Fitness Selection of Hyperfusogenic Measles Virus F Proteins Associated
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bioRxiv preprint doi: https://doi.org/10.1101/2020.12.22.423954; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title 2 Fitness selection of hyperfusogenic measles virus F proteins associated with 3 neuropathogenic phenotypes 4 5 Authors 6 Satoshi Ikegame1, Takao Hashiguchi2,3, Chuan-Tien Hung1, Kristina Dobrindt4, Kristen J 7 Brennand4, Makoto Takeda5, Benhur Lee1* 8 9 Affiliations 10 1. Department of Microbiology at the Icahn School of Medicine at Mount Sinai, New York, NY 11 10029, USA. 12 2. Laboratory of Medical virology, Institute for Frontier Life and Medical Sciences, Kyoto 13 University, Kyoto 606-8507, Japan. 14 3. Department of Virology, Faculty of Medicine, Kyushu University. 15 4. Pamela Sklar Division of Psychiatric Genomics, Department of Genetics and Genomics, Icahn 16 Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New 17 York, NY 10029, USA. 18 5. Department of Virology 3, National Institute of Infectious Diseases, Tokyo, Japan. 19 20 * Correspondence to: [email protected] 21 22 Authors contributions 23 S. I. and B. L. conceived this study. S.I. conducted library preparation, screening experiment, 24 fusion assay, and virus growth analysis. T. H. did the structural discussion of measles F protein. 25 C. H. conducted the surface expression analysis. K. R., and K. B. worked on human iPS cells 26 derived neuron experiment. M. T. provided measles genome coding plasmid in this study. B. L. 27 supervised this study. S.I. and B.L wrote the manuscript. 28 29 Competing interests: All authors declare no competing interests. 30 31 Classifications; Biological Sciences/Microbiology 32 33 Keywords 34 measles virus, fusion, mutagenesis 35 36 This PDF file includes: 37 Main Text 38 Figures 1 to 8 39 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.22.423954; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 40 Abstract 41 Measles virus (MeV) is resurgent and caused >200,000 deaths in 2019. MeV infection can 42 establish a chronic latent infection of the brain that can recrudesce months to years after recovery 43 from the primary infection. Recrudescent MeV leads to fatal subacute sclerosing panencephalitis 44 (SSPE) or measles inclusion body encephalitis (MIBE) as the virus spreads across multiple brain 45 regions. Most clinical isolates of SSPE/MIBE strains show mutations in the fusion (F) gene that 46 result in a hyperfusogenic phenotype in vitro and allow for efficient spread in primary human 47 neurons. Wild-type MeV receptor binding protein (RBP) is indispensable for manifesting these 48 mutant F phenotypes, even though neurons lack canonical MeV receptors (CD150/SLAMF1 or 49 Nectin-4). How such hyperfusogenic F mutants are selected for, and whether they confer a 50 fitness advantage for efficient neuronal spread is unresolved. To better understand the fitness 51 landscape that allows for the selection of such hyperfusogenic F mutants, we conducted a screen 52 of ≥3.1x105 MeV-F point mutants in their genomic context. We rescued and amplified our 53 genomic MeV-F mutant libraries in BSR-T7 cells under conditions where MeV-F-T461I (a 54 known SSPE mutant), but not wild-type MeV can spread. We recovered known SSPE mutants 55 but also characterized at least 15 novel hyperfusogenic F mutations with a SSPE phenotype. 56 Structural mapping of these mutants onto the pre-fusion MeV-F trimer confirm and extend our 57 understanding of the fusion regulatory domains in MeV-F. Our list of hyperfusogenic F mutants 58 is a valuable resource for future studies into MeV neuropathogenesis and the regulation of 59 paramyxovirus fusion. 60 61 Significance 62 Measles remains a major cause of infant death globally. On rare occasions, measles virus 63 infection of the central nervous system (CNS) leads to a fatal progressive inflammation of the 64 brain many years after the initial infection. MeV isolates from such CNS infections harbor fusion 65 (F) protein mutations that result in a hyperfusogenic phenotype. The small number of 66 hyperfusogenic MeV-F mutants identified thus far limits our ability to understand how these 67 mutations are selected in the context of CNS infections. We performed a saturating mutagenesis 68 screen of MeV-F to identify a large set of mutants that would mimic the hyperfusogenic 69 phenotype of MeV-F in CNS infection. Characterization of these mutants shed light on other 70 paramyxoviruses known to establish chronic CNS infections. 