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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. This suggests that receptor engagement and fusion protein triggering remain essential for 127 MeV spread in the CNS. The convergence of data indicates that the functional hallmark of 128 mutations from SSPE and MIBE strains is the gain of a hyperfusogenic phenotype mediated by 129 MeV-F. Importantly, these hyperfusogenic MeV-F mutants manifest their phenotype most 130 clearly in cells that do not express the canonical MeV receptors (CD150 and nectin-4). For 131 example, a single point mutant from the SSPE Osaka-2 strain (T461I) is able to confer upon wild 132 type (wt) MeV (IC323) the ability to form syncytia and replicate in Vero cells. Similarly, the 133 recombinant IC323-T461I virus can now infect neurons in culture, and cause substantial 134 neuropathology when injected into brains of suckling hamsters 21. 135 136 The MeV-F protein is functionally constrained26 and also well-conserved amongst all clinical 137 isolates27. The hyperfusogenic F mutations that neuropathogenic MeV acquires must therefore be 138 highly beneficial to its spread in the CNS. Our ability to understand the fitness landscape of such 139 MeV-F mutations is currently limited by the relatively small number of MeV-F mutations 140 reported to exhibit such a hyperfusogenic SSPE phenotype 6, 28. A comprehensive account of 141 the mutational spectrum that can give rise to this hyperfusogenic phenotype will facilitate a 142 better understanding of how these MeV-F mutations confer their fitness advantage. 143 In this study, we generated a saturation point mutagenesis library of MeV-F in its genomic 144 context. We then designed a fitness screen where only viral genomes bearing MeV-F mutations 145 that mimic the hyperfusogenic SSPE phenotype will have a selective advantage. We not only 146 identified a number of MeV-F mutations similar to ones that have already been reported 6, but 147 also numerous novel mutations that span all three structural domains of MeV-F 29. Structure- 148 function studies confirm the SSPE phenotype of these mutations and identified a new site on the 149 MeV-F trimer that regulates MeV fusion activity. Finally, we identified a hyperfusogenic 150 mutation in a highly conserved residue in the fusion peptide of MeV-F that was generalizable to 151 Nipah virus, a member of the only other paramyxovirus genus known to harbor viruses that 152 cause chronic latent CNS infections. 153 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.

154 Result 155 Saturation mutagenesis screen for MeV-F mutants with hyperfusogenic SSPE phenotypes 156 Rationale. The cardinal phenotype of hyperfusogenic MeV-F mutants from SSPE and MIBE 157 strains is the ability to pair with wild type MeV-RBP and form syncytia in cells that do not 158 express the canonical MeV receptors. In order to better understand the mechanistic features that 159 underlie the hyperfusogenic phenotype of such MeV-F mutants, we designed a screen of a 160 saturating genomic MeV-F mutant library using BSR-T7 cells where this phenotype was the 161 most apparent and rMeV could be rescued with high efficiencies 26. As shown in Fig. 1, a rMeV 162 bearing the well characterized F-T461I hyperfusogenic mutant derived from the Osaka-2 SSPE 163 strain30 was able to infect, spread and replicate in BSR-T7 cells whereas its isogenic wild type 164 (wt) rMeV (IC323-GFP) counterpart could not. The latter confirms that BSR-T7 cells, a 165 hamster-derived cell line, do not express the canonical MeV receptors as wt rMeV-IC323 166 replicates well and forms obvious syncytia in cells that express human CD150 or nectin-431. 167 Syncytia and spread (GFP counts) was readily observed with the control rMeV-F T461I 168 hyperfusogenic mutant but not its isogenic wt counterpart (Fig 1A and B). We observed 169 significant differences as early as 2 days post-transfection/rescue. RT-qPCR for genome copy 170 numbers confirmed productive replication of rMeV-F-T461I (Fig 1C), which produced several 171 thousand-fold more viral genomes than wild type rMeV at day 8 post-rescue. 172 173 Library Preparation. Next, we used error-prone PCR to generate a saturating mutagenesis library 174 that covered the entire F gene. We reasoned that only mutants that result in the hyperfusogenic 175 SSPE phenotype exemplified by rMeV-F-T461I would have a fitness advantage when rescued in 176 BSR-T7 cells (Fig. 1C). In order to rescue this MeV-F library in its genomic context and have 177 the requisite coverage (Table S1), we generated four independent mutagenesis libraries of 178 equivalent sizes (402-418 nucleotides) that altogether span the entire MeV-F gene (Fig. 2A). 179 The size limitation of each library (~400 nt) was imposed by the maximum amplicon size we 180 could sequence confidently on Illumina machines (250 nt paired-end reads). The cloning strategy 181 to shuttle each mutagenesis library into the genomic context of rMeV-IC323 is depicted in Fig 182 2B. Before embarking on making all four libraries, we randomly chose library 3 (MeV-F ORF, 183 nt 840-1241) to optimize our error-prone PCR conditions. Fig 2C summarizes the distribution of 184 mutation rates we observed when we sequenced library 3 generated from low, medium and high 185 error-prone PCR conditions. Sequencing the same region amplified with high fidelity DNA 186 polymerase served as our background control. The mutations were evenly distributed across this 187 library 3 region regardless of mutation rates (Fig S1A to D), which averaged 0.04% for plasmid 188 DNA (background), and 0.12%, 0.18%, and 0.27% for the low, medium, and high mutation 189 settings, respectively (Fig S1E). The mutational spectrum was also relatively unskewed with a 190 transition/transversion (Ts/Tv) ratio close to 1 (0.81). We chose the high error rate condition to 191 move forward with all 4 libraries because we aimed to introduce 1 mutation/400 bp (0.25%), 192 which was the size of each library segment. When we set the threshold detection limit for 193 mutations at 0.08% (double the sequencing error rate (0.04%) of the negative control in Fig S1E) 194 and used the high ‘mutational setting’ for our error-prone PCR conditions, 97.3% of the region 195 covered in library 3 were mutated. 196 197 When the rest of libraries (1, 2 and 4) in pCDNA3-MV-F were made with the same high 198 mutation setting, there was also no obvious skewing in the distribution and spectrum of 199 mutations (Fig S2A to C). The mutation rate of libraries 1 (0.33%), 2 (0.27%), and 4 (0.22%) 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.

200 were similar to that of library 3 (0.25%), and all had Ts/Tv ratios close to 1 (Fig S2D). Then, 201 each of the F gene mutated libraries were independently transferred into the genome coding 202 plasmid using the cloning strategy shown in Fig. 2B. Sequencing of the MeV-F libraries in the 203 MeV genome showed that the mutation rate and spectrum were maintained in all libraries with 204 the exception of library 4, where for unknown reasons, the average mutation rate dropped from 205 0.22% to 0.14% (Table S1). Given that we aimed for one mutation/400 bp and that saturation 206 point mutagenesis requires ~1600 distinct mutations/library, the number of independent genomic 207 clones we generated per library (range: 2.32 – 8.55 x104) ensured that every possible nucleotide 208 substitution at every position was adequately represented. This was confirmed by deep 209 sequencing our genomic libraries where as expected, the coverage was ≥99% for all libraries 210 except library 4 (Table S1). 211 212 Identification of putative hyperfusogenic MeV-F genomic clones in our library screens. We 213 then used our efficient reverse genetics system to rescue wt MeV (IC323-GFP), the four MeV-F 214 mutated libraries, and the hyperfusogenic rMeV-F-T461I (positive control) in BSR-T7 cells as 215 described in Fig. 126,32. The virus producer cells were grown for 8 days with one passage at day 216 4. The average number of rescue events in each of these screening trials were around 60,000- 217 90,000, which is sufficient to represent the mutational diversity present in each library (Table 218 S1). When wt rMeV-IC323 was rescued and passaged under these conditions, only single cells 219 turned EGFP+, which did not spread. In contrast, libraries 1-4 each gave rise to obvious foci of 220 spreading EGFP+ syncytia by day 8 (Fig. 3A). Each of these large spreading syncytia likely 221 represents a hyperfusogenic mutant similar to that seen with the MeV-F-T461I positive control. 222 These phenotypic observations were confirmed and extended upon analysis of F gene sequences 223 by next generation sequencing NGS at 8 days post rescue. While the genome plasmid libraries 224 contained a relatively even distribution of mutations, we detected a clear selection of fit mutants 225 in all four MeV-F libraries at day 8 post-rescue (Fig. 3B). 226 227 Each library was independently passaged three times for a total of 12 replicates that made up the 228 screen of the entire MeV-F gene. Some mutants were reproducibly selected in more than one 229 replicate (e.g. L137H, L137F) and/or can account for more than 50% of the library reads at that 230 position (e.g. G376V, T314P, L137F). Table S2 shows the top hit list from these screening 231 experiments. Mutants selected from each library in each replicate were ranked by their percent 232 representation at that position. Despite some stochasticity, it was clear that some aa positions or 233 microdomains were hotspots for mutations that putatively conferred a hyperfusogenic phenotype. 234 This was underscored by the G376V mutant in library 3, which not only dominated the 235 outgrowth of mutants in replicate 1 and 2, but also a ‘fourth’ replicate performed during our 236 preliminary optimization experiments (Table S3). The synonymous mutations that showed up as 237 top hits in Table S2 (e.g. nt 351 mutant in replicate 1 of library 1) were almost always on the 238 same reads of other putative hyperfusogenic mutants, suggesting that these were ‘passenger’ 239 mutations. 240 241 Library 4 appeared to be an outlier in that the top hits in all three replicates never exceeded 2% 242 except for N462K in replicate 2 (7.3%). The relatively low mutation rate in the genome library 243 (0.14% vs 0.25-0.35% for libraries 1-3, Table S1) might have reduced the efficiency and spread 244 of mutants that would have otherwise been detected as hyperfusogenic. Remarkably, we were 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.

