RNA viruses

VEEnext is an interactive resource to study the evolution and global spread of the Venezuelan equine encephalitis (VEE) serocomplex viruses within the Nextstrain platform. It features 165 virus isolates sampled between 1938 and 2017.

CHIKVnext is an interactive web resource to study the evolution and spread of Chikungunya virus (CHIKV), featuring almost 1400 whole genome CHIKV isolates sampled between 1953 and 2022. It is built on the #Nextstrain platform that makes phylogeny and phylogeographics easily accessible through an interactive web application.

Presentation given at the 7th 'Computational Approaches to RNA Structure and Function' conference, Benasque, Spain on 12 August 2022

Tick-borne encephalitis virus (TBEV) is the etiological agent of tick-borne encephalitis, an infectious disease of the central nervous system that is often associated with severe sequelae in humans. While TBEV is typically classified into three subtypes, recent evidence suggests a more varied range of TBEV subtypes and lineages that differ substantially in their 3’UTR architecture. Building on comparative genomics approaches and thermodynamic modeling, we characterize the TBEV 3’UTR structureome diversity and propose a unified picture of pervasive non-coding RNA (ncRNA) structure conservation. Moreover, we provide an updated phylogeny of TBEV, building on more than 220 publicly available complete genomes, and investigate the molecular epidemiology and phylodynamics with Nextstrain, a web-based visualization framework for real-time pathogen evolution.

The ability of RNA to fold into intricate structures in 2D and 3D is exploited by viruses to mediate tropism. Therefore, a deep understanding of RNA structure formation is crucial for the emerging field of virus bioinformatics. The ViennaRNA Package comes with a rich portfolio of tools to analyze RNA folding and the traits that are associated with particular folds. Single sequence modeling from thermodynamic principles and comparative studies of RNA structure provide a powerful approach to assess evolutionary conservation of viral genomes. This presentation introduces the ViennaRNA Package and examines how it can help virologists to address RNA structuredness by the example of a recent project that was focused around the characterization of alternative RNA structure conservation in different subtypes of tick-borne encephalitis virus (TBEV).

Objectives: Due to limited Hepatitis C sequence availability from patients in Russia, the relationship between subtypes and baseline resistance associated substitutions to direct antiretroviral treatment outcome is not fully understood. Methods: Deep sequencing of HCV NS3, NS5A and NS5B sequences was performed on plasma HCV samples from 412 DAA-naïve patients from Russia. Phylogenetic analysis was performed to investigate sequence similarities between HCV stains from Russia, Asia, Europe and North America. Pretreatment HCV RAS was assessed with a 15% cutoff. Results: HCV GT1b and GT3a sequences in Russia were related to strains in Europe and Asia. GT1a and GT2a had a low prevalence in Russia. In GT1b, the prevalence of NS5A Y93H was lower in Russia (6%) compared with Asia (15%). The prevalence of NS5B L159F was similar between Russia and Europe (26-39%). GT3a RAS prevalence was similar between Russia and Asia, Europe and North America. The 2k/1b recombinant strain in Russia were related to strains of Europe. A higher prevalence of the NS5A RAS L31M (10%) was observed in 2k/1b sequences compared to GT1b (1-6%). Conclusions: The prevalence of RASs and the phylogenetic analysis showed similarities in HCV strains between Russia, Europe and North America. This information may be useful for HCV regimens in Russia.

We provide a custom Nextstrain build to explore the molecular epidemiology of Tick-borne encephalitis virus, available at https://nextstrain.org/groups/ViennaRNA/TBEVnext/v1.0

The Internal Ribosome Entry Site (IRES) RNA of Bovine viral diarrhea virus (BVDV), an economically significant Pestivirus, is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modeling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA forms in solution a modular architecture composed of three distinct structural domains (I-III). Two regions within domain III are engaged in tertiary interactions to form an H-type pseudoknot. Computational modeling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.

Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3′-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3′-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3′-UTR of flaviviruses.

