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Musashi binding elements in Zika and related Flavivirus 3’UTRs: A comparative study in silico

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.


Introduction
Flaviviruses are an emerging group of arboviruses belonging to the Flaviviridae family. Researchers have been describing recent outbreaks of these viruses that have not been previously detected for decades [80,50,30].
Initially isolated in 1947 from a sentinel rhesus macaque in the Zika forest, Uganda, ZIKV has not been associated with severe disease, apart from skin rashes, body pain, and fever. Likewise, ZIKV has been circulating across equatorial zones in Africa and Asia for 60 years, until the first outbreak was reported in Yap Island, Micronesia in 2007. Subsequently, the virus spread eastwards to French Polynesia and other Pacific islands in 2013 and reached the Americas in 2015 [78,14]. There are two main ZIKV lineages, the original African (type strain MR766) and an Asian (type strain FSS13025) [24,77], the latter also comprising American strains such as PE243.
The 2015-2017 outbreak in the Americas raised the possibility of a link between ZIKV infection and congenital abnormalities, which included placental damage, intrauterine growth restrictions, eye diseases and microcephaly in children as well as acute motor axonal neuropathy-type Guillain-Barré syndrome in adults [44]. While MBFVs are typically transmitted by host-vector interaction, vertical transmission from mother to child during pregnancy via transplacental infection has been reported [63].
The neurotropic potential of ZIKV-related flaviviruses has been known since the 1970s, when Saint Louis encephalitis virus (SLEV) has been attributed to severe neurological disorder in infected mice [4,5]. Vertical transmission has been observed with JEV in mice [22] and human [11] and a case of human fetal infection has been reported after YFV vaccination [81]. Other transmission pathways of ZIKV include blood transfusions and sexual transmission [21,56]. Despite enormous efforts in studying ZIKV infections in the last years, the biological reasoning and mechanisms behind arbovirus congenital neurotropism remain elusive. (ORF) flanked by highly structured untranslated regions (UTRs). Upon translation of the ORF, a polyprotein is produced which is processed by viral and cellular enzymes, yielding structured (C, prM, E) and unstructured proteins (NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, NS5). Both flavivirus UTRs are crucially related to regulation of the viral life cycle, mediating processes such as genome circularization, viral replication and packaging [27,55,83,16].

Flaviviruses hijack the host mRNA degradation pathway
The central role of flavivirus 3'UTR in modulating cytopathicity and pathogenicity became apparent when an accumulation of both gRNA and viral long noncoding RNA (lncRNA) has been observed upon infection. These lncRNAs, also known as subgenomic flaviviral RNAs (sfRNAs) [62], are stable decay intermediates derived from exploiting the host's mRNA degradation machinery [1]. sfRNAs are produced by partial degradation of viral gRNA by Xrn1, a host 5'-3' exoribonuclease that is associated with the endogenous mRNA turnover machinery [36,6]. The enzyme stalls at highly conserved RNA structures in the viral 3'UTR, so-called Xrn1-resistant RNAs (xrRNAs), resulting in sfRNAs of variable lengths [23,10]. Xrn1-resistant RNAs and sfRNAs appear to be ubiquitously present in many flaviviruses. They have been described in MBFVs, including DENV [45], YFV [76], JEV [19], and ZIKV [2], TBFVs [62,74], and recently in ISFVs and NKVs [49]. There is typically more than one xrRNA, which is reflected in the varied molecular architecture of different flavivirus 3'UTRs. Earlier studies in our group have identified conserved RNA structural elements in viral 3'UTRs [66,28,87,29,79], some of which have later been attributed to xrRNA functionality [62]. Stem-loop (SL) as well as dumbbell (DB) structures are found in 3'UTRs of flaviviruses in single or double copies ( Figure 1) and have been associated with quantitative protection of downstream viral RNA [43].
The inhibition of Xrn1 by viral RNA yields sfRNAs that affect many cellular processes, both in the vector and the host [68]. In mosquitoes, sfRNA interacts directly with the predominant innate immune response pathway, RNA interference (RNAi), by serving as a template for microRNA (miRNA) biogenesis [32]. Conversely, in host cells sfRNA modulates the anti-viral interferon response [75], e.g., by binding proteins to inhibit the translation of interferonstimulated genes [51]. Moreover, sfRNA has been shown to inhibit Xrn1 and Dicer activity, thereby altering host mRNA levels [54,73].
At the same time, a variety of host proteins bind the 3'UTR of flaviviruses, thereby mediating viral replication, polyprotein translation or the anti-viral immune response (see Table 1 in ref. [68] for a comprehensive overview of host proteins that bind flavivirus 3'UTR/sfRNA). Although notoriously underrepresented in literature, one can expect that many of these proteins also bind sfRNA due to sequence and structure conservation.

