Viral RNA Degradation and Diffusion Act as a Bottleneck for the Influenza A Virus Infection Efficiency
[['Seasonal influenza epidemics and periodical pandemics remain a constant threat to the human population .', 'Influenza A virus ( IAV ) infection is a multi-step process that critically depends on the viral spike protein hemagglutinin ( HA ) , which mediates host cell adhesion by binding to sialic acid-containing receptors within the host cell plasma membrane ( Fig 1A ) .', 'Viruses are subsequently internalized into endosomes , which undergo a complex maturation process involving acidification and centripetal transport along microtubules [1] .', 'Endosomal acidification is highly critical since virus uncoating and genome release depend on membrane fusion mediated by a pH-dependent conformational change of the HA protein .', 'However , although microtubule-associated transport of IAV has been shown before [2–4] , the relevance of this directed transport for virus infection still remains largely unclear .', 'The genome of IAV consists of eight individual genome segments coding for a total of 11 viral proteins [5] .', 'The negative-sense single-stranded RNA is packaged together with the viral nucleoprotein ( NP ) and the polymerase complex ( PA , PB1 and PB2 ) forming rod-like ribonucleoprotein complexes ( vRNP ) .', 'After membrane fusion , released vRNPs travel to the nuclear membrane , most likely by passive diffusion [6] .', 'Since endosomal transport and acidification are concurrent the distance they need to overcome depends on the time and location of membrane fusion and thereby also on the pH-dependent conformational change of HA ( i . e . HA’s pH sensitivity ) .', 'Whether the eight different vRNPs stay in one complex or rather dissociate after being released is still under debate [7 , 8] .', 'In any case , the released vRNPs bind to importin-α by means of their nuclear localization signals and get shuttled across the nuclear membrane through nuclear pores [7 , 9 , 10] .', 'To establish a successful infection allowing the production of progeny viruses , it is essential that all eight genome segments are transported into the host nucleus where genome replication takes place .', 'Therefore , the early infection phase may represent a bottleneck for the infection that could potentially contribute to host cell specificity of the virus .', 'Indeed , it was suggested that adaptation to a different host does not only require binding to specific receptors but also modification of the pH sensitivity of HA , possibly to adapt to variations in the endosomal pH [11–13] .', 'Here , we address the question whether an altered pH sensitivity of HA modulates the residence time of diffusing vRNPs in the cytoplasm and thereby influences viral infectivity .', 'Mathematical models have been used before to foster a better understanding of IAV replication on the population [14 , 15] as well as on the single-cell level [16–19] .', 'However , none of those seem to be adequate for the analysis and comparison of different IAV strains with varying pH sensitivity .', 'We developed a model accounting for the first critical phase of IAV infection using a combination of ordinary differential equations ( ODE ) and a spatial modeling approach .', 'The model parameters were calibrated using experimental data from infection studies covering the individual steps of the infection process ( Fig 1A ) .', 'Our model enabled us to ( 1 ) predict differences in the infection efficiency ( i . e . delivered number of vRNPs in the nucleus ) of two IAV strains with altered pH sensitivity and to ( 2 ) simulate the diffusion of vRNPs as complex or individual particles utilizing a spatial stochastic model accounting for the specific cell geometry as well as the stochastic nature of the underlying processes .', 'Our simulations predicted that the pH sensitivity of HA critically controls the time of fusion and thereby the location of genome release .', 'We further concluded that a dissociation of released vRNP segments is highly unlikely to result in an efficient infection and that the viral RNA ( vRNA ) is subjected to degradation during cytosolic transport .', 'This in turn might affect the number of intact vRNPs arriving in the nucleus and thus the infection efficiency of a specific virus .', 'Using two HA variants with differing pH sensitivities , we complemented our simulations with experimental evidence validating the major model predictions .', 'Taken together , we propose that the pH dependence of influenza virus fusion resulting in the release of the viral RNP complex plays a determinative role for the initial phase of virus infection .', 'Our modeling data further suggest that diffusive transport and cytosolic stability of vRNPs represent limiting factors for efficient infection .'], ['To understand how a varying pH sensitivity can affect infection efficiency , we need to understand the dynamics of the entry process after virus binding .', 'To this end , we developed a dynamic ODE model that accounts for the early steps of the infection cycle and parameterized it using experimental time course