interaction with the lipid membrane. Unlike the electron microscopy, this technique enables the imaging of biological samples in a natural fluid environment with atomic resolution. Virus particles were adsorbed on a supported bilayer lipid membrane (sBLM) containing GD1a ganglioside, which served as a receptor for the influenza virus. The conversion of the conformation of HA at the threshold pH value leading to the activation of the fusion of the virus envelope with sBLM was registered by changing the domain structure of the lipid bilayer. We found that the HA conformational change of VN1203DNS1 occurred already at pH 5.8, whereas the VN1203DNS1-K58I mutant was stable under these conditions. The image confirming the conformational change of the mutant virus was observed at pH 5.0 (detailed data are presented as supporting information, Text S1, Fig.S1). Next, we investigated whether the virus VN1203DNS1-K58I, which was more resistant to an acidic pH, was also more stable at an increased temperature. VN1203DNS1 and VN1203DNS1- K58I viruses were incubated at a temperature ranging from 46uC to 58uC for 30 min with the subsequent measurement of the remaining infectious and hemagglutination titers. We found that the infectivity of the VN1203DNS1 virus was impaired already at 50uC, as indicated by a drop in the infectious titer more than 100 times. At the same time, this temperature did not affect the infectious titer of the mutant virus (Fig. 3). After incubation at 52uC, the VN1203DNS1 virus completely lost the infectious titer, whereas the titer of the mutant virus VN1203DNS1-K58I decreased by 1.0 log10 TCID50/ml. Analogous results were obtained by assessing HA titers indicating improved temperature stability of mutated surface glycoprotein. Mutation HA2 58KRI preserves virus infectivity at an acidic pH in cell culture The infectivity of reassortants VN1203DNS1 and VN1203DNS1- K58I under acidic conditions was compared in Vero cells at pH 5.4, 5.6, 5.8, or 7.4. The human BN/59/07 virus was taken as a control sample resembling a pH stable phenotype. As is shown in Fig. 4A, the mutant virus VN1203DNS1-K58I gained a resistance to low pH infecting Vero cells at pH 5.6, whereas the limit of infection was at pH 5.8 for the original virus VN1203DNS1. These results were Increased Infectivity of H5N1 Influenza Virus confirmed on MDCK cells estimated by the flow cytometry analysis under similar conditions (Fig. 4B). As expected, the human BN/59/ 07 (H1N1) virus was infectious on Vero cells at pH 5.4 and infected 32% of the MDCK cells even at pH 5.2 (Fig. 4C). Mutation HA2 58KRI increases virus sensitivity to lysosomotropic agents Avian influenza viruses in contrast to human strains are more resistant to the prevention of endosomal acidification mediated by lysosomotropic agents, such as chloroquine and NH4Cl [24]. Therefore, we compared the infectivity of both viruses in the presence of chloroquine or NH4Cl (Fig. 5). As a control, the seasonal influenza strain BN/59/07 (H1N1) was included in the study. The infection of Vero cells with VN1203DNS1-K58I was decreased by 10 mM chloroquine and completely inhibited by 50 mM. In contrast, the growth of the original virus was hardly affected even at 50 mM chloroquine. BN/59/07 (H1N1) was most sensitive to chloroquine as the infection was reduced by 1 mM. These results were confirmed by another lysosomotropic agent (1 mM NH4Cl) when the infectivity of the VN1203DNS1-K58I or BN/59/07 (H1N1) viruses was almost lost, whereas the VN1203DNS1 virus was not affected. Thus, the mutation HA2 58KRI converted the phenotype of the avian influenza virus to be more similar to that of the human isolate. Mutation HA2 58KRI decreases the replication efficiency of H5N1 viruses in cell culture To elucidate whether the changed stability of HA to an acidic pH has any influence on the replication efficiency in various cell lines, we compared the viral growth capacity in Vero, MDCK, A549 (human lung carcinoma cell), and in HBE (human bronchial epithelial) cells. As the DNS1 viruses are replication deficient in interferon competent cell lines (all mentioned above except Vero), two similar viruses expressing functional NS1 protein VN1203wtNS and VN1203wtNS-K58I were used in these experiments. Similar results of stability towards an acidic pH and elevated temperature were obtained for these viruses (results are not shown). Growth curves made for each cell line revealed that the introduced mutation HA2 58KRI impaired the yield of the mutant virus VN1203wtNS-K58I in HBE cells of about 150 Figure 3. Effect of elevated temperature on the viral HA and infectious titer. Virus preparations were incubated at indicated temperatures for 30 min. HA titers (bars, left Y-axis) and infectious titers measured as log10 TCID50 (graphs, right Y-axis) were determined for VN1203DNS1 (black bars and lines) and VN1203DNS1-K58I (gray bars and lines) viruses. The lower limit of detection for the TCID50 titer is 1.5 log10 TCID50/ml indicated by the horizontal dashed line. doi:10.1371/journal.pone.0018577.g003 PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18577