Subgenomic flaviviral RNA interacts with Musashi
One of these groups of host factors is the Musashi (Msi) protein family. Msi is a highly conserved family of proteins in vertebrates and invertebrates that act as a translational regulator of target mRNAs and is involved in cell proliferation and differentiation. While the two Msi paralogs in mammals, Musashi-1 (Msi1) and Musashi-2 (Msi2) are expressed in stem cells [69,70,33] and overexpressed in tumors and leukemias [42], they are absent in differentiated tissue. Moreover, Msi1 has been shown to be involved in the regulation of blood-testis barrier proteins and spermatogenesis in mice [18]. Musashi proteins have two RNA recognition motif (RRM) domains, whose sequence specificity has been determined by an in vitro selection method and NMR spectroscopy [33,59,34]. The trinucleotide sequence UAG has been identified as core Musashi binding element (MBE) and its thermodynamic binding specificity was determined by fluorescence polarization assays [91]. Uren et al. [82] performed iCLIP experiments with Msi1 in human glioblastoma cells and confirmed the preferential binding of Msi1 to single-stranded (stem-loop) UAG sequences in 3'UTRs. Conversely, they could not determine a preference for UAG binding in coding regions.

Musashi is involved in Flavivirus neurotropism
An interesting, yet understudied hypothesis is the possibility that the stem cell regulator protein Musashi could be related to ZIKV tropism. Based on the identification of a MBE in the 3'UTR of the ZIKV genome [44], de Bernardi Schneider et al. [14] reported the presence of the same element with a higher binding affinity for human Msi1 in all ZIKV sequences that belong to the Asia-Pacific-Americas clade in an in silico screen and implied that there could be a change of tropism for the viral lineage. Chavali et al. [12] tested the possibility of Msi1 interaction with the ZIKV genome in vivo and found that Msi1 not only interacts with ZIKV, but also enhances viral replication. They noted that ZIKV RNA could compete with endogenous targets for binding Msi1 in the brain of the developing fetus, thereby dysregulating the expression of genes required for neural stem cell development. Based on their data the authors concluded that Msi1 is involved in ZIKV neurotropism and pathology and raised the question whether MBEs present in other flavivirus genomes could exhibit similar functionality. In a recent study, Platt et at. [64] investigated whether ZIKV-related arboviruses can cause congenital infection and fetal pathology in utero in immunocompetent mice. They tested two emerging neurotropic flaviviruses, WNV, and Powassan virus (POWV), as well as two alphaviruses, Chikungunya virus (CHIKV) and Mayaro virus (MAYV). All four viruses caused placental infection, however, only WNV and POWV resulted in fetal demise, indicating that ZIKV is not unique among flaviviruses in its capacity to be transplacentally transmitted and cause fetal neuropathology.
In this contribution, we systematically analyze the Musashi-related neurotropic potential of well-curated flavivirus genomes in silico. We investigate structural features of MBEs in viral 3'UTRs by a thermodynamic model of RNA structure formation and work out the biophysical properties of conserved RNA structures harboring MBEs in order to build a theoretical ground for future in vivo studies.

Dataset
Sequence data for the present study was acquired from the public National Center for Biotechnology Information (NCBI) refseq database (https://www. ncbi.nlm.nih.gov/refseq/) on 15 December 2017. We filtered for all complete viral genomes under taxonomy ID 11051 (genus Flavivirus), resulting in 72 genomes, 51 of which had 3'UTR sequences and annotation available. Table 1 lists all viruses analyzed in this work and gives the number of Musashi binding elements (UAG trinucleotides) present in 3'UTR regions.
The refseq genome for Spondweni virus (SPONV, accession number NC 029055.1) does not include a 3'UTR sequence. Since SPONV is phylogenetically closely related to ZIKV [26], we were looking to include this sequence into our analysis. Nikos Vasilakis (Univ. of Texas Medical Branch, Galveston, TX) generously provided SPONV sequence data. The 338 nt 3'UTR sequence of the SA-Ar strain (see supplementary material) has been added to the set of flavivirus sequences analyzed here.
Kama virus (KAMV) does not contain UAG trinucleotides in the 3'UTRs, consequently it has been discarded from our dataset. The remaining virus species contain between 1 and 19 MBEs in their 3'UTRs.