data .', 'The model consists of a set of four coupled ODEs describing ( 1 ) the temporal behavior of the amount of surface bound virions , ( 2 ) the quantity of endocytosed viruses , and ( 3 ) the concentration of released vRNPs in the cytoplasm as well as ( 4 ) in the nucleus .', 'It describes the processes of endocytosis , endosome maturation , endosomal fusion and nuclear import of vRNPs .', 'Our experimental data led us to include cytosolic vRNP diffusion and degradation into the model .', 'The reaction network is depicted in Fig 1B , and the corresponding equations are introduced in the Materials and Methods section .', 'To calibrate our model , we measured the corresponding steps of virus infection—virus-endosome fusion , endosomal pH development , and nuclear NP accumulation—between 0 and 40 minutes post infection ( p . i . ) ( Fig 2 ) .', 'Intracellular fusion was measured by infecting MDCK cells using viruses labeled with R18 at self-quenched concentration .', 'Individual viruses were detected using confocal microscopy and fusion events were counted using automated image processing .', 'R18 dequenching is a commonly used method to detect IAV fusion in vitro [20] and a significant increase in R18 intensity was expected upon virus-endosome fusion ( S1 Fig ) .', 'Indeed , we observed that the intensity of internalized single viruses showed a strong increase between 10 and 20 min p . i . reaching a plateau after about 25 min ( S2 Fig ) .', 'Notably , we could reproduce the virus-endosome fusion kinetics using a FRET-based fluorescence readout as described previously [21] .', 'This trend correlated with the accumulation of viral NP inside the nucleus , which started to increase slightly delayed after 15–20 min ( Fig', '2 ) as detected by anti-NP immunostaining .', 'In order to identify pH-dependent effects on IAV infection , it was crucial to know the actual endosomal pH dynamics .', 'In the model , we defined the endosomal pH as an independent variable pHend , which served as an input to the coupled reaction system .', 'pHend followed an exponential decay , that we fitted to experimental data , with a decay parameter kATPase that corresponds to the endosomal V-ATPase activity .', 'To monitor the endosomal pH kinetics and calibrate kATPase , we used a double-labeled dextran as a pH-sensitive endosomal probe as described in Materials and Methods .', 'Briefly , after a short starvation period , cells were loaded with dextran for the indicated time points , washed and immediately analyzed by flow cytometry .', 'We observed an exponential decrease of the endosomal pH over the observed time period ( Fig 2 ) .', 'HA’s membrane fusion activity is characterized by a narrow pH regime of switching from zero to complete fusion [22] .', 'To accommodate for this behavior , in our model , endosomal fusion follows a Hill kinetics [23] with the threshold parameter kH+ and the Hill coefficient h .', 'We could determine these parameters by measuring the viral fusion kinetics in vitro using R18 fluorescence dequenching ( FDQ ) in the pH range from 5 . 0 to 7 . 4 .', 'We observed a steep increase of viral fusion with decreasing pH ( S3 Fig ) and could use our model to determine the fusion pH threshold value kpH ( i . e . the pH value where FDQ is half-maximal ) of the IAV X-31 strain to be at pH 5 . 6 from these data sets .', 'The combination of results from in vitro virus fusion with the endosomal fusion time course data allowed us to fit the parameters of the model with high confidence ( see Table 1 and S4 Fig ) further enabling us to introduce perturbations into the model .', 'During model development , it became evident that introduction of vRNP diffusion delay and degradation is necessary to fit all measured data at once .', 'In order to predict the system’s behavior for a higher value of kpH resulting in earlier virus-endosome fusion , we initially did not include vRNP diffusion and degradation into the model .', 'In that case an unchanged steady state of accumulated vRNPs in the nucleus vRNPnuc was predicted .', 'Hence , the pH sensitivity had no measurable effect on virus infection in that model .', 'To experimentally determine the effect of an altered pH sensitivity of HA on viral infectivity , we constructed a recombinant virus based on influenza virus A/WSN/33 ( H1N1 ) with the H1-subtype HA replaced by H3 HA of A/X-31 ( H3N2 ) yielding the so-called WSN H3 wild type virus ( WSN H3 wt ) [24] as well as a WSN H3 mutant virus carrying the destabilizing double mutation T212E-N216R in the HA protein ( WSN H3 mut ) [25] .', 'The fusion pH threshold of WSN wild type and mutant viruses was assessed in vitro as described above for A/X-31 .', 'Our experiments showed that , similar to the A/X-31 strain , a pH threshold of 5 . 6 triggers fusion of the WSN H3 wild type strain confirming that fusion mainly depends on the pH sensitivity of HA ( S3 Fig ) .', 'In contrast , for the WSN H3 mutant , this threshold was shifted to pH 5 . 