245 still able to detect and select for previously reported hyperfusogenic mutants such as N462K 33 246 and N465S 21 in addition to new mutations like G464W. 247 248 Selected MeV-F mutants have a hyperfusogenic phenotype. We developed a quantitative 249 image based fusion assay (QIFA) to evaluate the large number of potential hyperfusogenic MeV- 250 F mutants identified in our screen. QIFA is premised upon detecting syncytia frequency where 251 syncytia is defined by statistically robust criteria. We first transfected Lifeact-EGFP alone into 252 BSR-T7 cells and obtained a size distribution of single cells in the absence of any syncytia (Fig 253 S3A). We observed that 13 pixcel2 was the median size of single cell populations (n=2,605) and 254 GFP objects ≥260 pixcel2 (20X median single cell size) was rare in that more than 99% of cells 255 imaged were <260 pixcel2. So, we defined a bona fide syncytia as having ≥260 pixcel2 and 256 calculated syncytia frequency as a percent (%) of total GFP counts. To help us identify syncytia, 257 we transfected increasing amounts of plasmids expressing MeV-RBP and F-T481I along with a 258 fixed amount Lifeact-EGFP. Our QIFA was highly specific and quantitative, but the QIFA 259 metric (syncytia frequency %) had a dynamic range that plateaued around 8-10% (Fig S3 B and 260 C). The massive syncytia that formed in a finite area (e.g. between 50-100 ng MeV-RBP/F- 261 T461I transfected) limits the numerator. Nonetheless, the assay could be made more or less 262 sensitive by simply increasing or decreasing the hours post-transfection (hpt) before syncytia are 263 quantified (see below). 264 265 Next, we chose the best 2 non-synonymous mutants from each individual experiment in Table S2 266 (Library 1: Q96P, T132S, L137H, L137F; Library 2: P219T, S220I, G253E, L260S, S262N, 267 G264E; Library 3: A284T, H297Y, T314P, L354P, F375L, G376V; Library 4: G433E, S434G, 268 N462K, G464W, N465Y, D538G) plus two mutants (F375V, I393M) from the preliminary 269 screen of library 3 (Table S3). We also included D481G mutant (third best mutant in replicate 2 270 of library 4) because this corresponds to stalk region in which no previous hyper-fusion mutant 271 was reported. We tested fusion activity of these mutants in comparison with wt F protein which 272 remained mostly as single cells. Several mutants (L137H, L137F, S220I, G253E, S262N, 273 G264E, T314P, L354P, G376V, G464W) showed greatly increased fusion activity (≥ 8%) 274 comparable to the positive control T461I mutant (Fig 4A and 4B) at 30 hpt. Some (Q96P, 275 H297Y) showed moderately increased fusion activity (< 8%). Others (P219T, L260S, A284T, 276 F375V) showed only slightly increased syncytia frequency, which only became statistically 277 significant from wt at 48 hpt (Fig 4C and 4D). 278 279 To further differentiate between all the hyperfusogenic mutants that gave ≥ 8% syncytia 280 frequency, we repeated our QIFA on these mutants using half the amount of transfected MeV- 281 F/RBP (25 ng each). Under these limiting conditions, 6 of the 11 mutants still showed significant 282 syncytia formation above wt (Fig. S4A and B), as did our positive control T461I mutant. These 283 mutants were located in the C-terminus of the fusion peptide (L137F, L137H), at the protomer 284 interface (S262N), and in the neck region between the head and stalk domains (T314P, N462K, 285 G464W). 286 287 Although there was a general correlation between the relative mutational frequency of the 288 dominant mutants identified in the screening experiments and their fusogenicity as measured by 289 our QIFA, not all the mutants identified in the screening experiments were hyperfusogenic (Fig. 290 4E). Nonetheless, we were able to identify at least 15 novel MeV-F mutations that had a 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.

291 hyperfusogenic SSPE phenotype, effectively doubling the list of hyperfusogenic SSPE-like 292 mutants to date (Table S4). 293 294 A hyperfusogenic mutation in the conserved fusion peptide region is generalizable to the 295 Nipah virus F protein. L137F and L137H mutants repeatedly showed up and were often the 296 predominant mutations in all three replicates of library 1 screens (Table S2). In addition, our 297 quantitative image-based fusion assay (QIFA) revealed L137F and L137H to be as 298 hyperfusogenic as the positive control T461I mutant (Fig. 4A). L137 in MeV-F is located 299 towards the C-terminus of the fusion peptide. The homologous leucine is conserved amongst all 300 major paramyxoviruses (Fig 5A), as demonstrated by our alignment of the fusion peptide region 301 from the indicated viruses. We chose an overrepresentation of F proteins from morbilliviruses 302 and henipaviruses as paramyxoviruses that use protein-based receptors may have differential 303 features for fusion activation34. Nonetheless, a phylogenetic tree shows that we chose 304 prototypical viruses that span the diversity within the Paramyxoviridae (Fig 5B). We speculated 305 that mutation in this position may also change the fusion activity of other paramyxoviruses, 306 particularly those of henipaviruses, the only other genus of paramyxoviruses known to use 307 protein receptors and also cause chronic latent CNS infections35. Thus, we introduced these 308 mutations into NiV-F (L134H and L134F) and evaluated their fusion activity (when co- 309 transfected with NiV-RBP) by our QIFA. L134F markedly enhanced the fusion activity of NiV- 310 F beyond wt whereas L134H did not (Fig 5C and 5D). Interestingly, L137F was also 311 significantly more hyperfusogenic than L137H when fusion was evaluated under limiting 312 conditions (Fig S4A and B) (Tukey’s multiple comparisons test, adjusted p = 0.014). 313 314 Structural mapping of hyperfusogenic MeV-F mutations. Extant hyperfusogenic mutations in 315 MeV-F can be categorized into 3 sites based on how they mapped onto the crystal structure of 316 stabilized trimeric MeV-F 29. Site I mutations are in the region surrounding fusion peptide, site II 317 mutants localize to the interface of the protomers while site III mutations cluster in the neck 318 domain between head and stalk. Most of our hyperfusogenic mutants mapped to one of these 319 sites (Fig 6A). Mutants mapping to site I (Q96P, L137F, L137H, F375V, and G376V, Fig 6B) 320 can potentially affect fusion peptide exposure and the biophysical properties of the fusion 321 peptide itself. Site II mutants such as P219T, S220I, G253E, L260S, S262N, and G264E (Fig 322 6C) can disrupt the inter-protomer interactions that keep F from being pre-maturely triggered. 323 Site III mutants (T314P, L354P, G464W) at the base of the head domain (Fig 6D) can also 324 destabilize the F trimer resulting in lower activation energy for fusion triggering. Interestingly, 325 two of our hyperfusogenic mutants, A284T and H297Y, are located on the beta-sheet connecting 326 the head and neck region (corresponds to Y277 to G301). This region is structurally distinct from 327 sites I - III and no hyperfusogenic SSPE-derived mutants have been mapped to this beta-sheet. 328 We propose classifying this region as site IV, which may represent a novel fusion-regulatory 329 domain encompassing at least the two mutants identified in our study (Fig 6D, A284T and 330 H297Y). 331 332 Recombinant measles virus expressing selected hyperfusogenic F mutants recapitulate the 333 SSPE phenotype. To confirm that the hyperfusogenic MeV-F mutants identified in our screen 334 was necessary and sufficient to confer a SSPE phenotype, we chose 4 mutants (L137F, S262N, 335 H297Y, and G464W) to rescue as isogenic rMeV. We chose the best mutant from sites I-IV as 336 evaluated by our QIFA (Fig 4A and Fig S4A). For site III, we chose G464W instead of N462K 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.