Opening energy directly relates to single-strandedness
The biophysical model employed here is based on a description of RNA at the level of secondary structures, building upon the thermodynamic nearest neighbor energy model as implemented in the ViennaRNA Package [46]. This allows for computing equilibrium properties of RNA such as the single most stable, minimum free energy (MFE) structure, as well as the partition function Z. The latter makes an evaluation of the thermodynamic ensemble of RNA structures available and is defined as the sum over all Boltzmann factors of where E(s) is the free energy of the structure, R the universal gas constant and T the thermodynamic temperature of the system. The equilibrium probability of a secondary structure s is then defined as The partition function Z can be computed efficiently via dynamic programming [52] and allows calculation of individual base pair probabilities, even for large sequences [8]. In this line, the accessibility (i.e., the probability that a region i . . . j along the RNA is single-stranded) can be derived from the partition function (Eq. 1) [47]. Likewise, the opening energy (i.e., the free energy required to force the region to be single-stranded) can be computed as The opening energy of a region within an RNA is directly related to local RNA secondary structure. In this line, low opening energy is a reliable indicator for single-strandedness. We employ the sliding window approach of RNAplfold [8] to compute local pairing probabilities of UAG trinucleotide motifs to assess the likelihood of single-strandedness of and around MBEs. RNAplfold is part of the ViennaRNA Package [46] and can compute the accessibilities or single-strandedness of all intervals of an RNA in cubic time [9]. We select 97 nt windows upstream and downstream of MBEs in viral 3'UTRs and compute local pairing probabilities for base pairs within 100 nt windows. Opening energies for trinucleotides are then evaluated from averaged pairing probabilities with RNAplfold.
The significance of a calculated MBE opening energy is assessed by comparison with a large number of randomized sequences of the same length and same base or dinucleotide composition. We compute the opening energies of trinucleotides both in a genomic as well as a shuffled sequence context and apply a z score statistics. The normalized z score is defined as where E open (x) is the opening energy of trinucleotide x in its genomic context, µ and σ are the mean and standard deviations, respectively, of the opening energies of x computed over a large sample of randomized sequences. Randomization with regard to keeping sequence composition is achieved here by applying dinucleotide shuffling to the 97 nt windows upstream and downstream of MBEs, while keeping x in place.
The approach outlined above is implemented in the Perl utility plfoldz.pl, which is available from https://github.com/mtw/plfoldz. The script employs the ViennaRNA [46] scripting language interface for thermodynamics calculations, the ViennaNGS [88] suite for extraction of genomic loci and the uShuffle Perl bindings [35] for k-let shuffling. The tool reports for each requested trinucleotide the opening energy in a genomic context as well as an opening energy z score obtained from n shuffling events of upstream and downstream sequences. Here, n = 10, 000 dinucleotide shuffling events were used.

