1 A single amino-acid substitution in the M protein attenuates Japanese 1 encephalitis virus in ...

61 Pages·2015·1.35 MB·English

JVI Accepted Manuscript Posted Online 9 December 2015
J. Virol. doi:10.1128/JVI.01176-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 A single amino-acid substitution in the M protein attenuates Japanese
2 encephalitis virus in mammalian hosts
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4 Mélissanne de Wispelaere1,§, Cécile Khou1, Marie-Pascale Frenkiel1, Philippe
5 Desprès2, Nathalie Pardigon1,#
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7 1 Environment and Infectious Risks Unit, Infection and Epidemiology Department, lo
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8 Institut Pasteur, Paris, France e
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9 2 UMR PIMIT (I2T), Université de La Réunion, INSERM U1187, CNRS 9192, IRD ro
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10 249, technology platform CYROI, Saint-Clotilde, La Reunion, France
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11 § Current address: Department of Microbiology and Immunobiology, Harvard //
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12 Medical School, Boston, MA, United States of America s
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15 # Corresponding author: [email protected] p
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16 4
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17 Running title: A mutation in M affects JEV in mammalian hosts 1
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18 Abstract word count: 198 y

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19 Importance word count: 108 e
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20 Text word count: 7582
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21 Abstract
22 Japanese Encephalitis virus (JEV) membrane (M) protein plays important
23 structural roles in the processes of fusion and maturation of progeny virus during
24 cellular infection. The M protein is anchored in the viral membrane and its
25 ectodomain is composed of a flexible N-terminal loop and a perimembrane helix.
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26 In this study, we performed site-directed mutagenesis on the residue 36 of JEV o
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27 M protein and showed that the resulting mutation had little or no effect on the lo
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28 entry process but greatly affected virus assembly in mammalian cells. e
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29 Interestingly, this mutant virus had a host-dependent phenotype, and could o
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30 develop a wild-type infection in insect cells. Experiments performed on infectious
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31 virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant //
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32 expresses structural proteins but fails to form infectious particles in mammalian s
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33 cells. Using a mouse model for JEV pathogenesis, we showed that the mutation o
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34 conferred complete attenuation in vivo. The production of JEV neutralizing o
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35 antibodies in challenged mice was indicative of the immunogenicity of the mutant p
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36 virus in vivo. Together, our results indicate that the introduction of a single 4
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37 mutation in the M protein, while being tolerated in insect cells, strongly impacts 1
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38 JEV infection in mammalian hosts. y

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39 e
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40 Importance
41 JEV is a mosquito-transmitted Flavivirus and is a medically important pathogen in
42 Asia. The M protein is thought to be important for accommodating the structural
43 rearrangements undergone by the virion during viral assembly, and may play
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44 additional roles in the JEV infectious cycle. In the present study, we show that a
45 sole mutation in the M protein impairs JEV infection cycle in mammalian hosts,
46 but not in mosquito cells. This finding highlights differences in Flavivirus
47 assembly pathways amongst hosts. Moreover, infection of mice indicated that the
48 mutant was completely attenuated and triggered a strong immune response to
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49 JEV, thus providing new insights for further development of JEV vaccines. o
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50 Introduction
51 Flaviviruses such as Japanese encephalitis virus (JEV), are arthropod-borne
52 pathogens (arboviruses) that are transmitted through the bite of an infected
53 mosquito and cause serious human diseases worldwide (1). JEV is the causative
54 agent for Japanese encephalitis, one of the most important viral encephalitis of
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55 medical interest in Asia, with an incidence of approximately 67,900 human cases o
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56 per year (2). Up to 30% of the symptomatic cases are fatal, while 30 to 50% of lo
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57 survivors can develop long-term neurologic sequelae (3). e
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58 JEV has a positive-sense RNA genome encoding a single polyprotein. This o
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59 polyprotein is processed by host- and JEV-encoded proteases into 10 proteins:
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60 three structural proteins (core [C], pre-membrane [prM], and envelope [E]) and //
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61 seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and s
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62 NS5). Similarly to other Flaviviruses, JEV enters cells via receptor-mediated o
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63 endocytosis (4, 5). The acidic environment in the host endosome serves as the o
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64 physiological trigger for a major conformational change in the E protein that leads p
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65 to the insertion of its fusion loops in the host endosomal membrane (6, 7). This 4
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66 results in fusion of the viral membrane with the host membrane and delivery of 1
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67 the viral genome to the cytoplasm. The RNA replication occurs within y