8 due to the destabilizing double mutation in the HA protein ( Fig 3 ) .', 'We integrated these data into the dynamic model and again could not detect an effect on nuclear vRNP accumulation suggesting—at first glance—no effect of HA’s pH sensitivity on virus infection .', 'However , from measuring the infection efficiency of recombinant WSN viruses in MDCK cells under identical conditions ( MOI of 0 . 2 ) , we found that the amount of expressed viral NP 20h p . i . was 40–50% lower for the destabilized WSN H3 mutant virus than for the wild type ( Fig 3B ) .', 'Both , measuring the infection efficiency 5h p . i . as well as growth curves for both virus strains over 72h , could confirm the attenuation of WSN H3 mut ( S6 Fig ) .', 'If we considered a correlation between accumulated vRNPs in the nucleus and expressed NP protein 20h p . i . , these results clearly contradicted our model prediction .', 'Therefore , we extended our model to reproduce the infection efficiency observed in the experiment .', 'Based on our experimental observations of virus-endosome fusion taking place some μm away from the nucleus ( S7 Fig ) , we introduced a spatial component that accounts for the diffusion of released vRNPs through the cytosol .', 'We constructed a 3D reaction-diffusion model that allowed us to relate fusion distance to delay and efficiency of vRNP delivery into nucleus and to compare the different IAV strains quantitatively .', 'The simulation settings were adjusted to biologically relevant values ( see Materials and Methods section ) .', 'For the assumed cell geometry as well as the position of the nucleus , please see S8 and S9 Figs .', 'Due to the small number of vRNP particles ( 1 to about 80 ) in the cell and a binary outcome ( infection/no infection ) for each cell , we could not assume a mean-field approach to capture the full behavior of the system and thus decided to utilize a stochastic simulation environment .', 'To explain the experimental result of reduced infection efficiency , we established the hypothesis that free vRNPs are subjected to degradation while diffusing through the cytosol [26] .', 'We further allowed two forms of vRNP diffusion: ( 1 ) vRNPs are released from the virus as a complex consisting of all eight genome segments diffusing together .', '( 2 ) The released complex dissociates and the vRNP segments diffuse individually with a higher diffusion coefficient than the larger complexes .', 'The infection is only counted as successful if a full set of eight different vRNPs has reached the nuclear membrane where they bind to importins to be shuttled into the nucleus .', 'In our spatial model , three parameters were critical for the connection between infection efficiency and pH sensitivity: ( 1 ) vRNP degradation , ( 2 ) vRNP release distance from the nucleus and ( 3 ) vRNP dissociation .', 'Our computational approach made it possible to comprehensively sample a large sub-space of combinations of different dissociation rates of vRNP complexes ( kdiss ) and degradation rates of individual or complexed vRNPs ( kdeg ) .', 'Fig 4B shows the results of such two-dimensional parameter scans .', 'The x- and y-axes indicate the parameter values of kdiss and kdeg .', 'Each color-coded bin corresponds to the average number of successful infections after 40 min in a cell population of 1000 cells .', 'The populations were all simulated with the corresponding parameters on the axes .', 'The initial condition for the simulation is one complete vRNP package starting to diffuse at distance dnuc from the nucleus ( S10 Fig ) .', 'Interestingly , these parameter scans suggested that , given the measured diffusion rates , already small degradation rates would be sufficient to degrade the whole viral genome if virus-endosome fusion and thus vRNP release happens too far away from the nucleus .', 'Furthermore , although single particles have a much higher mean displacement due to their smaller size , our simulations clearly showed that the dissociation of complexes lowered the probability of a complete genome in the nucleus .', 'The larger the dissociation constant in the model was set , the lower the percentage of complete genomes .', 'This effect also increased with larger fusion distances from the nucleus , possibly because of an increased probability that the vRNPs were degraded before reaching the nucleus .', 'Our observations at the cell population level ( Fig 3B ) revealed that the infection efficiency of an IAV strain with a higher pH threshold was reduced as compared to the reference strain ( WSN H3 wt ) .', 'By adding a degradation term to the freely diffusing vRNP particles , we could establish a link between the pH sensitivity of HA and the viral infection efficiency: the larger the distance between the location of fusion and the nucleus , the higher the probability that the vRNPs are degraded and thus the lower the number of functional vRNPs arriving in the host cell’s nucleus .', 'To test whether vRNPs are subjected to degradation while diffusing in the cytoplasm , we measured the amount of cytosolic vRNA ( HA segment ) by RT-qPCR while blocking the nuclear import using 40 and 100 μM importazole , respectively [27 , 28] ( for experimental details see Materials and Methods ) .', 'The resulting