Chikungunya virus (CHIKV) is an emerging Alphavirus which causes millions of human infections every year. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages: West African, East Central South African, and Asian Urban. As new outbreaks occurred in the Americas, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. Building on a recently updated phylogeny of CHIKV, we report here the availability of an interactive CHIKV phylodynamics dataset, which is based on more than 900 publicly available CHIKV genomes. We provide an interactive view of CHIKV molecular epidemiology built on Nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution. CHIKV molecular epidemiology reveals single nucleotide variants that change the stability and fold of locally stable RNA structures. We propose alternative RNA structure formation in different CHIKV lineages by predicting more than a dozen RNA elements that are subject to perturbation of the structure ensemble upon variation of a single nucleotide.

Chikungunya virus (CHIKV) is an emerging Alphavirus which causes millions of human infections every year. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages: West African, East Central South African, and Asian Urban. As new outbreaks occurred in the Americas, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. Building on a recently updated phylogeny of CHIKV, we report here the availability of an interactive CHIKV phylodynamics dataset, which is based on more than 900 publicly available CHIKV genomes. We provide an interactive view of CHIKV molecular epidemiology built on Nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution. CHIKV molecular epidemiology reveals single nucleotide variants that change the stability and fold of locally stable RNA structures. We propose alternative RNA structure formation in different CHIKV lineages by predicting more than a dozen RNA elements that are subject to perturbation of the structure ensemble upon variation of a single nucleotide.

Recent experimental evidence revealed a thorough understanding of the involvement of functional RNA elements in the 3’ untranslated regions (UTRs) of flaviviruses with virus tropism. Comparative genomics and thermodynamic modeling allow for the prediction and functional characterization of homologous structures in phylogenetically related viruses. We provide here a comprehensive overview of evolutionarily conserved RNAs in the 3’UTRs of mosquito-borne flaviviruses.

Chikungunya virus (CHIKV) is an emerging alphavirus, which causes millions of human infections every year. Being the etiological agent of Chikungunya fever, a febrile disease associated with severe arthralgia, CHIKV has probably been circulating in sylvatic transmission cycles for many centuries. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages, West African, East Central South Africa (ECSA), and Asian Urban. As new outbreaks happened in South America, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. A recently updated phylogeny of CHIKV revealed a more fine-grained clade association of both ECSA and Asian lineages, particularly the presence of distinct lineages in South America (ex ECSA) and the Caribbean (ex Asian Urban). Building on the recently updated phylogeny of CHIKV, based on more than 700 publicly available CHIKV genomes, we report here the availability of an interactive CHIKV molecular epidemiology dataset. We provide an interactive view of CHIKV molecular phylogeny built on nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution (https://www.nextstrain.org). We clocked the phylogenetic tree using the timetree approach built into nextstrain, which allows for updated estimates of the time of the branching events of CHIKV lineages. Moreover, researchers can now visualize the spread of CHIKV over time and space utilizing nextstrain. The latest build is available at https://nextstrain.org/community/ViennaRNA/CHIKV

To monitor the arthropod-borne virus transmission in mosquitoes, we have attempted both to detect and isolate viruses from 3304 wild-caught female mosquitoes in the Livingstone (Southern Province) and Mongu (Western Province) regions in Zambia in 2017. A pan-flavivirus RT-PCR assay was performed to identify flavivirus genomes in total RNA extracted from mosquito lysates, followed by virus isolation and full genome sequence analysis using next-generation sequencing and rapid amplification of cDNA ends. We isolated a newly identified Barkedji virus (BJV Zambia) (10,899 nt) and a novel flavivirus, tentatively termed Barkedji-like virus (BJLV) (10,885 nt) from Culex spp. mosquitoes which shared 96% and 75% nucleotide identity with BJV which has been isolated in Israel, respectively. These viruses could replicate in C6/36 cells but not in mammalian and avian cell lines. In parallel, a comparative genomics screening was conducted to study evolutionary traits of the 5′-and 3′-untranslated regions (UTRs) of isolated viruses. Bioinformatic analyses of the secondary structures in the UTRs of both viruses revealed that the 5′-UTRs exhibit canonical stem-loop structures, while the 3′-UTRs contain structural homologs to exoribonuclease-resistant RNAs (xrRNAs), SL-III, dumbbell, and terminal stem-loop (3′SL) structures. The function of predicted xrRNA structures to stop RNA degradation by Xrn1 exoribonuclease was further proved by the in vitro Xrn1 resistance assay.