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68 invaginations of the endoplasmic reticulum (ER) (8-10). Following translation, the e
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69 viral RNA is encapsidated by C to form the nucleocapsid that interacts with the
70 prM and E proteins to bud at the ER membrane. The prM protein acts as a
71 chaperone for proper folding of JEV E protein (11), which leads to the formation
72 of an immature virion. In dengue virus (DV), a related Flavivirus, the structure of
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73 the immature virus is stabilized through interactions between the prM and E
74 proteins (12-16). The immature Flavivirus transits through the secretory pathway,
75 and the decrease in pH induces a rearrangement in the conformation of the
76 membrane proteins, that exposes the prM protein furin cleavage site (12, 17).
77 Subsequently prM is matured by furin into the pr and membrane (M) proteins in
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78 the trans-Golgi (12, 17-19). The pr fragment remains in close association with the o
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79 mature particle until secretion from the cells, and prevents premature fusion of lo
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80 viral and cellular membranes within cells (20). The prM cleavage is mandatory to e
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81 produce infectious particles, since immature particles containing solely uncleaved o
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82 prM are deficient in membrane fusion (21, 22). Yet, the prM cleavage site is
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83 suboptimal, leading to the secretion of partially mature particles (23, 24), that //
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84 were shown to interact uniquely with target cells and the host immune response s
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85 (25-29). The prM/M protein thus appears to play important roles in various o
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86 processes of the viral infection, and further studies are needed to identify the o
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87 molecular signatures associated to its diverse functions. p
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88 The M protein of Flaviviruses can be divided in three structurally distinct portions 4
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89 (13, 15): a flexible N-terminal loop (amino acids 1–20), an amphipathic 1
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90 perimembrane helix (amino acids 21–40) and a pair of transmembrane helices y

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91 (amino acids 41–75) (Figure 1A). The perimembrane and transmembrane helices e
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92 serve to anchor the M protein to the membrane. In the case of DV and JEV, the
93 perimembrane helix region was shown to be involved in virus assembly (30-33)
94 and entry (31, 32). Subsequent mutagenesis analysis of the M protein proved the
95 importance of an interactive network between E and M in those processes (16,
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96 34, 35). The Flavivirus prM/M protein was also shown to interact with mammalian
97 and mosquito host factors (36-41), thus indicating that this small protein may be
98 involved in additional non-structural aspects of the Flavivirus infectious cycle.
99 Notably, a peptide identified in Flaviviruses M proteins was shown to potently
100 trigger apoptosis in mammalian cells (42, 43). This pro-apoptotic phenotype
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101 could be alleviated once the residue located at position M-36 of wild-type yellow o
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102 fever virus (YFV) or DV2, respectively a leucine or an isoleucine (Figure 1B), was lo
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103 mutated to a phenylalanine (42). The residue M-36 lies on the hydrophobic side e
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104 of the amphipathic helix (Figure 1), and it is worth noting that phenylalanine, like o
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105 leucine or isoleucine, is a non-polar residue, and is not expected to modify the
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106 nature of the helix. Interestingly, the YFV vaccine strain 17D has a phenylalanine //
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107 at position M-36, and it is one of the 32 amino-acid differences between YFV- s
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108 17D and the wild-type YFV Asibi strain it was derived from (Figure 1B) (44). o
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109 While the contribution of this amino-acid change to YFV-17D vaccine properties o
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110 has not been fully evaluated, it is partly responsible for YFV-17D inability to infect p
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111 and disseminate in mosquitoes (45). Additionally, in DV4 the nature of the M-36 4
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112 residue is crucial for proper viral morphogenesis and subsequent entry, thus 1
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113 highlighting the importance of this residue in various aspects of the Flavivirus y

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114 infectious cycle (32). e
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115 In the present study, we examined the impact of a mutation of the isoleucine at
116 position 36 in JEV M into a phenylalanine (M-I36F) on the virus infectious cycle.
117 We show that this sole mutation impairs JEV infection in mammalian cells, but
118 not in mosquito cells. By using a virus-like particle system (VLP) we demonstrate
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119 that the introduction of the M-I36F mutation impairs assembly and/or secretion of
120 viral particles in mammalian cells. We also show that the JEV(M-I36F) mutant
121 virus is attenuated in vivo in a mouse model of JEV infection and that the mice
122 inoculated with the mutant virus produced JEV neutralizing antibodies. Thus in
123 vivo attenuation of JEV can be achieved through the introduction of a single
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124 mutation that affects viral assembly/egress, suggesting that such a mutation o
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125 could be used in the design of efficient new molecular JEV vaccines. lo
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126 Materials and Methods
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128 Cells
129 Mosquito Aedes albopictus C6/36 cells were maintained at 28°C in Leibovitz
130 medium (L15) supplemented with 10 % heat-inactivated fetal bovine serum
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131 (FBS). Baby hamster kidney-derived BHK-21, human neuroblastoma-derived SK- o
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132 N-SH, and human kidney-derived HEK293T cells were maintained at 37°C in lo
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133 DMEM supplemented with 10% FBS. e
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134 o
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135 Production of recombinant JEV
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136 A molecular cDNA clone of JEV genotype 3 strain RP-9 was kindly provided by //
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137 Yi-Lin Ling (46). This plasmid was modified as described previously (47). Briefly, s
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138 the plasmid was first modified to ensure correct propagation in bacteria, through o
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139 site directed mutagenesis of a bacterial cryptic promoter located between o
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140 positions 1787 and 1873 that had not yet been identified in the genome of JEV p
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141 RP-9 (48). We first used the primers pairs 5’- 4
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142 CAAGCTCAGTGAAGTTGACATCAGGCCACCTG-3’ / 5’- 1
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143 CAGGTGGCCTGATGTCAACTTCACTGAGCTTG-3’ and 5’- y

