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 adults10. While MBFVs are typically transmitted by host-vector interaction, vertical transmission from mother to child during pregnancy via transplacental infection has been reported11.

The neurotropic potential of ZIKV-related flaviviruses has been known since the 1970s, when Saint Louis encephalitis virus (SLEV) has been attributed to a severe neurological disorder in infected mice12,13. Vertical transmission has been observed with JEV in mice14 and human15 and a case of human fetal infection have been reported after YFV vaccination16. Other transmission pathways of ZIKV include blood transfusions and sexual transmission17,18. Despite enormous efforts in studying ZIKV infections in the last years, the biological reasoning and mechanisms behind arbovirus congenital neurotropism remain elusive.

Flavivirus genome organization

Flaviviruses have the structure of an enveloped sphere of approximately 50 nm diameter. They are single-stranded positive-sense RNA viruses of 10–12 kb in size, and their genomic RNA (gRNA) encodes a single open reading frame (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 packaging19,20,21,22.

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 non-coding RNA (lncRNA) has been observed upon infection. These lncRNAs, also known as subgenomic flaviviral RNAs (sfRNAs)23, are stable decay intermediates derived from exploiting the host’s mRNA degradation machinery24.

sfRNAs are produced by partial degradation of viral gRNA by Xrn1, a host 5′-3′ exoribonuclease that is associated with the endogenous mRNA turnover machinery25,26. The enzyme stalls at highly conserved RNA structures in the viral 3′UTR, so-called Xrn1-resistant RNAs (xrRNAs), resulting in sfRNAs of variable lengths27,28. Xrn1-resistant RNAs and sfRNAs appear to be ubiquitously present in many flaviviruses. They have been described in MBFVs, including DENV29, YFV30, JEV31, and ZIKV32, TBFVs23,33, and recently in ISFVs and NKVs34,35. There is typically more than one xrRNA, given the diverse molecular architecture of different flavivirus 3′UTRs. Pseudoknot interactions have been proposed in some, but not all flavivirus xrRNAs32,36. While they may form transiently under certain conditions28, conclusive validation of their ubiquitous presence is missing. Hence, we will exclude them in this work. Earlier studies in our group have identified conserved RNA structural elements in viral 3′UTRs37,38,39,40,41, some of which have later been attributed to xrRNA functionality23. Stem-loop (SL) as well as dumbbell (DB) structures are found in 3′UTRs of flaviviruses in single or double copies (Fig. 1) and have been associated with quantitative protection of downstream viral RNA42.

Figure 1 Schematic representation of the ZIKV 3′UTR. Conserved RNA elements include two stem-loop structures (SL1 and SL2), a \({\rm{\Psi }}\)DB and canonical DB element as well as the terminal 3′ stem-loop structure (3-SL). Positions of Musashi-binding UAG motifs in the Asian/American ZIKV lineage are highlighted in orange. Possible pseudoknot interaction sites (sketched in light blue) do not overlap with potential Musashi binding sites. Full size image

The inhibition of Xrn1 by viral RNA yields sfRNAs that affect many cellular processes, both in the vector and the host43. In mosquitoes, sfRNA interacts directly with the predominant innate immune response pathway, RNA interference (RNAi), by serving as a template for microRNA (miRNA) biogenesis44. Conversely, in host cells sfRNA modulates the anti-viral interferon response45, e.g., by binding proteins to inhibit the translation of interferon-stimulated genes46. Moreover, sfRNA has been shown to inhibit Xrn1 and Dicer activity, thereby altering host mRNA levels47,48.

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.43 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 cells49,50,51 and overexpressed in tumors and leukemias52, they are absent in differentiated tissue. Moreover, Msi1 is involved in the regulation of blood-testis barrier proteins and spermatogenesis in mice53. Musashi proteins have two RNA recognition motif (RRM) domains, whose sequence specificity has been determined by an in vitro selection method and NMR spectroscopy51,54,55. The trinucleotide sequence UAG, whose thermodynamic binding specificity was determined by fluorescence polarization assays, has been identified as core Musashi binding element (MBE). Nucleotides enclosing the main MBE recognition motif make minor contributions to binding affinity56. While earlier SELEX experiments identified the binding aptamer sequence (G/A)U n AGU (n = 1 − 3)51, iCLIP experiments with Msi1 in human glioblastoma cells confirmed the preferential binding of Msi1 to single-stranded (stem-loop) UAG sequences in 3′UTRs, but not in coding regions57. Zearfoss et al.56 observed that both GUAGU and AUAGU are recognized by mouse Msi1, whereas Drosophila Msi1 has a higher affinity for GUAGU. NMR-derived structures of the two Msi1 RNA recognition motifs in complex with RNA also show that both RNA-binding domains bind GUAGU (PDB IDs 2RS2 and 5X3Z).

In summary, there is a strong consensus in the literature that UAG is central to all proposed Musashi binding motifs. Therefore, we focus our calculations around this trinucleotide, and provide evidence that the availability of UAG in pentanucleotides expands to the accessibility of the entire motif.

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 genome10, de Bernardi Schneider et al.7 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.58 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 al.59 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.