MBEs are highly accessible in ZIKV 3'UTRs
The Musashi family of proteins preferentially bind single-stranded UAG motifs in 3'UTRs [82]. To evaluate the thermodynamics of Msi-UAG affinity more broadly, we set out to analyze the single-strandedness of all possible trinucleotides in ZIKV genomes. To this end, we computed the opening energies of all trinucleotide motifs present in the coding sequence (CDS) and 3'UTR of the African (ZIKV-UG) and Asian/American (ZIKV-BR) Zika strains. A z score was calculated for each occurrence of trinucleotide x according to Eq. 4, thereby normalizing the opening energy of x in its genomic context with n = 10, 000 dinucleotide-shuffled upstream and downstream regions of 97 nt, using 100 nt windows in RNAplfold.
Negative opening energy z scores indicate increased accessibility, i.e., UAG trinucleotides in viruses with overall low z scores are likely to occur in an unpaired structural context within the 3'UTR. Through the distribution of z scores, sorted by median z score ( Figure 2) we were able to see three aspects standing out. First, the distribution of z scores is markedly divergent among CDS and 3'UTR. The interquartile ranges of opening energy z scores are homogeneous within the CDS region, while dispersion is varied within the 3'UTR. We hypothesize that this is caused by a different sequence composition that manifests in highly variable opening energies. It could, however, also be an artifact of the different sample sizes based on the divergent trinucleotide count in CDS and 3'UTR, respectively. Second, UAG is the most accessible trinucleotide in the 3'UTR of ZIKV-BR and among the highest accessible trinucleotides in the 3'UTR of ZIKV-UG. This is striking as it corroborates previous experimental evidence of Musashi affinity to ZIKV [12] by means of a thermodynamic model, thus underlining a possible role of Msi1 in ZIKV neurotropism. Third, the canonical start codon AUG appears to the far right end of the scale in both ZIKB-BR and ZIKV-UG 3'UTRs, i.e., it is among the least accessible trinucleotides. This suggests evolutionary pressure on keeping the start codon in a paired structural context within the 3'UTR, thereby prohibiting accessibility to ribosomes and disabling undesirable leaky translation start from these AUG triplets.      GAA  AUU  AAC  CAA  CUC  UCA  UAA  UGU  AAG  GAU  AAU  GUA  GUC  AGC  AGG  GCU  UUC  CCU  CCC  GCG  GAG  CGU  UUA  CCA  CAC  GGG  AUG  GUU  CAG  UCG  CUU  GCA  UGC  CGG  ACG  CAU  ACU  CUG  GGA  GUG  ACC  UAC  GGU  UGA  AUC  GCC  UCU  GAC  CUA  UAG  UGG  CGC  AGU  UUU  UUG  GGC  UCC  CCG UGA  AAC  UUG  CUU  AUU  GUC  UGU  AGA  UCG  UAG  ACA  AGU  GAA  CUA  GAC  CCU  ACC  UUA  AUC  UUC  GUU  GAG  AGG  ACU  UUU  CCA  CUG  ACG  AAU  UCA  UGC  AGC  GAU  GCU  CUC  CCC  GCA  GCG  CCG  GGA  CAG  AUA  CGC  UCU  GCC  GUG  CAC  CGA  UGG  CAU  UCC  GGG  GGC  AAG  CGG GGU AUG Figure 2: Distribution of z scores of opening energies for trinucleotides found in the coding region (CDS, left) and 3'UTR (right) of ZIKV from Brazil (top) and Uganda (bottom), sorted by median opening energy z score. The MBE motif is highlighted in blue and shows low overall z scores in the 3'UTR, indicating that this trinucleotide is more likely to appear in a single-stranded structural context. Contrary, the canonical start codon AUG (highlighted in orange) shows high opening energy z scores, indicating reduced accessibility within the 3'UTR region. Data for trinucleotides AUU, UAA, UCG and UUU are omitted because they only occur once within the 3'UTR of ZIKV-BR. Likewise, trinucleotides CGU, GUA, UAA and UAU are omitted in the ZIKV-UG 3'UTR plot.

MBE accessibility in related viruses
To assess the Musashi-related neurotropic potential of other flaviviruses, we evaluated the accessibilities of MBEs in related species. To this end, all 435 UAG trinucleotide motifs within 3'UTRs in the refseq dataset were identified, grouped by vector specificity and subjected to the computational approach out-lined above (97 nt upstream/downstream windows, n = 10, 000 dinucleotide shufflings). Msi1 preferentially binds single stranded RNA [82], consequently UAG motifs that contribute with low z scores have a high affinity for Msi1 binding. Within the MBFV group, the Asian/American lineage Zika virus (ZIKV-BR) has the lowest median z score, followed by Saint Louis encephalitis virus (SLEV), Nounané virus (NOUV) and the African lineage Zika virus (ZIKV-UG). Among others, two lineages of West Nile virus (WNV1, WNV2) and Yellow fever virus (YFV) appear with a negative median z score. Interestingly, ZIKV-BR turns out to be the only isolate among MBFVs that has just negative z score values in our simulations, i.e., all UAG motifs within the 3'UTR of the Brazilian ZIKV isolate appear in an unpaired structural context. Likewise, Karshi virus (KSIV), Alkhumra hemorrhagic fever virus (ALKV) and Langat virus (LGTV) have a strictly negative z scores distribution among the TBFV group. POWV, Omsk hemorrhagic fever (OHFV) and Louping ill virus (LIV) have negative mean opening energy z scores. Among NKVs, Montana myotis leukoencephali-tis virus (MMLV) and Entebbe bat virus (ENTV) show negative mean opening energy z scores. Culex flavivirus (CxFV), Cell fusing agent virus (CFAG), Parramatta River virus (PaRV) and Ochlerotatus caspius flavivirus (OCFVPT) tend to have singe-stranded MBEs among the ISFVs. Here, OCFVPT is the only isolate with a strictly negative z score distribution (Figure 3).
The number of UAG trinucleotide motifs in 3'UTRs of the refseq dataset lies between 1 and 19 ( Table 1). The overall range of opening energy z scores is not equal among different flavivirus groups. While the lower bound is between −1.65 and −1.93 among all groups, MBFVs and ISFVs show markedly higher upper bounds than TBFVs and NKVs, respectively. Absolute values of computed MBE opening energy z scores are listed in Table 2.