337 because N462K has already been reported33. rMeV-IC323EGFP with the L137F, S262N, or 338 G464W F mutations formed huge syncytia (Fig 7A) and replicated several hundred-fold better 339 than wt even at day 6 post-rescue (Fig 7B). rMeV with the F-H297Y mutant showed a less 340 dramatic increase in the syncytia size at day 6, but nonetheless showed significant increase in 341 genome copy numbers by 8 days post-rescue compared to wt (Fig 7C). To substantiate the 342 biological relevance of our structural mapping efforts, we used a well-characterized fusion 343 inhibitory peptide (FIP) that not only blocks MeV fusion 36, but does so specifically by 344 interacting directly with residues in site III such as G464 29. We found that the G464W mutant 345 was resistant to FIP but not the other non-site III mutants (Fig 7D and 7E). To ensure that these 346 results were not due to differences in surface expression and/or F protein cleavage, we performed 347 cell surface biotinylation experiments. Briefly, surface expressing F proteins were pull downed 348 by streptavidin-beads after cell surface protein biotinylation, and the immunoprecipitated cell 349 surface MeV-F detected by Western blot using a MeV-F specific antibody (Fig 7F). 350 Densitometry showed that surface F protein expression was highest in wt F and the T461I 351 mutant, but all the hyperfusogenic mutants (L137F, S262N, and G464W, and H297Y) were 352 expressed at ~50% of wt levels mutants (Fig 7G). Cleavage efficiency was also evaluated by the 353 analyzing the ratio of F1/F0 (Fig 7H), which showed no significant differences in cleavage 354 efficiency between wt and any of the hyperfusogenic mutants. Altogether, these results show 355 that the hyperfusogenic phenotype was not due to overexpression or more efficient cleavage of 356 the indicated F mutants. 357 358 A cardinal feature of SSPE MeV strains is the ability to infect and spread in primary human 359 neurons, which do not express the canonical receptors for primary strains of MeV (CD150 and 360 nectin-4). Wild-type MeV can infect neurons, albeit inefficiently, but neurovirulent SSPE/MIBE 361 MeVstrains can infect and spread in cultured primary human neurons. To mimic primary human 362 neuronal infection, we used human iPSC-NPC-derived neurogenin 2(NGN2)-induced 363 glutamatergic neurons that are a well-characterized model of excitatory forebrain neurons37. 364 Upon wt rMeV-IC323EGFP infection, we detected a few GFP+ neurons at 2 days post-infection 365 (dpi). GFP appeared distributed in the cell bodies as well as the dendrities and axons that 366 outlined the neurons distinctly (Fig. 8A). On rare occasions, a small cluster of GFP+ neurons 367 could be found connected by GFP+ neurites, which suggest a slow cell-to-cell spread of MeV 368 (Fig. 8A, middle and bottom panels). This is reminiscent of what was reported by Sato et al. in 369 NT2-derived neurons22. In contrast, inoculation of the same neuronal cultures with the rMeV-F- 370 L137F mutant resulted in much larger clusters of GFP+ neurons that sometimes appeared to 371 coalesce (Fig. 8B, top panel). The latter show that novel hyperfusogenic F mutants identified in 372 our screen can recapitulate the SSPE phenotype involving efficient spread in primary human 373 neurons. 374 375 376 Discussion 377 Our saturating mutagenesis screen allowed us to interrogate the fitness landscape that allowed 378 for the selection of hyperfusogenic MeV-F mutants associated with SSPE/MIBE phenotypes, 379 which is the ability to pair with wt MeV-RBP and form syncytia in cells lacking the canonical 380 MeV receptors. We identified at least 15 such hyperfusogenic mutants, effectively doubling the 381 list of known hyperfusogenic SSPE/MIBE mutants curated in the past several decades (Table 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.

382 S4). These mutants are a community resource which can be used to shed further light on fusion 383 regulatory mechanism and aid in the search for the putative neuronal receptor for MeV. 384 385 The antigenic variability of MeV is highly constrained38. All vaccine strains are derived from 386 genotype A, yet sera from vaccinees can neutralize the broad range of circulating genotypes. 387 The monoserotypic nature of MeV implies the lack of antigenic drift and functional constraints 388 on its surface envelope glycoproteins (F and RBP). The latter is highlighted by our previous 389 study showing that MeV F and RBP are completely intolerant to 15 bp (5 aa) insertions during a 390 whole genome transposon mutagenesis screen 26. However, the unit of selection in neurovirulent 391 MeV strains appears to be single nucleotide mutations in MeV-F that confer the ability for 392 neuronal spread 6,21. This adaptation of MeV to spread within the central nervous system (CNS) 393 is correlated with in vitro syncytia formation and cell-to-cell spread in the absence of canonical 394 receptors. Therefore, we prepared saturation point mutagenesis libraries of MeV-F in its genomic 395 context and performed a reverse genetics rescue screen in a hamster derived cell line (BSR-T7), 396 where hyperfusogenic SSPE-like F mutants can spread and replicate, but not wt MeV (Fig 1). 397 The use of the well characterized F-T461I SSPE mutant helped us in choosing the optimal cell 398 line for the screen (Fig. 1) and served as a positive control for each replicate (Fig. 3) and 399 functional validation (Fig. 4). 400 401 For saturation coverage, we prepared four separate libraries of ~400 nt in size that altogether 402 span the entire F gene (Fig. 2). There are biological and technical reasons for this strategy. To 403 ascertain single mutations that can act independently in a given library, the library size is limited 404 by the maximal Illumina read depth (2x 250 bp paired end reads), which is effectively <450 bp 405 if one excludes the primer binding sites. To maximize the quality of our library and sensitivity of 406 our screen, we also wanted the biggest difference between the average mutation rate of our 407 libraries and the intrinsic error rate of Illumina sequencing, which we confirmed to be 0.04% in 408 our hands (Fig. 2C, Table S1E). Illumina’s reported error rate is between Q35 – Q40 (0.035% - 409 0.001%). If we had targeted the entire 1.6 kb F gene with an average of one point mutation, the 410 average mutation rate would be 0.063%, which will be statistically indistinguishable from 411 intrinsic error rate of Illumina sequencing. However, introducing a single point mutation in ~ 412 400 bp will result in an average mutation rate of 0.25%, which is comfortably 8-fold above the 413 background error rate. A similar strategy was also adopted by a previous influenza mutagenesis 414 screening study 39,40 where the authors separated the influenza genome into 52 parts comprising 415 of 250 bp segments. 416 417 Although some synonymous mutants appeared in our top hit list (Table S2), most were on the 418 same read of other non-synonymous mutations that were later confirmed to be hyperfuosgenic. 419 This suggests that these synonymous mutations are passenger mutations associated with bona 420 fide hyperfusogenic mutations. Separation of the F gene into four independent libraries thus 421 enabled us to designate these synonymous or neutral mutations as such. 422 423 424 The veracity of our functional screen for hyperfuosgenic MeV-F in its genomic context is 425 underscored by our ability to recover most of the extant hyperfusogenic F mutants that exhibit a 426 SSPE phenotype as long as that mutant was present in our original library. For hyperfuosgenic 427 mutants that we did not recover, close inspection of our data (Table S4) revealed that they were 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.