71 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.22.423954; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 Main text 73 Introduction 74 Measles is a highly contagious acute infectious disease caused by measles virus (MeV) (Genus 75 morbillivirus, Family Paramyxoviridae, Order Mononegavirales1). There has been a resurgence 76 of measles in recent years due to the lack or lapse of comprehensive vaccine coverage. The 77 global incidence of measles in 2019 of 120 per million represents a 6.7-fold increase from its 78 nadir in 2016 (18 per million). Primary MeV infections also caused an estimated 207,500 deaths 79 globally the same year2. These deaths occurred mostly in children under 5 years of age, who are 80 also most susceptible to complications of pneumonia, or diarrhea and dehydration. Measles 81 continue to exert its toll after recovery from acute infection. Due to virus-induced depletion of B- 82 cell memory pools— a form of immunological amnesia— recovered children can become newly 83 susceptible to common childhood infectious diseases 3–5. In the longer term, MeV can also cause 84 chronic latent central nervous system (CNS) infections such as measles inclusion body 85 encephalitis (MIBE) and subacute sclerosing panencephalitis (SSPE) 6. MIBE is restricted to 86 patients who are immunocompromised whereas SSPE can occur in fully immunocompetent 87 people 7-10 years after primary MeV infection7. The incidence of SSPE is rare; although more 88 recent estimates of its occurrence range from 22/100,000 to 30-59/100,000 in children that 89 acquire measles before the age of 5 8,9. That SSPE remains invariably fatal reflects our limited 90 understanding of the neuropathogenic complications of measles. 91 92 MeV is a non-segmented single-stranded negative sense RNA virus that is considered a 93 prototypical paramyxovirus10. Its genome encodes 6 genes that give rise to 8-9 proteins. The 94 nucleocapsid (N) encapsidates the RNA genome forming RNAse-resistant ribonucleoproteins 95 (RNPs) during viral replication. The phospho-(P) and large (L) proteins form the RNA- 96 dependent RNA polymerase (RdRp) complex that act as a viral transcriptase (P-L) or replicase 97 (N-P-L) at appropriate points in the viral life cycle. The matrix (M) protein facilitates the 98 assembly and budding of the RNP genome from the plasma membrane into virions that contain 99 the fusion (F) and receptor binding proteins (RBP, formerly termed H). All paramyxoviruses 100 require the co-ordinate action of F and RBP to mediate membrane fusion 11,12. Some 101 paramyxoviruses like MeV are preferentially cell-associated, can spread cell-to-cell, and 102 efficiently form multi-nucleated giant cell syncytia in appropriate receptor-positive cells13. 103 104 Primary MeV strains use CD150 and nectin-4 on immune and epithelial cells, respectively14,15, 105 neither of which are expressed on neurons or other brain parenchyma cells. This adds to the 106 mystery of how MeV establishes a chronic latent CNS infection that recrudesces many years 107 after recovery from the primary infection. However, characteristic mutations are known to arise 108 in CNS MeV isolates from patients with SSPE or MIBE. Nonsense mutations that result in a 109 non-functional M protein 16 and missense mutations that result in a hyperfusogenic F protein 17,18 110 are commonly found. Recombinant MeVs with a functional deletion of the M protein or 111 expressing the hypermutated M protein from an SSPE MeV isolate exhibit enhanced 112 fusogenicity and increased neurovirulence 19,20. Similarly, F mutants from neuropathogenic MeV 113 strains also show a hyperfusogenic phenotype in cells that do not express detectable amounts of 114 canonical MeV receptors (CD150 and nectin-4). This in vitro hyperfusogenic phenotype is 115 correlated with the ability of neuropathogenic MeV strains to initiate a spreading infection in the 116 CNS in vivo, and in human neuronal cell cultures in vitro 21,22 23. However, syncytia are never 117 observed in the brain or in human neuronal cells. It is unclear how neuropathogenic MeV spreads bioRxiv preprint doi: https://doi.org/10.1101/2020.12.22.423954; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 118 within the CNS and between neurons without forming syncytia. Proposed mechanisms include 119 the use of a MeV neuronal receptor (although a definitive candidate has not been identified) 6, or 120 host factors that could facilitate the putative trans-synaptic spread mediated by the 121 hyperfusogenic F protein 24. Nectin-elicited cytoplasmic transfer of MeV25 has been proposed as 122 a means to establish the initial transfer of infectious RNPs from epithelial cells to neurons, but 123 not subsequent CNS spread. 124 125 Regardless of the underlying mechanism, both MeV-F and -RBP are indispensable for neuronal 126 spread.