tropism and cellular effects. Here, we used simultaneous RNA sequencing and ribosome footprinting to define the transcriptional and translational dynamics of ZIKV and DENV infection in human neuronal progenitor cells (hNPCs). The gene expression data showed induction of aminoacyl tRNA synthetases (ARS) and the translation-activating PIM1 kinase indicating an increase in RNA translation capacity. The data also reveal activation of different cell stress reponses, with ZIKV triggering a BACH1/2 redox program, and DENV activating the ETF/CHOP endoplasmatic reticulum (ER) stress program. The RNA translation data highlight activation of polyamine metabolism through changes in key enzymes and their regulators. This pathway is needed for eIF5A hypusination and has been implicated viral translation and replication. Concerning the viral RNA genomes, ribosome occupancy readily identifies highly translated open reading frames and a novel upstream ORF (uORF) in the DENV genome. Together, our data highlight both the cellular stress response and also the activation of RNA translation and polyamine metabolism during DENV and ZIKV infection.
Project description:We use MeRIP-seq and PAR-CLIP in liver cell lines to detail the role of m6A in positive strand RNA virus infection. We find that hepatitis C virus, dengue virus, West Nile virus, yellow fever virus, and Zika virus all contain m6A within their genomes, with some conservation between viruses in methylation sites.
Project description:The RNA modification N6-methyladenosine (m6A) can modulate mRNA fate and thus affect many biological processes. We analyzed m6A modification across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters m6A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m6A to RIOK3 promotes its translation, while loss of m6A in CIRBP promotes alternative splicing. Importantly, we found that viral activation of innate immune sensing or the endoplasmic reticulum (ER) stress response contributes to the changes in m6A modification in RIOK3 and CIRBP, respectively. Further, several transcripts with infection-altered m6A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, and HCV infection. Overall, this work reveals that cellular signaling pathways activated during viral infection lead to alterations in m6A modification of host mRNAs to regulate infection.
Project description:Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.
Project description:Insect-borne flaviviruses produce a 300-500 base long noncoding RNA, termed sfRNA, by stalling the cellular 5’-3’ exoribonuclease XRN1 via structures located in their 3’ untranslated regions. In this study we demonstrate that the production of sfRNAs by Zika virus results in the repression of XRN1 similar to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay, we demonstrate that the sfRNA from both dengue type 2 and Zika viruses interact with a common set of 21 RNA binding proteins that influence post-transcriptional processes in the cell, including splicing, RNA stability and translation. We demonstrate that four of these interacting proteins - DDX6 and EDC3 (RNA decay factors), PHAX1 (a regulator of the biogenesis of the splicing machinery) and APOBEC3C (a nucleic acid editing deaminase) are inherently antiviral restriction factors for Zika virus infection. Furthermore, we demonstrate that cellular mRNA decay regulation as well as cellular RNA splicing are compromised during Zika virus infection. Collectively, these data demonstrate the large extent in which Zika virus sfRNAs interact with cellular RNA binding proteins as well as the potential for widespread dysregulation of post-transcriptional control which likely limits the effective response of the cell to viral infection.
Project description:Flavivirus infection is tightly connected to host lipid metabolism. Here, we performed shotgun lipidomics of cells infected with neurotropic Zika, West Nile, and tick-borne encephalitis viruses, as well as dengue and yellow fever virus. Early in infection specific lipids accumulated, e.g., neutral lipids in Zika and some lyso-phospholipids in all infections. Ceramide levels increased following infection with viruses that cause a cytopathic effect. In addition, fatty acid desaturation as well as glycerophospholipid metabolism were significantly altered. Importantly, depletion of enzymes involved in phosphatidylserine metabolism as well as phosphatidylinositol biosynthesis reduced orthoflavivirus titers and cytopathic effects while inhibition of fatty acid monounsaturation only rescued from virus-induced cell death. Interestingly, interfering with ceramide synthesis had opposing effects on virus replication and cytotoxicity depending on the targeted enzyme. Thus, lipid remodeling by orthoflaviviruses includes distinct changes but also common patterns shared by several viruses that are needed for efficient infection and replication.
Project description:Determination of miRNA profiles in most prominent mosquitoes will determine the potential targets for mosquito control Some of the most medically important viruses, such as dengue virus, West Nile virus, Zika virus, and yellow fever virus, are transmitted by mosquitoes. These aptly named arboviruses impose a tremendous cost to the health of populations around the world. As a result, much effort has gone into the study of the impact of these viruses in human infections. Comparatively less efforts, however, have been made to study the way these viruses interact with mosquitos themselves. It has long been held that these viruses are introduced into the midgut of mosquitoes upon ingestion of a blood meal before being transmitted within the saliva upon subsequent feeding. This sequence requires that the mosquito be able to defend itself from infection every step along the way-from ingesting bloodmeal to subsequent feeding. The main defense mechanisms employed by the mosquitoes to control viruses is RNA interference (RNAi). Modulation of this facet of the mosquito’s immune system would thereby suggest a practical strategy for vector control. This paper will provide an up to date overview of the mosquito’s immune system along with novel data describing miRNA profiles for Aedes aegypti and Culex quinquefasiatus in Grenada, West Indies.
Project description:Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. ZIKV infections are associated with neurodevelopmental deficiencies termed Congenital Zika Syndrome. ZIKV strains are grouped into three phylogenetic lineages: East African, West African, and Asian, which contains the American lineage. RNA virus genomes exist as genetically-related sequences. The heterogeneity of these viral populations is implicated in viral fitness, and genome diversity is correlated to virulence. This study examines genetic diversity of representative ZIKV strains from all lineages utilizing next generation sequencing (NGS). Inter-lineage diversity results indicate that ZIKV lineages differ broadly from each other; however, intra-lineage comparisons of American ZIKV strains isolated from human serum or placenta show differences in diversity when compared to ZIKVs from Asia and West Africa. This study describes the first comprehensive NGS analysis of all ZIKV lineages and posits that sub-consensus-level diversity may provide a framework for understanding ZIKV fitness during infection.