428 either not present in the original genomic mutant library (e.g. L454W), or present but 429 outcompeted by a distinct mutant at the same amino acid residue (e.g. S262N>>S262R, 430 L354P>>L354M, T461A>>T461I, G464W>>G464E), or known to be hypefusogenic only when 431 present together (G168R, E170G, AS440P, R520C and L550P). This lends greater support to 432 the 16 hyperfusogenic mutants we validated from our screening experiments (lib1: Q96P, 433 L137H, L137F; lib2: P219T, S220I, G253E, L260S, S262N, G264E; lib3: A284T, H297Y, 434 T314P, L354P, G376V; lib4: N462K, G464W). Except for the previously characterized N462K 435 mutant, others were all novel mutants. 436 437 L137 mutants are particularly interesting. L137 is located in the C-terminus of fusion peptide and 438 is highly conserved among all paramyxoviruses. The homologous L134F mutation in the NiV-F 439 protein also made NiV-F hyperfusogenic in the presence of NiV-RBP (Fig 5C and 5D). How far 440 this extends to other paramyxoviruses is a subject for future studies. 441 442 In addition to validating their phenotype in our fusion assays, we rescued four mutants (one from 443 each library) as independent rMeV clones and showed that they exhibited the expected 444 hyperfusogenic SSPE phenotype (Fig. 7). Altogether, these data indicate our saturation 445 mutagenesis screening system is robust and efficiently identifies mutants dictated by the 446 screening conditions. This system can also be applied to genes of interest in other 447 paramyxoviruses to find interferon vulnerable mutations for better vaccine development as was 448 done for influenza 39. 449 450 451 A majority of the previously reported hyperfusogenic mutations (Table S4) are located on site III 452 (part of library 3 and library 4 in our study) in the prefusion structure of trimeric MeV-F (Fig.6, 453 and Table S4), which might have overemphasized the contribution of this site (base of the head 454 domain) to the neurovirulent hyperfusogenic phenotype. However, our screening found many 455 novel and strong phenotypic mutations in site 1 and site 2, suggesting that mutations in all sites 456 found across all three structural domains of F can contribute to the hyperfusogenic phenotype. In 457 addition, we found new mutants – A284T and H297Y— which are located in the beta-sheet 458 (Y277-G301) that connect the head and neck domains (Fig 6D). These two mutants in this 459 structurally distinct site IV do not appear to have as strong a phenotype but are nonetheless real 460 as rMeV-F-H297Y can grow and replicate in BSR-T7 cells. 461 462 As expected from crystal structure, G464W may resist FIP inhibition because G464 directly 463 interacts with FIP in the complex crystal structure 29. Our data showed that G464W was indeed 464 resistant to FIP inhibition (Fig 7E) just as G464E was also reported as a FIP-resistant hyper 465 fusiogenic mutation 41. This suggest that our screen can be judiciously applied to study the 466 barriers to drug or antibody resistance. 467 468 469 Our reverse genetics rescue screen based on a particular phenotype is made possible by the 470 transformative improvements in the efficiency of paramyxovirus rescue. This can now be 471 applied to any gene in any paramyxoviruses for which rescue efficiency is high enough. 472 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.

473 Method 474 Cell lines. 475 293T cells (ATCC),Vero cells (CCL-81; ATCC), and BSR T7 cells 42 were propagated in 476 Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum 477 (Atlanta Biologicals) at 37°C. Vero cells constitutively expressing human SLAM (Vero-hSLAM 478 cells) were gifted from Dr. Yusuke Yanagi 43. 479 480 Plasmids and viruses. 481 We modified the genome coding plasmid for MeV (p(+) MV323-AcGFP) 44 and generated 482 pEMC-MV323-EGFP; AcGFP gene was deleted and GFP gene were inserted at the head of 483 genome, and hammer head ribozyme was introduced to increase rescue efficiency. This clinical 484 isolate is well characterized and based on the clinical isolate of H4 strain 45. Helper plasmid for 485 virus (N, P, and L protein expression plasmid) rescue were gifted from Dr. Richard Plemper 32. 486 The plasmid coding full length viral genome were maintained in Stbl2 E. coli (Thermo Fisher 487 Scientific) with growth at 30°C. 488 DNA fragment of measles RBP and F gene were amplified from p(+) MV323-AcGFP and 489 cloned into pCDNA3 or pCAGGS vector, creating pCDNA3-MV-F, pCAGGS-MV-RBP, and 490 pCAGGS-MV-F. We introduced mutation to the plasmid or modified the plasmid with the site 491 directed mutagenesis by overlapping PCR, followed by in-fusion (Clontech) reaction to fuse the 492 mutated PCR fragment with backbone cut by restriction enzyme. Primer set used for making 493 each mutant are listed in Table S5. 494 495 Error prone PCR based mutagenesis. 496 Point mutations in F gene were introduced by Genemorph II random mutagenesis kit (Agilent) 497 which uses error-prone PCR to introduce mutations. We divided F gene into 4 part with 402 -418 498 bps size; library 1 (418 bp), library 2 (418 bp), library 3 (402 bp), and library 4 (409 bp) as 499 written in Fig 2A. Mutation rate can be adjusted by adjusting the amount of initial template. We 500 used DNA fragments from pCDNA3-MV-F cut by Nru I-HF (NEB) and Pac I (NEB) restriction 501 enzyme as an error-prone PCR template. Amplified PCR fragments were fused with backbone 502 prepared by high-fidelity PCR of Cloneamp (Clonetech) using NEBuilder (NEB), then 503 transformed into stellar competent cells (Clonetech). We scaled up so that each library generate 504 > 20,000 colonies. Bacterial colonies were collected 24 hours after plating and then amplified in 505 Terrific Broth medium (Research Products International) for several hours. DNA were extracted 506 by Plasmid DNA Maxi Prep Kits (Thermo Fisher Scientific). 507 Next, the insert for genome plasmid was made by treating pCDNA3-MV323-F mutated libraries 508 with Nru I and Pac I. The insert and genome backbone (pEMC-MV323-EGFP cut by Nru I-HF 509 and Pac I) were ligated with T4 DNA ligase (NEB), followed by transformation into 510 electrocompetent cells with ElectroMAX stbl4 (Thermo Fisher Scientific), securing > 20,000 511 colonies in each library. Bacterial colonies were collected 24 hours after plating and then 512 amplified in Terrific Broth medium for several hours. DNA (F gene mutated genome coding 513 plasmid) were extracted by Plasmid DNA Maxi Prep Kits. 514 515 Recovery of recombinant measles virus from cDNA. 516 For the recovery of recombinant MeV, 4 × 105 BSR-T7 cells were seeded in 6-well plates. The 517 next day, the indicated amounts (detailed below) of antigenomic construct, helper plasmids (-N, - 518 P and -L), T7 construct, and Lipofectamine LTX / PLUS reagent (Invitrogen) were combined in 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.

519 200 uL Opti-MEM (Invitrogen). After incubation at room temperature for 30 minutes, the DNA - 520 lipofectamine mixture was added dropwise onto cells. Transfected cells were incubated at 37°C 521 with medium replacement every day. The amount of measles plasmids used for rescue follows 522 our previous study 32: 5 ug antigenomic construct, 1.2 ug MeV-N, 1.2 ug MeV-P, 0.4 ug MeV-L, 523 3 ug of a plasmid encoding a codon-optimized T7 polymerase, 5.8 uL PLUS reagent, and 9.3 uL 524 Lipofectamine LTX. 525 526 Growth comparison between wild type and F T461I mutant. 527 Plasmid coding wild type measles or F T461I were transfected to one well (6 well plate) of BSR- 528 T7 cells, then cells were grown with medium replacement every day. At day 4, BSR-T7 cells 529 were treated with trypsin, and 1 / 6 cells were passed into one well in new 6 well plate, then 530 grown for additional 4 days. The number of GFP positive cells was counted at day 2, day 4, day 531 8 by Celigo Imaging Cytometer (Nexcelom). Supernatant was collected for titration at day 4 and 532 day 8. We modified waiting time down to 6 days (cells were passaged at day 3 once) when 533 comparing the growth of L137F, S262N, and G464W, because syncytia became quite huge and 534 detached from dishes for 8 days waiting time. 535 536 Tittering viral supernatants by plaque assay. 537 Rescued MeV were grown in Vero-hSLAM cells, then supernatants were collected. For tittering 538 virus, monolayer of Vero-hSLAM cells in 12 well was infected by 500 ul of serially diluted 539 samples for 1 hour, followed by medium replacement with methylcellulose containing DMEM. 4 540 days later, the number of GFP positive plaque was counted under fluoro-microscope to decide 541 titer. 542 543 Passaging virus in the screening experiment. 544 MeV genome coding plasmid with mutations in F protein (genome libraries) were transfected to 545 BSR-T7 cells with helper plasmid and T7 expressing plasmid (as indicated at ‘Recovery of 546 recombinant MeV from cDNA’). Rescued viruses were grown in BSR-T7 cells for 4 days with 547 medium replacement every day. At day4, BSR-T7 cells in one well of 6-well plate were treated 548 by trypsin and passed into 6 wells of 6-well plate. Then BSR-T7 cells with MeV were grown for 549 additional 4 days (total 8 days). Cytosolic RNA was extracted, reverse transcribed, and 550 amplified, then sequenced by next-generation sequencer to evaluate variation of F gene sequence 551 after growth in BSR-T7 cells. 552 553 RT-PCR and Illumina sequencing. 554 The cytosolic RNA was extracted by phenol-chloroform method with Trizol (Thermo Fisher 555 Scientific). The entire F gene sequence was amplified by RT-PCR reaction using Superscript III 556 (Thermo Fisher Scientific) with a primer set to detect F gene, creating 1.7 K bp fragment. The 557 RT-PCR fragment were used as a template for nested PCR by Cloneamp to amplify the sequence 558 of mutated part (402 – 418 bp, excluding primer sequence). The information of primer is 559 available in Table S5. 560 DNA library preparations, sequencing reactions, and initial bioinformatics analysis were 561 conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). DNA Library Preparation, clustering, 562 and sequencing reagents were used throughout the process using NEBNext Ultra DNA Library 563 Prep kit following the manufacturer’s recommendations (Illumina, San Diego, CA, USA). End 564 repaired adapters were ligated after adenylation of the 3’ends. The pooled DNA libraries were 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.