Conserved xrRNAs contain MBEs
Several species appear to the right of the plots in Figure 3 due to the sorting by median z score. However, they comprise a non-negligible number of accessible MBEs, as indicated by negative opening energy z scores. Examples are (re-) emerging species like JEV and Usutu virus (USUV), which contain 15 and 19 MBEs, respectively.
To investigate this further, we assigned each MBE in our dataset to one of the conserved elements stem loop (SL), dumbbell (DB) and 3' stem loop (3SL) (Figure 1) by means of covariance models. Analysis of RNA sequence and structure conservation revealed that the majority of virus isolates in our dataset contain only a single UAG motif within their SL and 3SL elements. Conversely, DB elements, which are conserved in MBFVs and NKVs (Figure 4), stand out among conserved RNA structures in flavivirus 3'UTRs. They contain a pair of MBEs, separated by a 4 nt spacer, within a perfectly conserved sequence motif of approx. 20 nt length in their distal stem loop structure. We hypothesize that this pair of conserved UAG motifs interacts with the two RNA-binding domains in Musashi proteins. Figure 5 shows the consensus secondary structure of flavivirus DB elements.

DISCUSSION
Our findings lead to the conclusion that the accessibility of UAG motifs calculated through opening energies in Flavivirus 3'UTRs is indicative of the Musashirelated neurotropic potential of virus species. Our computational analyses show that there is little difference in the distribution of opening energies for all trinucleotides within the polyprotein (CDS) region of ZIKV. When comparing CDS and 3'UTR regions, we see a difference in behavior, as different trinucleotides do possess different opening energies, UAG being highly accessible in ZIKV. Although not possible to quantify the impact of the accessibility on the patient phenotype, it is interesting to see the UAG motifs on the Brazilian ZIKV isolate more accessible than on the Ugandan ZIKV isolate. This result raises the ((( (((((....(...(((((((.)))))..))).......))....... ((((......)))))))))) YOKV_NC_005039.1|DB.1|211-292| GGGAGCCUCCGCC  question once again if the increased pathogenicity seen in ZIKV today is due to changes in the sequence over time or simply lack of better surveillance [15]. Previous experiments lead toward the idea that ZIKV is unique among flaviviruses regarding the clinical outcomes resulting from congenital infection [67,39]. Although our results indicate that this may be true for well-studied viruses such as DENV and WNV, other viruses which have not caused recent outbreaks may have been neglected.
Looking in depth at other viruses, Nounané virus (NOUV), a dual-host affiliated insect-specific flavivirus is found among the viruses with high MBE accessibility. NOUV was isolated in Cote d'Ivoire in 2004 from Uranotaenia mashonaensis, a Culicidae mosquito not known to harbor flaviviruses before [38]. While replication has been tested on human and non-human cell lines, vertebrate infection and pathogenesis could not be observed [31].
Within the TBFV serocomplex, KSIV has the lowest overall MBE opening energies. Originally isolated from Ornithodoros papillipes ticks in Uzbekistan in 1972 [48] it currently does not present history of infection in humans. Conversely, Powassan virus (POWV), another TBFV with negative MBE opening  Figure 4 with R2R [85]. Structure and color annotation inferred by R2R. Nucleotide symbols represent conserved nucleotides. Circles represent columns in the MSA that are typically or always present but do not conserve nucleotide identity. Red, black and gray colors indicate the level of nucleotide conservation, in decreasing order. energy, originally isolated in Powassan, Ontario, Canada in 1959 from a child who died of acute encephalitis [53] can cause transplacental infection [64] and has been associated with severe neuropathology and death in mice and human [61].
Given that UAG can be regarded the primary Msi1 binding motif, we can argue that ZIKV has the highest affinity for binding Msi1 among all MBFVs. Platt et al. [64] showed that besides ZIKV, the neurotropic flaviviruses WNV and POWV, as well as the alphaviruses Chikungunya (CHIKV) and Mayaro (MAYV) infect placenta and fetus in immunocompetent, wild-type mice. However, only WNV was shown to infect the placenta and the fetal central nervous system, causing injury to the developing brain.
Bizarre neurological manifestations were also observed in patients infected by Ntaya virus (NTAV) [89], a neurotropic viruses from the Japanese encephalitis serocomplex, as well as WNV in humans [20,3] and in mice [37], USUV [72,7] and DENV [90,65]. The fact that these viruses line up more on the positive side of the opening energy plots in Fig. 3 does not mean that they should not be neurotropic. It merely highlights that there might be additional mechanisms causing neuropathogenicity.