data are shown in Fig 2 .', 'As also reported by Kublun et al . [28] , we could observe a higher inhibitory effect on nuclear import using 40 μM importazole than using the higher concentration of 100 μM which might be due to cell damage or the activation of other import pathways for the higher dose of the drug .', 'In any case , for both importazole concentrations the results indicate a fast drop of cytosolic ( including intact viral particles ) vRNA in the first 10 min p . i . .', 'We also compared the total ( nuclear + cytosolic ) vRNP concentration with and without importazole 60 min p . i . showing that with importazole the vRNP levels are significantly lower ( for control experiments without importazole see S11 Fig ) .', 'By including these data into the fit of our ODE model , we could identify the model parameters accounting for vRNA degradation ( kdeg ) and diffusion time ( kτ ) as well as their confidence intervals ( S4 Fig , Table 1 ) .', 'With these parameters we could use the spatial model to further characterize the impact of fusion distance on infection .', 'Combining the results of the ODE and the 3D diffusion model enabled us to determine the mean fusion distance of vRNP particles in our simulations .', 'Based on the calibration data for the ODE model , we could estimate an optimal delay time between membrane fusion and vRNP arrival at the nucleus τ of 4 minutes for IAV X-31 ( Fig 4A , upper panel ) .', 'Using the spatial model simulations , we could then translate this value into a distance of fusion from the nucleus of ≈ 3 μm for the complex and ≈ 5 μm for individually diffusing vRNPs ( Fig 4A , lower panel ) .', 'In the simulations , the estimated diffusion delay parameter kτ for IAV X-31 represents the delay between the half maximum times of fusion and import into the nucleus which we denote as τeff ( Fig 5 ) .', 'It is reasonable to assume that the vRNPs of WSN H3 wt and H3 mut have the same diffusion coefficient and import rate ( kimp ) as the one fitted for IAV X-31 .', 'Furthermore , for the higher pH threshold of WSN H3 mut , an increase of the vRNP concentration in the nucleus can at best happen as early as for the wild type because we assume active transport to be faster than diffusion .', 'Using our model , we can now combine endosome acidification data and HA pH sensitivity measurements to predict the dynamics of the mutant infection .', 'Simulations showed the mutant fusing about three minutes earlier than the WSN H3 wt ( Δτeff ≈ 3 min ) which corresponds to a fusion distance of ≈ 6 μm for the complex .', 'If we simulated the model for the mutant with a prolonged delay τmut ≈ τwt + Δτeff we could predict a strong reduction in the amount of vRNPs reaching the nucleus , which could be a possible explanation for the reduced infection efficiency observed for the mutant .', 'Next , we used the estimated degradation rate ( kdeg = 0 . 19 min−1 ) in the stochastic model to estimate upper limits for fusion distances from the nucleus for a successful virus infection by simulating the diffusion of cytosolic complexed vRNPs from different distances .', 'We found that for a fusion distance greater than dnuc = 10 μm from the nucleus , on average only 0 . 4 complete sets of viral genomes would arrive in the nucleus when running the simulation with one complete set .', 'The number of complete sets quickly reached zero with larger distances .', 'Simultaneously , we could scan through the parameter kdiss to obtain a parameter landscape of successful infection ( Fig 4B ) .', 'Again , the dissociation of vRNPs affected transport to and import into the nucleus and proved to be detrimental for successful infection: for kdiss > 0 . 1 nearly no vRNPs would reach the nucleus even for small distances .'], ['Viral escape from cellular endosomes is an essential step of the IAV replication cycle .', 'To allow uncoating and vRNP release , IAV utilizes the complex maturation program of the endocytic system .', 'Among other changes , it involves two inherently connected processes: endosomal motility and lumenal acidification [1] .', 'Endosomal motility is dominated by the association with and movement along microtubules .', 'This transport is mediated by two motor protein families , kinesins and dyneins , which operate in opposite directions .', 'Although endosomes typically associate with both motors , which can lead to a bidirectional movement , they perform a net translocation towards the microtubule organizing center ( MTOC ) in the nuclear periphery [1] .', 'Indeed , live-cell tracking of fluorescently labeled IAV showed that the particles are transported towards the perinuclear region of the cell [2 , 4] .', 'During the directed transport inside endosomal vesicles , the virus finally reaches late endosomal compartments ( pH 5 . 0-6 . 