565 loaded on the Illumina instrument according to manufacturer’s instructions. The samples were 566 sequenced by MiSeq on a 2x 250 paired-end (PE) configuration. Base calling was conducted by 567 the Illumina Control Software (HCS) on the Illumina instrument. 568 569 Data analysis and selection of the top list. 570 Paired-end Fastq files were merged by BBtools to make single read. We trimmed low quality 571 nucleotides in edge of sequence to by PRINSEQ because 5’ and 3’end of sequence tend to show 572 low quality value 46. We trimmed nucleotides until PRINSEQ found the first nucleotide of 573 quality value (QV) => 33, then we selected the read with the average QV >= 33. 574 WIG file (include nucleotide counts at each position) were generated from processed SAM and 575 BAM files with IGV tools. Mutation rate were calculated from nucleotides counts at each 576 position using reference sequence file. 577 578 Genome quantification by qPCR 579 Extracted RNA was reverse transcribed by genome specific primer (written in Table S5) with 580 Tetro Reverse Transcriptase (Bioline), then the number of genome was quantified by 581 SensiFAST™ SYBR® & Fluorescein Kit (Bioline) and CFX96 Touch Real-Time PCR 582 Detection System (Biorad). 583 584 Image based fusion assay 585 BSR-T7 cells were seeded at a density of 40,000 cells/well onto 48-well dish at 24 hours before 586 transfection. Then, cells were transfected with 200 ug of pCAGSS-MV-RBP, 200 ug of 587 pCAGGS-MV-F or F-mutants, and 50 ug of pEGFP C1 Lifeact-EGFP (Addgene, plasmid 588 #58470) with 1.25ul of Lipofectamine 2000 (Invitrogen). At 30 hours (for the evaluation of 589 major mutants) or 48 hours (for the evaluation of minor mutants) post transfection, GFP+ cells 590 and syncytia were captured via an imaging cytometer (Celigo™, Nexcelom) using the green 591 channel (Ex/Em: 483/536 nm). Images were exported at the resolution of 5 um / pixel. The GFP- 592 positive foci (single cell or syncytia by fusion) were analyzed by ImageJ (developed by NIH), 593 creating the profile of individual GFP-positive foci with size information. 594 For quantification of syncytia formation that reflects a hyperfusogenic phenotype, we first 595 measured the size distribution of single cells. Over 5 replicates of cells transfected with MV- 596 RBP and LifeActGFP only (8:1), which should not result in any syncytia, the median size of 597 putative single cells was 13 pixels2 (n= 2,605 objects). We then counted all GFP-positive foci ≥ 598 7 pixel2 (half of the median size of single cells). Then we calculated the frequency of syncytia 599 which is defined as the GFP foci of ≥ 260 pixel2 (20 times of average median size of single 600 cells) / total GFP counts of ≥7 pixcel2. 601 For Nipah virus fusion assay, we used 100 ng of pCAGGS-Nipah-RBP-HA and 100 ng of 602 pCAGGS-Nipah-F-AU1 (both from Malaysia strain) and waited 30 hours post transfection. 603 For NDV fusion assay, we used 100 ng of pCAGSS-NDV-RBP, 100 ng of pCAGGS-NDV-F 604 (cloned from lasota strain) and waited 48 hours post transfection. 605 606 Fusion inhibitory peptide (FIP) 607 FIP (Z-D-Phe-Phe-Gly-OH) was purchased from BACHEM (#4015768). FIP was dissolved into 608 10mM solution by DMSO. FIP solution was added to the medium at the final concentration of 609 2% (200 uM) at 3 hours post transfection. DMSO was used as a negative control of FIP. 610 Cell surface expression analysis of F protein 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.

611 12 ug pCAGGS-IC323-F or F mutants (L137F, S262N, G464W, H297Y, and T461I) were 612 transfected to 293T cells (8x 10^6 cells in 10 cm dish) with X-tremeGENE™ HP DNA 613 Transfection Reagent (Roche). 48 hours after transfection, cell surface proteins were labeled with 614 biotin, then pulled down by streptavidin beads by using cell surface biotinylation and isolation 615 kit (ThermoFisher, #A44390). Collected surface expressed proteins were run on 4 - 15% poly 616 polyacrylamide gel (Bio-rad. #4561086) and transferred onto PVDF membrane (FisherScientific, 617 #45-004-113), followed by primary antibody reaction and secondary antibody reaction. For the 618 detection of measles virus F protein, rabbit polyclonal antibody (Abcom, ab203023) was used as 619 the primary antibody. For the detection of internal control, pan-cadherin antibody (Cell 620 signaling, #4068) and GAPDH Rabbit monoclonal Antibody (Cell signaling technology, #2118) 621 were chosen as cell surface and cytosolic protein, respectively. Alexa Fluor 647-conjugated anti- 622 rabbit antibody (Invitrogen, #A-21245) were used as secondary antibody appropriately. Image 623 capturing and signal intensity analysis were done by ChemidocTM MP (Biorad). 624 625 Neuron infection experiment 626 On day -1 NPCs were dissociated with Accutase Cell Detachment Solution for 5min at 37°C, 627 counted and seeded at a density of at 6x104 cells/well on Matrigel coated 24-well plates in hNPC 628 media (DMEM/F12 (Life Technologies #10565), 1x N-2 (Life Technologies #17502-048), 1x B- 629 27-RA, 20 ng/mL FGF2 (Life Technologies)) on Matrigel (Corning, #354230). On day 0, cells 630 were transduced with rtTA and NGN2 lentiviruses as well as desired shRNA viruses in NPC media 631 containing 10 M Thiazovivin and spinfected (centrifuged for 1 hour at 1000g). On day 1, media 632 was replaced and dox was added with 1ug/mL working concentration. On day 2, transduced 633 hNPCs were treated with the corresponding antibiotic to the lentivirus (1 μg/mL puromycin for 634 NGN2-Puro). On day 4, medium was switched to Brainphys neuron medium (Brainphys 635 (STEMCELL, # 05790), 1% N-2, 2% B-27-RA, 1 μg/mL Natural Mouse Laminin (Thermofisher, 636 # 23017015), 20 ng/mL BDNF (R&D, #248), 20 ng/mL GDNF (R&D, #212), 250 μg/mL 637 Dibutyryl cyclic-AMP (Sigma, #D0627), 200 µM L-ascorbic acid (Sigma, # A4403) plus 1 μg/mL 638 dox. From day 6 to day 15, 200nM cytosine arabinoside (Ara-C) was added to Brainphys neuron 639 medium to prevent extended proliferation, medium was changed half every second day. For 640 maturation, medium was replaced half with Brainphys neuron medium every second day until 641 infection on day 21. 642 5 x 103 PFU /well (tittered in Vero-hSLAM cells) of wild type MeV and MeV-F L137F were 643 added to infect the neurons for one hour, then virus reagent was removed and replaced by fresh 644 medium. Cells were incubated for 2 days, then fixed by 2%-PFA at 2 days post infection. Infected 645 cells were visualized by Cytation 3 (Biotek). 646 647 648 Data and materials availability: The raw next generation sequencing results are uploaded at

649 NCBI GEO: GSE14767.

650 651 Ethics declaration 652 All hiPSC research was conducted under the oversight of the Institutional Review Board (IRB) 653 and Embryonic Stem Cell Research Overview (ESCRO) committees at the Icahn School of 654 Medicine at Mount Sinai (ISSMS). Informed consent was obtained from all skin cell donors as 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.

