Effect of site-directed mutagenesis within the 5′UTR and VP1 regions on temperature sensitivity of Enterovirus 71 in vitro. Natallia Lazouskaya, Enzo Palombo, Tony Barton. Environment and Biotechnology Centre, Swinburne University of Technology, Hawthorn VIC 3122 Australia Introduction • Enterovirus 71 (EV71) is a human pathogen associated both with sporadic cases and large outbreaks of hand, foot and mouth disease (HFMD) throughout the world (1). Young children under 5 are most susceptible to EV71 infection, although, adult related cases have also been reported (2, 3). The clinical manifestation in EV71 patients may vary from mild and self-limited conditions to severe neurological complications, such as meningitis, brain-stem encephalitis, meningoencephalitis, poliomyelitis-like paralysis or pulmonary oedema with lethal outcome. Due to the lack of anti-EV71 specific inhibitors or vaccines, treatment of EV71-associated complications relies on symptomatic and supportive therapies (4). In the current situation, the major research efforts are being focused on the development of vaccination strategies against EV71. An attenuated virus which carries several neutralizing epitopes, both linear and conformational, and which can induce an immune response similar to natural infection is the most efficient approach. • The aim of this study was to explore EV71 molecular determinants within the 5′UTR and VP1 capsid protein, which can lead to the virus attenuation. Temperature sensitivity of EV71 in vitro was used as a phenotypic marker of attenuation. Methods • Site-directed mutagenesis (SDM) was employed in order to introduce mutations within the 5′UTR and VP1 regions of an infectious cDNA clone of EV71: • The 5′UTR mutations corresponded to the Sabin 3 attenuating determinant of PV (473U in EV71), and the compensatory nucleotide change (538A in EV71) stabilizing the stem-loop V secondary structure within the Internal Ribosome Entry Site (IRES); • The VP1 mutations were alanine (A) substitutions of single charged amino acids within the charged clusters. The cluster was defined as a stretch of 4 to 9 amino acids with 50% of them charged and separated within the cluster, at most, by 1 hydrophobic amino acid. • Mutant viruses were rescued in cell culture from the in vitro RNA transcripts. • In vitro phenotypes of the parent and mutant viruses were studied in Vero cell culture. • Positions of the alanine substitutions within the three-dimensional (3D) structure of VP1 were analysed in Cn3D software. Results • 3. Growth kinetics and efficiency of plating (EOP) of the EV71 mutants • Virus titres (log10 TCID50) obtained at 12 h (single-cycle growth) and 96 h (multiple-cycle growth) post-infection of Vero cells were used to calculate EOP of the parental and mutant EV71 strains. Only mutant viruses with statistically different EOP, when compared to that of the parental strain, are shown: • 5. Temperature-dependent reduction in virus titres of EV71 mutants • 1. Mutations within the 5′UTR of EV71 • Secondary structure of EV71 RNA within the 5′UTR (IRES, stem-loop V) was predicted with UNAFold software (5). Mutated nucleotides are boxed. • 2. Mutations within the VP1 protein of EV71 • 2.1. Clusters of charged amino acids targeted in SDM: Vero cells were infected with EV71 at an m.o.i of 0.1 and incubated at indicated temperatures for 72 h. The virus titres were determined by TCID50 assay. Reduction in virus titre (∆TCID50) at each temperature was calculated versus the TCID50 at 37˚C. • Amino acids with charged side chains are underlined. Amino acids substituted with alanine are highlighted with grey background. Amino acids constituting β-strands and α-helix are highlighted in red and blue, respectively. • 2.2. Location of the alanine substitutions on the VP1 structure: EOP expressed as the mean ratio of the virus tire at 39.5˚C versus the virus titre at 37.0˚C (7); ±SEM (standard error mean) of three experiments; P-values of the “unpaired” version of the Student’s t-test, with the unequal variance and one-tailed distribution; EOP, statistically different (P<0.05) from that of the parental virus, is highlighted in blue. * Reduction in virus titres is expressed as the mean ± SEM of the data from three experiments relative to 37˚C. The lower limit of virus detection was 1.45 log10 TCID50/ml. The symbol “>” indicates that a reduction in virus titre is greater than the indicated value (when virus titre at indicated temperature is below the detection limit) and therefore the SEM is not applicable; ** Temperature at which TCID50 titre is reduced by 1.00 log10 when compared to viral titre at 37˚C; a – P<0.05 of the “paired” Student’s t-test (statistical significance of the reduction in a virus titre against 37˚C); b – P<0.05 of the “unpaired” Student’s t-test (statistical significance of the difference between the shut-off temperature of the query virus and that of the parental strain); n/d – not determined. • 4. Combination of the 5′UTR mutations with alanine substitutions (164A and 213A) within the VP1 protein • Substitutions with alanine resulting in infectious (A) or non-infectious (B) mutant EV71 are mapped onto EV71 VP1 crystal structure, 3VBF_A (6). References Conclusions • SDM within the stem-loop V of the 5′UTR led to temperature-dependant inhibition of EV71 in cell culture; • SDM within the structural elements (α-helix and β-sheet) of the VP1 abolished infectivity of EV71 mutants; • Out of eleven infectious VP1-mutant strains, six demonstrated growth kinetics impaired at the elevated temperature of 39.5˚C; • Further inhibition of virus growth in vitro was demonstrated as a result of a combination of the VP1 (164A and 213A) and 5′UTR (473U/538A) mutations. • Bible, JM et al. 2007. 'Genetic evolution of enterovirus 71: epidemiological and pathological implications', Rev Med Virol, vol. 17, no. 6, pp. 371-379. • Chen, SC et al. 2007. 'An eight-year study of epidemiologic features of enterovirus 71 infection in Taiwan', Am J Trop Med Hyg, vol. 77, no. 1, pp. 188-191. • Hamaguchi, T et al. 2008. 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