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144 GGCCACCTGAAATGCAGGCTGAAAATGG-3’ / 5’- e
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145 CCATTTTCAGCCTGCATTTCAGGTGGCC-3’ to introduce silent mutations
146 predicted to disrupt the bacterial promoter (mutations are underlined), then we
147 reintroduced a missing nucleotide at position A1915 using the primers 5’-
148 AGAAAAATTCTCGTTCGCAAAAAATCCGGCGGACAC-3’ and 5’-
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149 GTGTCCGCCGGATTTTTTGCGAACGAGAATTTTTCT-3’. A non-silent mutation
150 at position 3216, that changed the isoleucine at position 247 in the NS1 protein to
151 a valine, was also reverted to the wild-type sequence using primers 5’-
152 CATCATTCCGCATACCATAGCCGGACCAAAAAGCAA-3’ and 5’-
153 ttgctttttggtccggctatggtatgcggaatgatg-3’. The resulting plasmid, pBR322(CMV)-
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154 JEV-RP9, could then be stably propagated at 30°C in Stbl2 cells (Life o
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155 Technologies, catalog no. 10268-019). lo
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156 The M-I36F mutation was introduced directly in pBR322(CMV)-JEV-RP9 through e
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157 PCR mutagenesis using primers 5’- o
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158 CATGAAAACTGAGAACTGGTTCATAAGGAATCCTGGCTA-3’ and 5’-
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159 TAGCCAGGATTCCTTATGAACCAGTTCTCAGTTTTCATG-3’ (the mutation is //
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160 underlined). s
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161 To produce infectious virus, the molecular clones were transfected into C6/36 o
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162 cells using Lipofectamine 2000 (Life Technologies, catalog no. 11668-019). Once o
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163 a cytopathic effect was visible, viral supernatants were collected and used as p
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164 final virus stocks for experiments. The structural part of the JEV(M-I36F) genome 4
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165 was amplified by RT-PCR using SuperScript® III One-Step RT-PCR System with 1
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166 Platinum® Taq DNA Polymerase (Life technologies, catalog no. 12574-018) and y

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167 the primers 5’-ACGGAAGATAACCATGACTAAAAAACCAGGA-3’ and 5’- e
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168 TTCTGCAGTCAAGCATGCACATTGGTCGCTAAGA-3’. The PCR fragments
169 were then sequenced by Eurofins Genomics.
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171 Virus infections
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172 For infections, SK-N-SH or C6/36 cells were seeded in 24-well tissue culture
173 plates in respectively DMEM or L15, supplemented with 2% FBS. Aliquots of
174 virus were diluted in 200 μl of medium and added to the cells. Plates were
175 incubated for 1 h at 37°C or 28°C. Unadsorbed virus was removed by two
176 washes with Dulbecco's phosphate-buffered saline (DPBS) and then 1 ml of
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177 DMEM or L15 supplemented with 2% FBS was added to the cells, followed by o
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178 incubation at 37°C or 28°C until collection. lo
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179 e
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180 Recombinant virus transfections o
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181 For transfections, HEK293T cells were seeded in 24-well tissue culture plates in t
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182 DMEM supplemented with 2% FBS. Transfections were performed using /jv
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183 Lipofectamine 2000 (Life Technologies, catalog no. 11668-019) according to the s
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184 manufacturer’s instructions. The cells were incubated at 37°C until collection.
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185 o
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186 Antibodies p
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187 Mouse hybridomas producing the monoclonal antibody 4G2 anti-Flavivirus E ,

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188 were purchased from ATCC (catalog no. HB-112) and a highly purified antibody 1
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189 preparation was produced by RD Biotech (Besançon, France). Rabbit polyclonal y

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190 antibody anti-JEV C was kindly provided by Yoshiharu Matsuura (49). Rabbit e
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191 polyclonal antibody anti-JEV M was kindly provided by Young-Min Lee (50). The
192 antibody against prM was obtained by collecting sera from mice immunized
193 against JEV-RP-9. The antibodies against Calnexin and Actin were respectively
194 purchased from Enzo Life Sciences (catalog no. ADI-SPA-865) and Abnova
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