655 part of a study directed by Judith Rapoport MD at the National Institute of Mental Health 656 (NIMH). 657 658 Acknowledgement 659 This work was supported by Japan Agenct for Medical Research and Development (AMED) 660 Grant 20wm0325002h (to T.H.), JSPS KAKENHI Grant Numbers 20H03497 (to T.H) and Joint 661 Usage/Research Center program of Institute for Frontier Life and Medical Sciences, Kyoto 662 University.” 663 664 Funding information 665 S.I., and C.H. were supported by Fukuoka University’s Clinical Hematology and Oncology 666 Study Group (CHOT-SG) Fellowship and a post-doctiral fellowship from the Ministry of 667 Science and Technology (MoST, Taiwan), respectively. This study was supported in part by 668 NIH grants AI115226 and AI123449 to B.L. This work was also supported by Japan Agent for 669 Medical Research and Development (AMED) Grant 20wm0325002h (to T.H.), JSPS KAKENHI 670 Grant Numbers 20H03497 (to T.H) and Joint Usage/Research Center program of Institute for 671 Frontier Life and Medical Sciences, Kyoto University. 672 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.

673 References 674 1. Amarasinghe, G. K. et al. Taxonomy of the order Mononegavirales: update 2017. Arch. 675 Virol. 162, 2493–2504 (2017). 676 2. Patel, M. K. et al. Progress Toward Regional Measles Elimination — Worldwide , 2000 – 677 2019. MMWR 67, 1323–1329 (2020). 678 3. Mina, M. J. et al. Measles virus infection diminishes preexisting antibodies that offer 679 protection from other pathogens. Science. 366, 599–606 (2019). 680 4. Mina, M., Metacalf, C. J., Swart, R., Osterhaus, A. D. M. E. & Grenfell, B. T. Childhood 681 Infectious Disease Mortality. Science. 348, 694–700 (2015). 682 5. Petrova, V. N. et al. Incomplete genetic reconstitution of B cell pools contributes to 683 prolonged immunosuppression after measles. Sci. Immunol. 4, (2019). 684 6. Watanabe, S., Shirogane, Y., Sato, Y., Hashiguchi, T. & Yanagi, Y. New Insights into 685 Measles Virus Brain Infections. Trends Microbiol. 27, 164–175 (2019). 686 7. Ludlow, M., McQuaid, S., Milner, D., De Swart, R. L. D. & Duprex, W. P. Pathological 687 consequences of systemic measles virus infection. J. Pathol. 235, 253–265 (2015). 688 8. Bellini, W. J. et al. Subacute sclerosing panencephalitis: More cases of this fatal disease 689 are prevented by measles immunization than was previously recognized. J. Infect. Dis. 690 192, 1686–1693 (2005). 691 9. Schönberger, K., Ludwig, M. S., Wildner, M. & Weissbrich, B. Epidemiology of 692 Subacute Sclerosing Panencephalitis (SSPE) in Germany from 2003 to 2009: A Risk 693 Estimation. PLoS One 8, 1–8 (2013). 694 10. Plemper, R. K. & Lamb, R. A. Paramyxoviridae: The viruses and their replication. in 695 Fields Virology: Emerging Viruses (eds. Howley, P. M. & Knipe, D. M.) (Wolters 696 Kluwer, 2020). 697 11. Jardetzky, T. S. & Lamb, R. A. Activation of paramyxovirus membrane fusion and virus 698 entry. Curr. Opin. Virol. 5, 24–33 (2014). 699 12. Plattet, P., Alves, L., Herren, M. & Aguilar, H. C. Measles virus fusion protein: Structure, 700 function and inhibition. Viruses 8, (2016). 701 13. Rota, P. A. et al. Measles. Nat. Rev. Dis. Prim. 2, (2016). 702 14. Mateo, M., Navaratnarajah, C. K. & Cattaneo, R. Structural basis of efficient contagion: 703 Measles variations on a theme by parainfluenza viruses. Curr. Opin. Virol. 5, 16–23 704 (2014). 705 15. Lin, L. T. & Richardson, C. D. The host cell receptors for measles virus and their 706 interaction with the viral Hemagglutinin (H) Protein. Viruses 8, 1–29 (2016). 707 16. Cattaneo, R. et al. Biased hypermutation and other genetic changes in defective measles 708 viruses in human brain infections. Cell 55, 255–265 (1988). 709 17. Ning, X. et al. Alterations and diversity in the cytoplasmic tail of the fusion protein of 710 subacute sclerosing panencephalitis virus strains isolated in Osaka, Japan. Virus Res. 86, 711 123–131 (2002). 712 18. Schmid, A. et al. Subacute sclerosing panencephalitis is typically characterized by 713 alterations in the fusion protein cytoplasmic domain of the persisting measles virus. 714 Virology 188, 910–915 (1992). 715 19. Cathomen, T. et al. A matrix-less measles virus is infectious and elicits extensive cell 716 fusion: Consequences for propagation in the brain. EMBO J. 17, 3899–3908 (1998). 717 20. Patterson, J. B. et al. Evidence that the hypermutated M protein of a subacute sclerosing 718 panencephalitis measles virus actively contributes to the chronic progressive CNS disease. 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.

719 Virology 291, 215–225 (2001). 720 21. Watanabe, S. et al. Mutant Fusion Proteins with Enhanced Fusion Activity Promote 721 Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters. J. 722 Virol. 87, (2013). 723 22. Sato, Y. et al. Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent 724 on Hemagglutinin and Hyperfusogenic Fusion Protein. J. Virol. 92, 1–11 (2018). 725 23. E.M., J. et al. Measles fusion machinery is dysregulated in neuropathogenic variants. 726 MBio 6, 1–12 (2015). 727 24. Makhortova, N. et al. Neurokinin-1 enables measles virus trans-synaptic spread in 728 neurons. Virology 362, 235–244 (2007). 729 25. Generous, A. R. et al. Trans-endocytosis elicited by nectins transfers cytoplasmic cargo, 730 including infectious material, between cells. J. Cell Sci. 132, (2019). 731 26. Fulton, B. O. et al. Mutational Analysis of Measles Virus Suggests Constraints on 732 Antigenic Variation of the Glycoproteins. Cell Rep. 11, 1331–1338 (2015). 733 27. Rota, J. S., Hummel, K. B., Rota, P. A. & Bellini, W. J. Genetic variability of the 734 glycoprotein genes of current wild-type measles isolates. Virology 188, 135–142 (1992). 735 28. Angius, F. et al. Analysis of a Subacute Sclerosing Panencephalitis Genotype B3 Virus 736 from the 2009-2010 South African Measles Epidemic Shows That Hyperfusogenic F 737 Proteins Contribute to Measles Virus Infection in the Brain. J. Virol. 93, 1–13 (2018). 738 29. Hashiguchi, T. et al. Structures of the prefusion form of measles virus fusion protein in 739 complex with inhibitors. Proc. Natl. Acad. Sci. 115, 2496–2501 (2018). 740 30. Ayata, M. et al. The F Gene of the Osaka-2 Strain of Measles Virus Derived from a Case 741 of Subacute Sclerosing Panencephalitis Is a Major Determinant of Neurovirulence. J. 742 Virol. 84, 11189–11199 (2010). 743 31. Sugiyama, T. et al. Measles virus selectively blind to signaling lymphocyte activation 744 molecule as a novel oncolytic virus for breast cancer treatment. Gene Ther. 20, 338–347 745 (2013). 746 32. Beaty, S. M. et al. Efficient and Robust Paramyxoviridae Reverse Genetics Systems. 747 mSphere 2, 1–14 (2017). 748 33. Doyle, J. et al. Two Domains That Control Prefusion Stability and Transport Competence 749 of the Measles Virus Fusion Protein. J. Virol. 80, 1524–1536 (2006). 750 34. Azarm, K. D. & Lee, B. Differential features of fusion activation within the 751 paramyxoviridae. Viruses 12, (2020). 752 35. Dawes, B. E. & Freiberg, A. N. Henipavirus infection of the central nervous system. 753 Pathog. Dis. 77, 1–10 (2019). 754 36. Richardson, C. D., Scheid, A. & Choppin, P. W. Specific inhibition of paramyxovirus and 755 myxovirus replication by oligopeptides with amino acid sequences similar to those at the 756 N-termini of the Fl or HA2 viral polypeptides. Virology 105, 205–222 (1980). 757 37. Ho, S. M. et al. Rapid Ngn2-induction of excitatory neurons from hiPSC-derived neural 758 progenitor cells. Methods 101, 113–124 (2016). 759 38. Beaty, S. M. & Lee, B. Constraints on the genetic and antigenic variability of measles 760 virus. Viruses 8, 1–20 (2016). 761 39. Du, Y. et al. Genome-wide identification of interferon-sensitive mutations enables 762 influenza vaccine design. Science. 359, 290–296 (2018). 763 40. Ye, J. et al. Error-prone pcr-based mutagenesis strategy for rapidly generating high-yield 764 influenza vaccine candidates. Virology 482, 234–243 (2015). 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.

