Introduction

1. The Ser299Thr substitution within the 3Dpol confers a temperature resistant phenotype in Enterovirus 71. Natallia Lazouskaya, CharmiTrivedi, EnzoPalombo, Tony Barton. Environment and Biotechnology Centre, Swinburne University of Technology, Hawthorn VIC 3122 Australia Introduction • The molecular basis and mechanism determining the pathogenicity of Enterovirus 71 (EV71) are not well understood. The earliest symptom of the EV71 infection is fever which is followed (in 1-2 days) by a skin rash and herpes-like mouth ulcers (1). At the beginning, the fever is usually mild (around 38˚C) but the temperature can reach over 39˚C and become persistent in patients with severe complications, such as pulmonary oedema, brainstem encephalitis and meningitis (2). According to clinical observations, neurological symptoms develop approximately 3-5 days after an increase in body temperature and an appearance of skin lesions (3). Analysis of the virus in CNS samples several days after the onset of fever indicates a possible role, that the elevated body temperature may play in evolution of the originally transmitted viral strain/s that may affect viral replication and disease progression. Correlation between temperature resistance (TR) of the virus and CNS-involvement has been reported for EV71 clinical isolates in Taiwan (4). Identification of the molecular determinants responsible for virus adaptation to increased temperatures in field isolates is difficult due to the original sequence variation between circulating viral strains. Additionally, multiple selective forces applied upon the immune system response would direct viral evolution in situ to emergence of adaptive mutations in response to a number of factors. To eliminate these other factors and study virus adaptation to an elevated temperature experiments were conducted in vitro. The aim of this study was to describe molecular determinants associated with TR phenotype of EV71. Methods • Two viral stocks were prepared by passaging EV71 in Vero cells at a permissive or potentially restrictive temperature: • the temperature sensitive stock (tss) was obtained after multiple passages at 37°C • temperature resistant stock (TRs) was derived from the ts stock after 4 passages at 39°C • cDNA clones of the full-length genome of EV71 were constructed and designated as ts clone (tsc) and TR clone (TRc) • Full-length genome sequences were obtained from the both viral stocks and viral clones and analysed at nucleotide and amino acid levels • Amino acid changes were mapped on the three-dimensional (3d) structures of the reference proteins obtained from MMDB • Site-directed mutagenesis (SDM) was employed to substitute mutations observed in the TR EV71 with the reciprocal positions of the ts virus • Mutant viruses were rescued from the in vitro RNA transcripts transfected into Vero cells • Replication of the TRc, tsc and mutant strains was examined in temperature sensitivity assay Results • 3) Temperature dependent reduction in virus titres of EV71 mutants in comparison to the TRc and tsc: • 4) Structure modelling: interactions formed within the active centre of the 3Dpol during RNA synthesis • 1) Mutations observed in the conversion of the tss isolate of EV71 to TRs: • * Invariant positions are in bold • ** Nucleotide variation (indicated by the arrow in the picture below) observed within the VP1 gene of the tss Vero cells were infected with EV71 at an m.o.i of 0.1 and incubated at indicated temperatures. The virus titres were determined by TCID50 assay. Reduction in virus titre (∆TCID50) at each temperature was calculated versus the TCID50 at 37˚C. The figure is a result of the alignment of the crystal structures 3N6M (5), 3OL6 and 3OL7 (6). The RNA template strand and some residues within 3Dpol are omitted for clarity. The open state of the 3Dpol structure is coloured according to the element scheme, where carbon is grey, nitrogen is blue, oxygen is red and phosphorus is green. Structure in closed state is coloured in yellow. Amino acid residues forming interactions within the core region of the 3Dpol are highlighted in pink on the grey backbone. Interactions formed prior to the active site closure and after that are marked with dash lines in grey and yellow, respectively. Position of the incoming NTP directly above the catalytic site (G328, D329 and D330) is coordinated by interactions with R163, K167, R174 and L175. In this position the incoming NTP is base-paired with the template nucleotide (6). The NTP recognition leads to formation of the extensive network of new interactions between the 2’ and 3’-ribose hydroxyls and D238, S289 and N298 of the 3Dpol. As a result, the NTP is sinking towards the active site. These conformational changes position D233 for metal ion binding which, in turn, triggers the active site closure and catalysis. The 299th amino acid (highlighted in turquoise) is located within the αH-helix, and is adjacent to the N298, which selects for the sugar in the incoming nucleotide. • 2) Schematic representation of the EV71 mutants obtained after SDM of the TRc: * Reduction in virus titres is expressed as the mean ± SEM of the data from three experiments relative to 37˚C ** 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 b – P<0.05 of the “unpaired” Student’s t-test n/d – not determined • Genome of the tssserves as a reference. Silent and non-silent nucleotide changes identified within the TR EV71 variant are indicated with dark and white stars, respectively. Conclusions References • Selection of the TR phenotype led to a decrease in the genetic diversity of the viral population; • Growth of the naturally selected TR variant was suppressed at temperatures higher than that used for its selection; • A single amino acid change (Ser299Thr) within the 3Dpol was associated with the acquired temperature resistance; • Location of the 299th amino acid within the catalytic centre of the 3Dpol suggests its possible role in virus replication; • The amino acid changes within the capsid proteins VP1 and VP3 had very little or no effect on temperature resistance, and were most likely co-selected with the fittest EV71 genome. • Tran, C. B., H. T. Nguyen, et al. (2011). "The seroprevalence and seroincidence of enterovirus71 infection in infants and children in Ho Chi Minh City, Viet Nam." PLoS One6(7): e21116. • McMinn, P., I. Stratov, et al. (2001). 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