RNA viruses cause millions of infections in humans and animals every year, thereby challenging health systems and economies globally. Large-scale outbreaks of emerging and re-emerging agents, such as Dengue, Zika, Chikungunya, and Ebola virus in human and African swine fever virus in animals have been reported over the last years. These outbreaks can reveal new clinical manifestations, e.g., the accumulation of congenital microcephaly cases during the Zika virus epidemic in the Americas 2015-2016. Very recently, the emergence of a novel pneumonia (COVID-19) in the city of Wuhan, China, and its subsequent worldwide spread, has been associated with a novel coronavirus, SARS- CoV-2. Thanks to the rapid availability of dozens of full-length SARS-CoV-2 genomic sequences, phylogenetic proximity to known pathogens like SARS and MERS could be established. On a more fine-grained scale, SARS-CoV-2 shares ancestral roots with coronaviruses isolated from bats and pangolins. While vaccines are only available for a few viruses, and the biochemical mechanisms that mediate tropism and pathology are often poorly understood, experimental data suggest an involvement of RNA structure in many of these viral pathologies. Comparative studies of RNA structure combine thermodynamic and kinetic modeling, phylogenomics, and homology search, thereby providing a powerful approach for characterizing the evolutionary conservation of viral genomes. Here, the availability of large amounts of genome data allows building fine-grained models of homologous RNA structures in phylogenetically related viruses. Typically, the untranslated regions (UTRs) of virus genomes harbor functional RNA elements, which play vital roles in viral translation, replication, pathogenesis, and host adaptation. Novel data from comparative RNA bioinformatics screens also provide evidence for the existence of many evolutionarily conserved RNA structures within the coding regions of SARS-CoV-2 and related coronaviruses. While many of these structures have not been previously described, they reveal characteristic patterns of evolutionarily conserved RNAs inside and outside of coronavirus coding regions. In this line, they represent potential targets for vaccine development and novel antiviral drugs.

In silico prediction of evolutionarily conserved RNA secondary structures in the upstream 500 nucleotides of Wuhan seafood market pneumonia virus (Wuhan-nCoV, GenBank MN908947.3, top) and severe acute respiratory syndrome virus (SARS, RefSeq NC_004718.3, bottom). The genomic regions shown here comprise the 5'UTR and the amino-terminal region of the nonstructural protein 1 (highlighted in orange). A small upstream ORF (uORF) is found in both 5'UTRs and is highlighted in blue. Phylogenetic proximity of Wuhan-nCoV and SARS is reflected by several homologous stem-loop structures, as inferred from covariance model analysis. In particular, stem-loops SL1, SL2, SL3, SL4, SL5, SL5a, SL5b, SL5c, SL6, and SL7 show a high degree of structural similarity. Wuhan-nCov is predicted to form an additional small stem-loop structure SL4a, as well as a slightly extended SL8. Structures were predicted and plotted with the ViennaRNA Package v2.4.14 (https://www.tbi.univie.ac.at/RNA/)

Arboviruses are a group of viruses transmitted by arthropods, mostly ticks, and mosquitoes. Among the members of this group are the families Togaviridae and Flaviviridae, (+)ssRNA viruses, such as Chikungunya, Dengue, and Zika. Multiple of these viruses have demonstrated their ability to cause neuropathology in humans. In Zika, a protein binding motif known as Musashi binding element (MBE) has been attributed to promoting replication, neurotropism, and pathology. Musashi-1 (MSI1) is an RNA-binding protein involved in the maintenance and self-renewal of stem cells and a translational regulator in many biological systems. MSI1 pre-dominantly binds single-stranded UAG motifs in the 3’ untranslated region (UTR) of RNA. We have recently analyzed Musashi binding elements (MBEs) in the 3’UTR of flaviviruses (FV) in silico. In this study, we could show that MBEs in the 3’UTR of neurotropic viruses such as Zika, West Nile, and Powassan virus are highly accessible, and mostly occur in an unpaired structural context, which renders them optimal Musashi binding targets and corroborates previous experimental studies by a theoretical model.