765 41. Ha, M. N. et al. Mutations in the Fusion Protein of Measles Virus That Confer Resistance 766 to the Membrane Fusion Inhibitors Carbobenzoxy-d-Phe-l-Phe-Gly and 4-Nitro-2- 767 Phenylacetyl Amino-Benzamide. J. Virol. 91, 1–19 (2017). 768 42. Buchholz, U. J., Finke, S. & Conzelmann, K. K. Generation of bovine respiratory 769 syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in 770 tissue culture, and the human RSV leader region acts as a functional BRSV genome 771 promoter. J. Virol. 73, 251–259 (1999). 772 43. Ono, N. et al. Measles Viruses on Throat Swabs from Measles Patients Use Signaling 773 Lymphocytic Activation Molecule (CDw150) but Not CD46 as a Cellular Receptor. J. 774 Virol. 75, 4399–4401 (2001). 775 44. Seki, F. et al. The SI Strain of Measles Virus Derived from a Patient with Subacute 776 Sclerosing Panencephalitis Possesses Typical Genome Alterations and Unique Amino 777 Acid Changes That Modulate Receptor Specificity and Reduce Membrane Fusion 778 Activity. J. Virol. 85, 11871–11882 (2011). 779 45. Kobune, F., Sakata, H. & Sugiura, A. Marmoset lymphoblastoid cells as a sensitive host 780 for isolation of measles virus. J. Virol. 64, 700-705. J. Virol. 64, 700–5 (1990). 781 46. Ma, X. et al. Analysis of error profiles in deep next-generation sequencing data. Genome 782 Biol. 20, 1–15 (2019). 783 784 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.

785 Figures and Tables.

786 787 Figure 1. BSR-T7 cells allowed efficient spread of hyperfusogenic (F-T461I mutant) but not wild 788 type (wt) MeV. Recombinant MeV genomes (IC323-GFP) bearing wt F or F-T461I were rescued via 789 reverse genetics in BSR-T7 cells using a transfection-only protocol (see Methods). (A) At 2 days post- 790 transfection (dpt), many GFP+ single cells were seen in both wt and F-T461I rMeV rescue wells. By 8 dpt, 791 most of the GFP+ cells in the wt rMeV rescue wells had disappeared while the GFP+ cells in the rMeV-F- 792 T461I rescue wells showed an increase in both number and size. The latter is indicative of a hyper 793 fusogenic SSPE phenotype. (B) GFP+ cells/syncytia in both the wt and T461I rMeV rescue wells were 794 imaged and quantified on 2, 4 and 8 dpt. Results shown are mean values (+/- standard deviation) from 5 795 independent experiments. (C) Genome copy numbers in the cytoplasm for both viruses were quantified 796 by RT-qPCR and normalized to the mean value of wt rMeV. Results shown are mean relative genome 797 copy numbers (+/- standard deviation) from 5 independent experiments. Statistically significant 798 differences in the growth and spread of rMeV bearing wt versus F-T461I are indicated (*, p<0.01; **, p< 799 0.005; ***, p < 0.001, unpaired Student’s t-test). All images shown were captured on the Celigo imaging 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.

800 cytometer (Nexcelom) and GFP+ counts enumerated with the manufacturer’s software. Images are 801 computational composites from an identical number of fields in each rescue well. White scale bar equals 802 2 millimeters. 803 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.

804 805 Figure 2. Preparation of genomic MeV-F saturation mutagenesis libraries. (A) Schematic of MeV-F 806 showing the relevant structural and functional domains. Regions targeted by libraries of 1-4 are indicated 807 below the schematic. Nucleotide (nt) and amino acid (aa) positions in the MeV-F open reading frame 808 (ORF) are indicated counting from the initiator methionine. (B) The strategy for using error-prone PCR to 809 construct the MeV-F saturating mutagenesis library in its genomic context is shown. Point mutations in a 810 given library region of F gene were introduced first by error-prone PCR (red X box) and transferred back 811 into the pcDNA3-MV323-F shuttle plasmid via NEB builder. The full F ORF, now containing the mutated F 812 library region, and flanked by NruI/PacI restriction sites, was then transferred to the MeV genome coding 813 plasmid via direct ligation using the same unique restriction sites in the UTRs flanking the MeV-F gene. In 814 this way, four independent measles genome plasmid libraries were generated with saturating F mutations 815 that altogether cover the entire gene. (C) The distribution of mutation rates in pcDNA-MV323-F libraries 816 (library 3) generated under low, medium or high error -prone PCR conditions, or amplified with high 817 fidelity DNA polymerase from the parental ‘plasmid’ is shown. Box and whiskers plot shows the 10/90 818 percentile (whiskers), 25/75 percentile (box), and median (horizontal bar). 819 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.

820 821 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.

822 Figure 3. Fitness screen of MeV-F mutagenesis libraries identifies hyperfusogenic F mutants. (A) 823 Representative images from rescue of MeV-F libraries at 8 dpt in BSR-T7 cells show spreading GFP+ 824 syncytia of varying sizes in all libraries except for wt MeV, suggesting that hyperfusogenic mutants are 825 being positively selected. The T461I rescue well showed multiple giant syncytia as expected since only a 826 single clone was transfected. Images were captured by the Celigo Imaging Cytometer (Nexcelom) and is 827 a computational composite of several identical fields of view taken in each well. White scale bar equals 2 828 millimeters. (B) Mutation rate across the F gene in our saturation mutagenesis genome plasmid libraries 829 before transfection and rescue are indicated by the solid blue circles. The horizontal line at 0.08% 830 represents the threshold we used to define a genuine mutation at any given position. Distribution statistics 831 of the mutants in each genomic library is given in Table S1. Salmon, grey, and green solid circles 832 represents the mutation rate for each library rescued three independent times (replicates 1-3 for library 1- 833 4 = 12X total) based on MeV-F genomic RNA extracted from BSR-T7 cells at 8 days post-rescue. The 834 graph is a concatenation of deep sequencing results of library 1, 2, 3, and 4. The salmon, grey and green 835 circles outlined in black represent the two most predominant mutants in each library and replicate as 836 indicated in Table S2. These mutants were chosen for functional validation in Fig. 4. The open circles 837 correspond to synonymous passenger mutations that were associated with their cognate hyperfusogenic 838 mutants (detailed in Table S2 notes). A scaled schematic of the MeV-F protein is shown below the graph 839 for interpretative convenience. 840 841 842 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.

843 844 Figure 4. Validation of selected hyperfusogenic MeV-F mutants by a quantitative image based 845 fusion assay (QIFA). We selected the top two mutants from each replicate experiment across all 4 846 libraries (Table S2) to assess their fusion phenotype (see text for details). We transfected MeV-F/RBP-F 847 and Lifeact-EGFP into BSRT-T7 cells and quantified syncytia formation via our QIFA as described in Fig. 848 S3. (A) Standard QIFA using 200 ng each of MeV RBP and the indicated MeV-F. Images were captured 849 at 30 hours post-transfection (hpt) and analyzed. Data shown are mean (+/- S.D.) syncytia frequency (%) 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.