0 ) where the acid-induced conformational change of HA triggers membrane fusion and vRNP release into the cytosol .', 'Due to the connection between lumenal acidification and endosome movement towards the nucleus , timing of fusion affects the location of the fusion event inside the cell .', 'Several studies suggested that the pH sensitivity of HA needs to be adapted to the endosomal properties of the respective host cell in order to allow for efficient virus replication [11–13] .', 'However , so far it remained largely unclear how HA’s pH sensitivity affects time and location of fusion and as a consequence , the infection efficiency of the virus .', 'Here , we present a mathematical model that quantitatively describes IAV cell entry .', 'We combined cell-specific parameters like geometry , endosomal uptake and acidification with virus-specific determinants such as the pH sensitivity of HA and were able to estimate the distance of endosomal fusion from the nucleus for different HA variants .', 'Furthermore , we showed that vRNA is subjected to cytosolic degradation and thereby demonstrated the importance of the exact timing of fusion for the efficiency of infection in a given host cell .', 'Measuring the intracellular fusion kinetics of IAV X-31 , we found a mean fusion time of 10 min in MDCK cells which is in good agreement with Lakadamyali et al . [2] who reported a mean fusion time of 8 min in CHO cells .', 'Our result also correlates well with the study of Sakai et al . [21] , who used the same intracellular fusion assay , although in a different cell line .', 'As reported previously [2 , 4] , fusion events were detected close to the nucleus , which we could demonstrate to be on average located in the center of the cell ( S7 and S8 Figs ) .', 'Using our combined stochastic and spatial model , we could predict that IAV X-31 and a recombinant WSN virus carrying the H3 HA of IAV X-31 fuse at an estimated distance of 3 μm from the nucleus in MDCK cells , whereas a more pH sensitive mutant ( kpH = 5 . 8 compared to 5 . 6 for the wild type ) fuses earlier at a distance of 6 μm from the nucleus .', 'We proposed that the released vRNPs are subjected to degradation , thereby strongly attenuating the infection as well as the replication efficiency of the WSN H3 mutant virus as observed experimentally ( Fig 3 and S6 Fig ) .', 'Indeed , we could show that vRNAs , complexed in diffusing vRNPs after being released from the virus , are degraded within a few minutes in the cytosol ( degradation rate of 0 . 19 min−1 ) .', 'Adding this limiting factor to our model allowed us to predict that the earlier release of the viral genome , i . e . in larger distance to the nucleus ( due to a higher pH sensitivity of HA ) , results in a lower level of nuclear vRNPs and thus , in a lower infection efficiency .', 'It was shown previously that IAV propagation over multiple passages can lead to accumulation of defective interfering RNAs , which give rise to defective interfering particles ( DIP ) that interfere with the replication of non-defective viruses [29 , 30] .', 'Since the presence of DIPs and in particular deletions in HA and NP could affect our results , we performed a segment-specific PCR .', 'We included the PB2 segment since DI RNAs predominantly originate from the large polymerase genes ( segments 1–3 ) [31] .', 'In addition to all three full length segments , for PB2 we found a smaller PCR product indicating the presence of DIPs in our virus sample ( S12 Fig ) .', 'However , for both the HA as well as the NP segment , we could not detect a significant amplification of smaller PCR products , thereby ensuring the unperturbed detection of HA vRNA as well as incoming NP protein during our virus-entry measurements .', 'With our model , we were also able to challenge existing hypotheses on the different strategies of how vRNP segments reach the nucleus ( i . e . as a complex or as individual vRNPs ) .', 'Hence , we simulated various degrees of dissociation of vRNP segments after fusion and compared the effect on infection efficiency .', 'Our model suggests that diffusion of complexed vRNPs is more favorable for infection than dissociation of vRNPs in the cytosol .', 'Transport as a complex might assure the arrival of all eight vRNPs in the nucleus simultaneously and thus be much more efficient and reliable than gating single vRNPs one-by-one .', 'Our model thus supports the tight association of released vRNP segments during cytosolic diffusion as it was also recently suggested from single-molecule fluorescence in situ hybridization ( smFISH ) data [8] .', 'Our mathematical model integrates cell-specific as well as virus-specific parameters .', 'Therefore , it is useful to predict the infection of a given virus in a specific host cell .', 'Mathematical models of virus entry have been presented for Semliki Forest virus in BHK-21 cells and of the baculovirus-insect cell system [32 , 33] .', 'These viruses—similar to influenza—require endocytic uptake and , after release of the viral genome by membrane merger , transport to a specific compartment for replication .', 'Our model extends the current understanding of the role of the fusion protein’s pH sensitivity on viral entry dynamics and the impact of vRNA degradation as well as vRNP dissociation for the success of infection .', 'These newly determined parameters might also be relevant for other models of viral entry .', 'Still , the complex nature of a