Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

de Vries, Erik; Tscherne, Donna M.; Wienholts, Marleen J.; Cobos-Jiménez, Viviana; Scholte, Florine; García-Sastre, Adolfo; Rottier, Peter J. M.; de Haan, Cornelis A. M.

To confirm our results and to obtain further proof for the utilization of DYNA-DEP and DYNA-IND entry routes by IAV, we additionally used an IAV virus-like particle (VLP) direct entry assay [26] . These VLPs contain IAV HA and NA in their envelope and harbor a beta-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1), which allows the rapid and direct detection of entry, independent of virus replication. Upon fusion of viral and endosomal membrane, BlaM1 gains access to the cytoplasmically retained fluorigenic substrate CCF-2 that, after cleavage by BlaM1, shifts to a shorter fluorescent emission wavelength that can be detected by flow cytometry. Entry into HeLa cells was performed in the absence or presence of 10% FCS using VLPs containing HA and NA either from IAV-WSN (having a strict alpha 2-3 linked sialic acid binding specificity) or from the pandemic 1918 IAV (HA from A/ NewYork/1/18, binding to alpha 2-3 and alpha 2-6 linked sialic acids; NA from A/BrevigMission/1/18). Entry of VLPs of both IAV strains was severely inhibited by dynasore when no serum was added to the inoculum (Fig. 4A, 4D) , whereas the presence of 10% FCS rendered entry completely dynasore resistant. (Fig. 4B, 4E ). Quantification of VLP entry is shown in Fig. 4C and F. Importantly, to confirm the existence of the serum-inducible entry pathway by a method that is independent of dynasore, we used siRNA induced silencing of dynamin 2. Fig. 4G shows that two different siRNAs had a significant inhibitory effect (48 hrs after siRNA transfection) on entry of the Renilla luciferaseencoding pseudovirus WSN-Ren [27] in HeLa cells in the absence of FCS, whereas the presence of 10% serum no reduction in entry levels was observed, confirming the results obtained with dynasore. Knockdown of dynamin 2 protein levels (48 hrs after siRNA transfection) was analyzed by western We conclude that a DYNA-IND entry pathway can be induced by serum in different cell types from several species. The evidence was obtained using both replication-dependent (Gluc-entry assay and immunodetection of infected cells) and replication-independent assays (entry of VLPs), the latter allowing immediate detection of the fusion-mediated delivery of viral M1 protein into the cytoplasm. Cascade Blue). In the histograms entry is displayed by a shift to higher fluorescence (the grey area represents background fluorescence of noninfected cells). (C and F) Quantification of FACS results. Background fluorescence was subtracted from each measurement (geometric mean) and data were normalized to VLP entry in Optimem without dynasore (DY) (red curve of panel A and and D). VLP entry was not inhibited by dynasore in presence of 10% FCS whereas the access of BlaM1 to its CCF2 substrate in the cytoplasm was blocked by dynasore in PBS. VLP entry was more efficient in the presence of serum. (G) Effect of downregulation of dynamin 2 by siRNA silencing. Serum-inducible DYNA-IND entry was analyzed in HeLa cells that were transfected 48 hrs prior to infection with two different siRNAs targeting dynamin-2 (dyna). siRNA treated cells were infected with the pseudovirus WSN-Ren in PBS (grey bars) or in PBS containing 10% FCS (black bars) and luciferase activity was determined after 16 hrs post infection (y-axis; RLU relative to infection of cells transfected with a scrambled siRNA). Entry of pseudovirus WSN-Ren (MOI 0.5) was reduced by 50% to 70% when entry was performed in PBS (grey bars) whereas entry wa