Arthropod-borne RNA viruses (arboviruses) represent a global health threat, causing millions of human and animal infections every year. Recent outbreaks of emerging and re-emerging viruses have revealed new clinical manifestations, e.g., the accumulation of congenital microcephaly cases during the Zika virus epidemic in the Americas 2015-2016. While vaccines against many of these viruses are still under development, experimental data suggest an involvement of RNA structure in viral pathogenesis. Comparative studies of RNA structure combine thermodynamic modeling, phylogenomics and homology search, thereby providing a powerful approach for characterizing the evolutionary conservation of viral genomes. In particular, untranslated regions (UTRs) of arboviruses harbor functional RNA elements, which play vital roles in virus replication and often mediate tropism. Here, the availability of large amounts of genome data allows building fine-grained models of homologous RNA structures in phylogenetically related viruses. On a broader scale, these data reveal common patterns and evolutionary traits of non-coding viral RNAs.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus of the family Togaviridae, has recently emerged in the Americas from lineages from two continents: Asia and Africa. Historically, CHIKV circulated as at least four lineages worldwide with both enzootic and epidemic transmission cycles. To understand the recent patterns of emergence and the current status of the CHIKV spread, updated analyses of the viral genetic data and metadata are needed. Here, we performed phylogenetic and comparative genomics screens of CHIKV genomes, taking advantage of the public availability of many recently sequenced isolates. Based on these new data and analyses, we derive a revised phylogeny from nucleotide sequences in coding regions. Using this phylogeny, we uncover the presence of several distinct lineages in Africa that were previously considered a single one. In parallel, we performed thermodynamic modeling of CHIKV untranslated regions (UTRs), which revealed evolutionarily conserved structured and unstructured RNA elements in the 3'UTR. We provide evidence for duplication events in recently emerged American isolates of the Asian CHIKV lineage and propose the existence of a flexible 3'UTR architecture among different CHIKV lineages.

Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3'UTR of tick-borne, insect-specific and no-known-vector flaviviruses in silico. Our data support the wide distribution of previously experimentally characterized exoribonuclease resistant RNAs xrRNAs within tick-borne and no-known-vector flaviviruses and provide evidence for the existence of a cascade of duplicated RNA structures within insect-specific flaviviruses. On a broader scale, our findings indicate that viral 3'UTRs represent a flexible scaffold for evolution to come up with novel xrRNAs