given host cell and the possible presence of other yet unknown factors of the cellular immune response may lead to deviations from our model predictions .', 'For example , the activation of signaling pathways upon viral binding can interfere with the uptake of the virus what would considerably influence the dynamics of viral entry .', 'The impact of such factors on infection can vary greatly between different viruses and cell lines and thus is difficult to predict .', 'Furthermore , in this study we only focused on the early steps of IAV infection , which limits its predictive power for viral replication .', 'To improve predictions for this case the model could be combined with existing models that describe the whole infection cycle [34] , which might be very useful for several applications e . g . vaccine production [16] .', 'Taken together , we describe the first critical phase of IAV infection using mathematical and 3D diffusion modeling , which accounts for the pH sensitivity of HA as well as cell-specific parameters such as endosomal acidification , cell geometry and the degradation rate of vRNPs , a newly identified host factor of the cellular immune system .', 'Using recombinant viruses with differing HA pH sensitivities we could validate our model experimentally and at the same time provide evidence that the interplay between pH-dependent fusion of HA and degradation of viral RNA by the cellular immune system represents a bottleneck for IAV entry determining the success of viral infection .']]
['Introduction', 'Results', 'Discussion']
['After endocytic uptake , influenza viruses transit early endosomal compartments and eventually reach late endosomes .', 'There , the viral glycoprotein hemagglutinin ( HA ) triggers fusion between endosomal and viral membrane , a critical step that leads to release of the viral segmented genome destined to reach the cell nucleus .', 'Endosomal maturation is a complex process involving acidification of the endosomal lumen as well as endosome motility along microtubules .', 'While the pH drop is clearly critical for the conformational change and membrane fusion activity of HA , the effect of intracellular transport dynamics on the progress of infection remains largely unclear .', 'In this study , we developed a comprehensive mathematical model accounting for the first steps of influenza virus infection .', 'We calibrated our model with experimental data and challenged its predictions using recombinant viruses with altered pH sensitivity of HA .', 'We identified the time point of virus-endosome fusion and thereby the diffusion distance of the released viral genome to the nucleus as a critical bottleneck for efficient virus infection .', 'Further , we concluded and supported experimentally that the viral RNA is subjected to cytosolic degradation strongly limiting the probability of a successful genome import into the nucleus .']
['Influenza A virus carries its segmented genome inside a lipid envelope .', 'Since genome replication occurs inside the nucleus , the main goal of virus infection is to deliver all genome segments through the cytoplasm into the nucleus .', 'After endocytic uptake , influenza viruses transit early endosomal compartments and eventually reach late endosomes .', 'Within a complex maturation process , the endosomal lumen acidifies while the vesicles are transported trough the cytosol .', 'If and how these early processes affect virus infection remained mostly speculative .', 'To reach a better understanding and to quantify the physical interplay between membrane fusion , genome diffusion and infection , we developed a mathematical model that comprises all initial steps of virus infection until genome delivery .', 'We calibrated our model using experimental data and challenged its predictions using recombinant viruses to introduce perturbations .', 'Our results provide a theoretical framework to understand the importance of the endosomal virus passage before membrane fusion and genome release .', 'We further unraveled RNA degradation as a previously unknown limiting factor for virus infection .', 'Our work will help to make predictions and evaluate newly occurring virus strains , regarding their infection efficiency in a given host cell , by simply considering their pH sensitivity .']
['cell physiology', 'medicine and health sciences', 'vesicles', 'pathology and laboratory medicine', 'pathogens', 'microbiology', 'signaling networks', 'orthomyxoviruses', 'viruses', 'wireless sensor networks', 'membrane fusion', 'rna viruses', 'network analysis', 'molecular biology techniques', 'cellular structures and organelles', 'microbial genomics', 'endosomes', 'research and analysis methods', 'viral genomics', 'influenza a virus', 'computer and information sciences', 'genome complexity', 'artificial gene amplification and extension', 'medical microbiology', 'microbial pathogens', 'molecular biology', 'cell membranes', 'cell biology', 'influenza viruses', 'polymerase chain reaction', 'virology', 'viral pathogens', 'genetics', 'biology and life sciences', 'genomics', 'computational biology', 'organisms', 'cell fusion']