Influenza virus genome reaches the plasma membrane via a modified endoplasmic reticulum and Rab11-dependent vesicles

Abstract

Transport of neo-synthesized influenza A virus (IAV) viral ribonucleoproteins (vRNPs) from the nucleus to the plasma membrane involves Rab 11 but the precise mechanism remains poorly understood. We used metal-tagging and immunolabeling to visualize viral proteins and cellular endomembrane markers by electron microscopy of IAV-infected cells. Unexpectedly, we provide evidence that the vRNP components and the Rab11 protein are present at the membrane of a modified, tubulated endoplasmic reticulum (ER) that extends all throughout the cell, and on irregularly coated vesicles (ICVs). Some ICVs are found very close to the ER and to the plasma membrane. ICV formation is observed only in infected cells and requires an active Rab11 GTPase. Against the currently accepted model in which vRNPs are carried onto Rab11-positive recycling endosomes across the cytoplasm, our findings reveal that the endomembrane organelle that is primarily involved in the transport of vRNPs is the ER.

Fig. 1

WSN-infected A549 cells recruit rough ER cisternae and a new type of organelle around the MTOC. a, b Immunofluorescence and confocal microscopy images (lateral and frontal merge, respectively) of A549 cells expressing HA-tagged Rab11 and infected with the WSN virus (8 hpi). Rab11 (red) and viral NP (green) co-exist in discreet spots near the nucleus (arrows). (c) Ultrathin-section of a mock-infected A549 cell as visualized by transmission electron microscopy (TEM). The MTOC area near the nucleus is shown. Mitochondria (mi) surround the centrioles (c) near the nucleus (N). d Equivalent MTOC region in a WSN-infected A549 cell at 8 hpi showing swollen rough ER cisternae (arrowheads) and numerous irregularly coated vesicles (ICVs, arrows) that are a new type of organelle. e Close-up of a pair of ICVs. f General view of perinuclear region in a WSN-infected A549 cell at 8 hpi showing modified rough ER (black arrowheads), groups of ribosomes (white arrowheads), and an ICV (arrow). Scale bars, 10 μm a, b; 0.5 μm c, d, f; 100 nm e

Fig. 2

IAV induces a general remodeling of the ER. a Serial sections of a WSN-infected A549 cell at 8 hpi showing long ER elements (arrows) expanding from the nucleus to the cell periphery. b Serial sections of a mock-infected A549 cell. ER cisternae near the nucleus (N) are marked with arrows; mi, mitochondria. Scale bars, 0.5 μm

Fig. 3

Time-course of influenza virus infection shows a progressive remodeling/tubulation of ER in A549 cells. a TEM of a mock-infected A549 cell. Mitochondria (mi) and rough endoplasmic reticulum (white arrows) have a random distribution. N, nucleus. b WSN-infected A549 cell at 4 hpi. The cell contains intranuclear dense structures (white arrowheads). Black arrowheads mark tubular, swollen ER membranes near the nuclear envelope (ne). c, d WSN-infected A549 cells at 6 hpi. Long ER-like tubular membranes (black arrowheads) expand from the nuclear envelope (ne) to the plasma membrane (pm). Arrows point to ICVs. e, f Immunofluorescence and confocal microscopy images showing viral NP (green) and the ER marker PDI (red) in mock- and WSN-infected A549 cells, respectively. Swollen/tubulated ER expands from the nucleus in WSN-infected cells at 8 hpi (arrows in f). Scale bars, 0.5 μm a−d; 10 μm e, f

Fig. 4

WSN vRNPs are detected in the ER and ICVs by METTEM. A549 cells infected with the recombinant virus WSN-PB2-MT were treated with gold salts at 8 hpi, fixed and incubated with silver to increase the diameter of gold nanoclusters built by the MT tag. Cells were post-fixed with osmium tetroxide, sectioned, stained and studied by TEM. a Cell showing labeled vRNPs in nuclear envelope (ne), in ER (arrowheads), ICVs (arrows) and close to plasma membrane (pm). b Higher magnification from a different cell showing labeled vRNPs in ER membranes (arrowhead). c Cell section through the MTOC showing label in ICVs (arrows) around the centriole (dashed circle). d Cell sectioned through the nuclear envelope showing vRNPs in nuclear pores (arrows) and close to filaments (arrowhead). e Signal associated with vRNPs in ICVs (arrows) and close to filaments (arrowheads). f Signal associated with vRNPs in a region close to plasma membrane. An ICV (arrow) and budding viruses (arrowhead) are labeled. Scale bars, 0.5 μm a–c; 200 nm d–f