Zika virus (ZIKV) belongs to a class of neurotropic viruses that have the ability to cause congenital infection, which can result in microcephaly or fetal demise. Recently, the RNA-binding protein Musashi-1 (Msi1), which mediates the maintenance and self-renewal of stem cells and acts as a translational regulator, has been associated with promoting ZIKV replication, neurotropism, and pathology. Msi1 predominantly binds to single-stranded UAG motifs in the 3'UTR of RNA. We systematically analyzed the properties of Musashi binding elements (MBEs) in the 3'UTR of flaviviruses based on a thermodynamic model for RNA folding. Our results indicate that MBEs in ZIKV 3'UTRs occur predominantly in unpaired, single-stranded structural context, thus corroborating experimental observations by a biophysical model of RNA structure formation. Statistical analysis and comparison with related viruses shows that ZIKV MBEs are maximally accessible among all mosquito-borne flaviviruses. Our study addresses the broader question whether other emerging arboviruses can cause similar neurotropic effects through the same mechanism in the developing fetus. In this line, we establish a link between the biophysical properties of viral RNA and teratogenicity. Moreover, our thermodynamic model can explain recent experimental findings and predict the Msi1-related neurotropic potential of other viruses.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus of the family Togaviridae, has recently emerged in the Americas from lineages from two continents, Asia and Africa. Historically, CHIKV circulated as at least four lineages worldwide with both enzootic and epidemic transmission cycles. To understand the recent patterns of emergence and the current status of the CHIKV spread, updated analyses of the viral genetic data and metadata are needed. Here, we performed phylogenetic and comparative genomics screens of CHIKV genomes, taking advantage of the public availability of many recently sequenced isolates. Based on these new data and analyses, we derive a revised phylogeny from nucleotide sequences in coding regions. Using this phylogeny, we uncover the presence of several distinct lineages in Africa that were previously considered a single one. In parallel, we performed thermodynamic modeling of CHIKV untranslated regions (UTRs), which revealed evolutionarily conserved structured and unstructured RNA elements in the 3'UTR. We provide evidence for duplication events in recently emerged American isolates of the Asian CHIKV lineage and propose the existence of a flexible 3'UTR architecture among different CHIKV lineages.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus of the Togaviridae family, has recently emerged in the Americas from two originating continents, Asia and Africa. Historically, CHIKV circulated as at least four lineages worldwide with both enzootic and epidemic transmission cycles. To understand the recent re-emergence patterns, and the current status of the CHIKV spread, an updated phylogeny of the virus is needed. Here, we performed phylogenetic and comparative genomics screens of CHIKV genomes, taking advantage of the public availability of many recently sequenced isolates. Based on these new data and analyses, we derive a revised phylogeny from full open reading frame nucleotide sequences. Within this phylogeny, we propose the presence of several distinct lineages in Africa which were once considered a single lineage. In parallel, thermodynamic modeling of CHIKV untranslated regions (UTRs) reveals a cascade of evolutionarily conserved RNA structures, in particular in the 3' UTR. We provide evidence for the duplication of non-coding RNAs (ncRNAs) in recently emerged Carribean CHIKV isolates and propose the existence of lineage-specific duplicated ncRNAs in different CHIKV lineages.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus of the family Togaviridae, has recently emerged in the Americas from lineages from two continents, Asia and Africa. Historically, CHIKV circulated as at least four lineages worldwide with both enzootic and epidemic transmission cycles. To understand the recent patterns of emergence and the current status of the CHIKV spread, updated analyses of the viral genetic data and metadata are needed. Here, we performed phylogenetic and comparative genomics screens of CHIKV genomes, taking advantage of the public availability of many recently sequenced isolates. Based on these new data and analyses, we derive a revised phylogeny from nucleotide sequences in coding regions. Using this phylogeny, we uncover the presence of several distinct lineages in Africa that were previously considered a single one. In parallel, we performed thermodynamic modeling of CHIKV untranslated regions (UTRs), which revealed evolutionarily conserved structured and unstructured RNA elements in the 3'UTR. We provide evidence for duplication events in recently emerged American isolates of the Asian CHIKV lineage and propose the existence of a flexible 3'UTR architecture among different CHIKV lineages.

Zika virus (ZIKV) belongs to a class of neurotropic viruses that have the ability to cause congenital infection, which can result in microcephaly or fetal demise. Recently, the RNA-binding protein Musashi-1 (Msi1), which mediates the maintenance and self-renewal of stem cells and acts as a translational regulator, has been associated with promoting ZIKV replication, neurotropism, and pathology. Msi1 predominantly binds to single-stranded motifs in the 3′ untranslated region (UTR) of RNA that contain a UAG trinucleotide in their core. We systematically analyzed the properties of Musashi binding elements (MBEs) in the 3′UTR of flaviviruses with a thermodynamic model for RNA folding. Our results indicate that MBEs in ZIKV 3′UtRs occur predominantly in unpaired, single-stranded structural context, thus corroborating experimental observations by a biophysical model of RNA structure formation. statistical analysis and comparison with related viruses show that ZIKV MBEs are maximally accessible among mosquito-borne flaviviruses. Our study addresses the broader question of whether other emerging arboviruses can cause similar neurotropic effects through the same mechanism in the developing fetus by establishing a link between the biophysical properties of viral RNA and teratogenicity. Moreover, our thermodynamic model can explain recent experimental findings and predict the Msi1-related neurotropic potential of other viruses.

Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3 UTR of tick-borne, insect-specific and no-known-vector flaviviruses in silico. Our data support the wide distribution of previously experimentally characterized exoribonuclease resistant RNAs (xrRNAs) within tick-borne and no-known-vector flaviviruses and provide evidence for the existence of a cascade of duplicated RNA structures within insect-specific flaviviruses. On a broader scale, our findings indicate that viral 3 UTRs represent a flexible scaffold for evolution to come up with novel xrRNAs.

Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3'UTR of tick-borne, insect-specific and no-known-vector flaviviruses in silico. Our data support the wide distribution of previously experimentally characterized exoribonuclease resistant RNAs (xrRNAs) within tick-borne and no-known-vector flaviviruses and provide evidence for the existence of a cascade of duplicated RNA structures within insect-specific flaviviruses. On a broader scale, our findings indicate that viral 3'UTRs represent a flexible scaffold for evolution to come up with novel xrRNAs.

Emerging and re-emerging arthropod-borne viruses such as Japanese encephalitis (JEV), Dengue (DENV), Yellow fever (YFV), and Chikungunya (CHIKV) viruses are a growing global health threat. Zika virus (ZIKV) is a neurotropic flavivirus (FV) that can cause congenital infection, which can result in microcephaly and fetal demise. Recently, the translational regulator protein Musashi-1 (Msi1) has been attributed to promoting ZIKV replication, neurotropism, and pathology. Msi1 predominantly binds single-stranded UAG motifs in the 3'UTR of RNA. Here we systematically analyzed the thermodynamic properties of Musashi binding elements (MBEs) in the 3'UTR of 76 arbovirus genomes in silico. Our results indicate that MBEs in the ZIKV 3'UTR occur predominantly in unpaired, single-stranded structural context, thus supporting experimental observations of Msi1 binding affinity with a thermodynamic model of RNA structure formation.

Flaviviruses represent a global health threat and are responsible for millions of infections in humans every year. During the 2015-2017 outbreak of Zika virus (ZIKV) in the Americas accumulation of congenital microcephaly cases was observed, which has been associated with ZIKV infection. However, the underlying biological mechanisms remain elusive. Recently, experimental studies showed that the host protein Musashi-1 (Msi1) mediates ZIKV replication. Msi1, which is expressed in neuronal progenitor cells and tumors, typically binds single-standed UAG motifs in the 3'UTR of RNA. Here we employ a biophysical model of RNA structure formation to show that UAGs within ZIKV 3'UTRs are highly accessible and therefore represent optimal Msi1 binding targets. More generally, the theoretical approach presented here allows to predict the Msi-mediated teratogenic potential of related arboviruses.

Purpose: Arboviruses are a group of viruses transmitted by arthropods, mostly ticks, and mosquitoes. Among the members of this group are the families Togaviridae and Flaviviridae, (+)ssRNA viruses, such as Chikungunya, Dengue, and Zika. Multiple of these viruses have demonstrated their ability to cause neuropathology in humans. In Zika, a protein binding motif known as Musashi binding element (MBE) has been attributed to promoting replication, neurotropism, and pathology. Musashi-1 (MSI1) is an RNA-binding protein involved in the maintenance and self-renewal of stem cells and a translational regulator in many biological systems. MSI1 predominantly binds single-stranded UAG motifs in the 3’ untranslated region (UTR) of RNA. We have recently analyzed Musashi binding elements (MBEs) in the 3’UTR of flaviviruses in silico. In this study, we could show that MBEs in the 3’UTR of neurotropic viruses such as Zika, West Nile, and Powassan virus are highly accessible, and mostly occur in an unpaired structural context, which renders them optimal Musashi binding targets and corroborates previous experimental studies by a theoretical model. Methods & Materials: To expand to other related viruses, we systematically analyzed the properties of Musashi binding elements (MBEs) in the 3’UTR of alphaviruses based on a thermodynamic model for RNA folding and correlated to the currently described pathogenicity in literature. Results: Our results indicate that MBEs in the 3’UTR of alphaviruses are not as accessible as in flaviviruses, suggesting that the MBE role in neuropathology might be an exclusive feature of flaviviruses. Conclusion: Our study addresses the broader question whether other emerging arboviruses can cause similar neurotropic effects. We expanded our study from an initial flavivirus dataset to all (+)ssRNA flavi- and alphaviruses to evaluate the link between MBE accessibility and currently described neuropathology. Moreover, our thermodynamic model can be the initial indicative for in vivo studies to evaluate the potential neurotropic effect of MBEs on different viral families.