850 per total GFP counts from five independent experiments. Red, yellow and green bars indicate syncytia 851 formation at the levels indicated in the key. Dunnet’s multiple comparison test was used for the detection 852 of statistically significant differences above wild type MeV-RBP and F. Mutants from each library were 853 assayed with MeV-F-T461I always serving as a positive internal control. Representative images of the 854 summary data in A are shown in (B). For mutants that did not show significant syncytia at 30 hpt, we re- 855 evaluated them at 48 hpt (C). Representative fusion images of C are shown in (D). A heat map 856 summarizes how well the putative hyperfusogenic mutants identified in our library screens correspond to 857 their fusogenic activity (E). Since each library had a different average mutation rate (Table S1) and each 858 replicate was independently rescued and passaged, we first calculated the relative mutation frequency 859 (RMF) for each of the top-ranked mutants (Table S3). RMF = the mutation rate for a given 860 mutation/highest mutation rate for that experiment. In this way, the highest ranked mutant in each library 861 for a given replicate was always 100% and all mutants in that experiment were enriched relative to that 862 highest ranked mutant. The RMF for the indicated mutants from all three replicates can thus be averaged 863 and shown as mean RMF (left column). The fusion activity for the same mutants was categorized into 864 five groups (right column): 0, same as wild typ; 1+, syncytia visible only after 48 hours; 2+, 4-8% at 30 865 hpt; 3+, ≥8 % at 30 hpt; 4+, significantly different from wt at low expression conditions (25 ng MeV-RBP/F 866 transfected) (Fig. S4). Images shown were generated by the Celigo Imaging Cytometer as described in 867 Fig. 3. White scale bar (B and D) equals 500 micrometers. 868 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.

869 870 Figure 5. The homologous mutation of MeV-F-L137F in Nipah virus F (NiV-F-L134F) renders NiV-F 871 hyperfusogenic. 872 (A) Amino acid sequence alignment of the fusion peptide region flanking the L137 residue in MeV (arrow) 873 shows that it is highly conserved amongst paramyoviruses. The F1/F2 protease cleavage site (arrowhead) 874 is indicated as a point of reference. Prototypic viruses were chosen to represent paramyxoviruses from 875 all three subfamilies and the major genera within each subfamily. (B) A phylogenetic tree of the F protein 876 sequence demonstrates that the selected F proteins span the diversity within Paramyxoviridae. Amino 877 acid sequences were aligned by clustalw, and the phylogeny was generated by the maximum likelihood 878 method using MEGA 10 (version 10.1.8). The numbers at the node indicates the fidelity by bootstrap test 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.

879 (1,000 times). The scale indicates substitutions per site. (C) Introduction of the homologous mutation at 880 this position (L134F) made the NiV-F protein hyperfusogenic. Fusion activity In NiV-RBP and F 881 transfected cells was evaluated by our quantitative image based fusion assay (QIFA) as described in Fig. 882 4. Mean values of 5 independent experiments are shown with error bar indicating standard deviation. 883 Dunnet’s multiple comparison test was used for tests of significance for the indicated comparisons. (D) 884 Representative images of data presented in (B). Images were generated by the Celigo Imaging 885 Cytometer as described in previous figures. White scale bar equals 500 micrometer. Canine morbillivirus 886 (Canine MV); NP_047205, Rinder morbillivirus (Rinder MV); YP_087124, Small ruminant morbillivirus 887 (Small ruminant MV); YP_133826, Phocine morbillivirus (Phocine MV); YP_009177602, Cetacean 888 morbillivirus (Cetacean MV); NP_945028, Feline morbillivirus (Feline MV); YP_009512962, Sendai virus 889 (Sendai); NP_056877, mumps virus (mumps); NP_054711, Nipah virus (Nipah); NP_112026, Hendra 890 virus (Hendra); NP_047111, Cedar virus (Cedar); YP_009094085, Newcastle disease virus (NDV); 891 YP_009513197, Nariva virus (Nariva); YP_006347587, Beilong virus (Beilong); YP_512250. 892 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.

893 894 Figure 6. Structural mapping of hyperfusogenic F mutants. (A) Amino acid residues (spheres) whose 895 substitutions confer hyperfusogenicity were mapped onto the trimeric pre-fusion MeV-F structure 896 (PDB:5YXW) using Pymol. A ribbon model is shown where each of the protomers is colored rainbow, 897 dark gray and light gray, respectively. (B-D) The majority of mutants discovered in this study, while novel, 898 mapped to the three sites (I, II and III) that were previously used to classify extant hyperfusogenic 899 mutants by Hashiguchi et al. (E) Two mutants, A284T and H297Y, mapped to structurally distinct beta- 900 sheet that connects the head and neck domains, which we term site IV. In the aggregate model showing 901 all the mutants (A), Site I-IV mutants are represented by orange, yellow, green, and blue spheres, 902 respectively. 903 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.

904 905 906 Figure 7. Recombinant MeV expressing the hyperfusogenic F mutants from each of the four sites 907 exhibit the expected phenotype. Recombinant MeV with wt F, T461I, and representative F mutations 908 (L137F, S262N, G464W, H297Y) from sites I-IV (Fig. 6) were rescued in Bsr-T7 cells. Virus growth was 909 monitored via fluorescence microscopy (A). At day 6 post rescue (with one passage at day 3), cytosolic 910 RNA was collected and genome copy number was quantified by RT-qPCR (B). Genome copy numbers 911 for all the mutants were normalized to that for wt MeV, which was set to 1 (B and C). Growth of H297Y 912 mutants was further evaluated for 8 days incubation period (with one passage at day 4) (C). Data shown 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.

913 are mean relative genome copy numbers (+/- S.D) from 5 independent experiments (B and C). 914 Statistically significant differences from wt were determined by Dunnet’s multiple comparison test (B) and 915 t-test (C). Syncytia images were generated by the imaging cytometer as in previous figures. White scale 916 bar equals 2 milimeters. MeV-specific fusion-inhibitory peptide (FIP) inhibited the fusion activity of all the 917 hyperfusogenic mutants except the G464W mutant, as evaluated by our QIFA (D). Data shown are mean 918 (+/- S.D.) from 5 independent experiments. Representative images of the fusion inhibitory data are shown 919 in (E). P<0.05 or <0.01 for the indicated comparisons (Student’s t-test). Images generated on the image 920 cytometer as before. White scale bar equals 500 micrometers. (F and G) Cell surface biotinylation 921 experiments show that the L137F, S262N, G464W, and H297Y hyperfusogenic mutants were expressed 922 at lower levels than wt F or F-T461I. At 48 hpt, biotinylated cell surface proteins on MeV-F transfected 923 293T cells were pulled down by streptavidin-beads and western-blotted with a MeV-F specific antibody. 924 Cadherin and GAPDH served as respective cell surface and cytosolic protein controls. The upper and 925 lower blots in (F) show the input and surface protein (streptavidin pull-down) fraction, respectively. The 926 full-length F0 and cleaved F1 products are marked (arrowhead). * indicates the non-specific band from the 927 cytosol (upper blot) which disappeared in the cell surface pull-down fraction (lower blot). (G) shows the 928 relative F1 surface expression levels of the various F proteins normalized to wt F set at 1 (based on 929 densitometric measurements). (H) shows the F1 / F0 ratio of cell surface F, which indicates cleavage 930 efficiency. Data shown are the average and range of two independent experiments. 931 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.

932 933 934 Figure 8. Recombinant MeV with hyperfusogenic F-L137F mutant infects and spreads efficiently in 935 primary human neurons. Homogenous iPSC-derived NGN2-induced glutamatergic neurons, seeded 936 and differentiated in 24-well plates, were infected with 5000 PFU of wild type MeV (A) or MeV-F-L137F 937 (B) per well. (A) At 2 dpi, rare GFP+ neurons could be seen in wild type MeV infected wells. GFP was 938 localized to the cell body as well as the axon and dendrites. Occasionally, small clusters of several 939 neurons could be seen (middle panel). (B) In contrast, MeV-F-L137F infected wells showed numerous 940 GFP positive neurons in large clusters at 2 dpi. 3 representative images from 3 independent infections 941 are shown. Images were taken on the Cytation 3. White scale bar equals 200 micrometers. 943