MBEs are conserved in flavivirus 3'UTR elements
Flavivirus DB elements do not only show structural conservation over the MBFV and NKV serocomplexes, but even maintain their primary sequence within a region of approx. 20 nt of the distal stem-loop (Figs. 4 and 5). The combination of covariation and primary sequence conservation within a single RNA element underlines the importance of DB elements in flavivirus pathogenicity. It could also be indicative of a special role of DB element regions in the minus-strand synthesis during flavivirus replication.
Although the Musashi proteins could bind motifs other than UAG, UAG has been shown to be the motif contributing the highest binding free energy [91]. Our current analysis underlines that there seems to be evolutionary pressure on keeping UAG motifs within the DB elements unpaired. In ZIKV we see that not only the UAGs within DB elements, but also those that overlap with SL elements show a negative opening energy z-score.

Msi1 presence in different cells
The presence of Msi1 proteins in both sperm and neural precursor cells highlights the importance of studying the Msi1-MBE interaction in flaviviruses. Given that Msi1 has been shown to enhance ZIKV replication [12], this interaction could be a critical reason why ZIKV persists in sperm for a long time after the individual has been infected [71,17], allowing the virus to be transmitted sexually and also why the virus would harbor itself in neuronal cells, allowing it to interfere with and dysregulate neurodevelopment.

A possible role of Musashi in the flavivirus life cycle
Msi1, which binds to the 3'UTR of target mRNAs, has been shown to repress translation initiation by competing with translation initiation factor eIF4G for binding to poly(A)-binding protein (PABP), thereby inhibiting the assembly of the 80S ribosomal unit [41]. Ribosome profiling experiments have corroborated this down-regulatory effect of Msi1, while keeping mRNA levels [40]. This allows for a speculative explanation of the findings by Chavali et al. [12], i.e., that Msi1 enhances ZIKV replication, and a possible role of Msi1 in the viral life cycle: Flaviviruses need to "donate" a few copies of the quasispecies ensemble for Xrn1 degradation and subsequent sfRNA production. In this line, Msi1 could serve as agent that provides a reasonable amount of gRNAs that are not translated but subject to Xrn1 degradation. The resulting sfRNAs can then down-regulate the host response [13,51].

CONCLUSION
We studied a specific aspect of flavivirus congenital pathogenicity, i.e., the neurotropic effect inferred by the presence of MBEs in the 3'UTR of flavivirus genomes. Employing an established biophysical model of RNA structure formation, we analyzed the thermodynamic properties of MBEs in silico. Our results underline experimental studies suggesting that ZIKV is not alone in its capacity to cause severe neuropathology to infants through the MBE mechanism. While several tick-borne and mosquito-borne flavivirus species like Karshi virus (KSIV), Alkhumra hemorrhagic fever virus (ALKV) or Nounané virus (NOUV) line up with ZIKV in our theoretical model, their tropism might have been overseen due to the lack of reported significant outbreaks. However, some of them appear to have a similar neurotropic potential and thus might be potent emerging pathogens.

FUNDING
This work was partly funded by the Austrian science fund FWF project F43 "RNA regulation of the transcriptome".  Table 2: Distribution ranges of opening energy z scores for MBEs in the 3'UTR of flaviviruses. The minimal and maximal z score is listed for all UAG motifs (total) within a the 3'UTR, and only for those that overlap one of the conserved xrRNA elements SL and DB. Dashes indicate that SL/DB elements are not conserved.