Fig. 5

Localization of Rab11 and PDI in WSN-infected A549 cells. a, b Immunofluorescence and confocal microscopy images of two A549 cells (one optical section per cell) infected with the WSN virus and labeled at 8 hpi with antibodies specific for Rab11 (red) and the ER marker PDI (green). c, d Two optical sections of a control mock-infected cells. Arrows point to co-localization spots. Scale bar, 5 μm

Fig. 6

Ultrastructural localization of HA-MT-Rab11 and HA-MT-Rab11-S25N by METTEM upon IAV infection. a Distribution of stably expressed HA-MT-Rab11 in WSN-infected A549 cells at 8 hpi. Cells were incubated with gold and silver before processing. Signals reveal the location of HA-MT-Rab11 in remodeled ER (arrowheads) and ICVs (arrows). N, nucleus. b Distribution of stably expressed HA-MT-Rab11-S25N in WSN-infected A549 cells at 8 hpi. Signals show the presence of HA-MT-Rab11-S25N in small vesicles (arrows). The arrowheads label remodeled ER with groups of ribosomes. No ICVs are detected. Insets are enlargements of labeled vesicles. The inset on the top corresponds to the vesicle marked with a double arrow in the middle of the mainfield. After irradiation in the microscope the silver particles decrease in size and the electron lucent center of the vesicle is better seen. The inset on the bottom corresponds to a labeled vesicle from another cell. Scale bars, 0.5 μm a; 200 nm b. 100 nm (insets)

Fig. 7

Rab11 co-localizes with NP on ICVs and is required for ICV formation. A549 cells expressing HA-MT-Rab11 a, b or HA-MT-Rab11-S25N c, d were infected with the WSN virus and fixed for immuno-EM at 16 hpi. Thawed cryosections were labeled for HA (5 nm gold) and NP (10 nm gold). a Two ICVs in the cytoplasm that are positive for both NP and Rab11. The insert shows in detail an ICV with the characteristic irregular electron dense coat and label for Rab11 and NP. b An area underneath the plasma membrane (pm) with viral particles outside the cell and a tubular ICV (arrow) close to it. Label for Rab11 and NP are found on the ICV. The irregular electron dense coat of the ICV can be clearly distinguished against the regular coat of clathrin, present on a clathrin-coated pit (arrowhead). c, d In cells expressing HA-MT-Rab11-S25N ICVs are absent. Instead, NP labeling is present on small vesicles and tubules (arrows). These tubules show no visible coat but often have an electron dense lumen. Scale bars, 200 nm

Fig. 8

Remodeled ER and ICVs expands from nuclear envelope to plasma membrane in WSN-infected A549 cells. a WSN-induced membranous compartment at 8 hpi as visualized by TEM of serial sections, 3D reconstruction and image processing. Tubular ER membranes (yellow) with attached mitochondria (red) and ICVs (gray) contact with the nuclear envelope (blue) and the plasma membrane (brown). Viruses are shown in black. b−d Collection of 2D images showing the assembly of ICVs from modified ER membranes and the ultrastructure of ICVs at 8 hpi. b The membrane of ICVs is continuous with that of altered ER (arrows). c ICVs near a virus budding area. The irregular coat of ICVs (arrows) is very similar to the layer of short filaments under the plasma membrane (arrowheads). d Close-ups of ICVs very close to the plasma membrane (pm). V, viruses. Scale bars, 200 nm

Fig. 9

Characterization of purified ICVs. A549 cells were infected with the WSN virus and submitted to subcellular fractionation at 14 hpi. a Western-blot analysis of subcellular fractions of mock-infected (left panel) and IAV-infected (right panel) A549 cells, upon sucrose gradient ultracentrifugation. The same amount of total protein was analyzed for each fraction. TfR: transferrin receptor; PDI: protein disulfide isomerase; Cal: calreticulin; Clathrin HC: Clathrin Heavy Chain. For gel source data, Supplementary Fig. 17. b TEM analysis of an aliquot of fraction 60–40% from IAV-infected cells, upon fixation and negative staining. Black arrows: ICVs. White arrows: vRNPs. Scale bar: large panel 200 nm, small pannel 100 nm

Fig. 10

Model for the trafficking of vRNPs across the cytoplasm in an IAV-infected cell. The remodeled tubulo-vesicular ER, positive for the PDI marker (in blue) extends around the MTOC (in gray) and from the nuclear envelope (ne) to the plasma membrane (pm). After their exit from the nucleus, individual vRNPs and/or sub-bundles of vRNPs are targeted to the modified ER. ICVs loaded with vRNPs and with the Rab11 molecule (in red) might bud from the ER and ensure the transport of vRNPs to the plasma membrane. The frequently observed pairing of ICVs could favor RNA−RNA interactions among vRNPs and the progressive assembly of sets of 8 distinct vRNPs. vRNPs are released from ICVs and possibly transferred to the plasma membrane in a touch-and-go process