Fig 1.
Influenza A Virus structure, genome, and replication cycle.
(A) Schematic representation of the IAV virion. HA, NA, and M2 are included in the lipid envelope, oligomeric M1 provides structure to the internal membrane leaflet, while the eight viral ribonucleoparticles (vRNPs) reside in the inner core. (B) Organisation of the vRNP. The trimeric influenza RNA polymerase (FluPol), formed by PB2, PB1, and PA, encasing the termini of the viral genome (vRNA), itself coated with nucleoprotein (NP). (C) Organisation of the IAV genome, highlighting the major alternatively spliced segments, M and NS. (D) Overview of the viral replication cycle, focussing on the major RNA trafficking events. After endocytosis, vRNPs escape in the cytoplasm and are transported to the nucleus, where they can generate viral mRNAs and replicate new vRNA segments through a full-length positive sense intermediate (cRNA). The former are efficiently exported and translated into viral proteins, while the latter is trafficked to budding sites at the plasma membrane. Graphical representations of M1, M2, HA, NA, envelope, cell membrane, nucleus, arrows, and ribosomes are adapted from Servier medical art repository (https://smart.servier.com).
Fig 2.
Viral entry and host endomembrane systems.
On the right side, the major endomembrane systems of the host cell are represented. At the top, endocytosed material (e.g., signalling receptors, in blue) is trafficked to early endosomes (EE), where it can be sorted for fast (directly to the membrane) or slow (transiting through the endocytic recycling compartment, ERC) recycling, or for degradation. In the latter case, it is directed to late endosomes (LE) which then fuse with lysosomes (L). On the bottom, the secretory pathway involves protein production at the endoplasmic reticulum (ER) and trafficking through the Golgi apparatus to acquire post-translational modifications, before reaching the membrane. On the left side, endocytosed virions reach EE, and then LE. Acidification of the vesicle triggers vRNP release. The viral genome is subsequently imported into the nucleus. Graphical representations of cell membrane, nucleus, arrows, virion, actin, scission proteins, receptors, vesicles, ER Golgi, and NPC are adapted from Servier medical art repository (https://smart.servier.com).
Fig 3.
Trafficking through the nuclear pore.
(A) In the cytosol, NLSs on the vRNP are targeted by the importin system for translocation through the NPC. (B) In the nucleus, vRNPs localise to sites of active host transcription by binding to the RNA polymerase II (RNAPII) C terminal domain (CTD), and perform cap snatching. (C) M1 mRNA is spliced into M2 by trafficking to the nuclear speckle by interaction with viral NS1, and host NS1-BP and hnRNPK. (D) NS1 mRNA is spliced into NS2/NEP in the nucleoplasm by the concerted action of several splicing factors, initiated by binding of SF2 to an exonic splicing enhancer (ESE). Both NS2 and M2 are translocated to the cytosol at the end of the splicing process. (E) The precise mechanisms with which unspliced viral mRNAs reach the cytosol are currently unclear. (F) Viral genome replication is segregated to the dense chromatin, by interaction of vRNPs with nucleosomes. (G) Proximity to the chromatin-associated RCC1 provides newly replicated vRNPs with privileged access to the export machinery. (H) After dissociation of the export machinery, vRNPs are loaded (possibly by YB-1) on ERC vesicles at the MTOC. Graphical representations of the NPC, nuclear membrane, microtubulses, vesicles, M1, arrows, DNA, host proteins, and the spliceosome are adapted from Servier medical art repository (https://smart.servier.com).
Fig 4.
Proposed model for cytosolic vRNP trafficking.
After viral genome replication, vRNPs are exported to the cytosol where they are loaded onto Rab11-positive vesicles at the MTOC. Extensive remodelling of the endomembrane systems of the cells upon infection produces ER tubulation and enlarged Rab11-/vRNP-positive puncta. MT-associated vRNP-clad Rab11+ vesicles travel towards the plasma membrane by action of molecular motors as KIF13a, and reach ER exit sites (ERES) where they are unloaded by action of ATG9A. ERES are also sites of viral inclusion phase separation. The means of vRNP delivery to budding sites is still incompletely elucidated. Graphical representations of cell membrane, nucleus, virion, microtubules, host proteins, NPC, and arrows are adapted from Servier medical art repository (https://smart.servier.com).
Fig 5.
(A) Schematic representation of cis-acting packaging and bundling sequences in IAV RNA segments. Adapted from Noda et al. [93]. (B) Representation of secondary structures on the vRNA. (C) Model of inter-segment RNA-RNA interactions. (D) Inter-segment interaction maps have been generated to explore the interaction networks established during infection. (E) Representation of the main models being investigated to explain the formation of genomic bundles: on the right, the dispersed model argues that collisions between vRNP-containing vesicles could lead to the formation of vesicles containing multiple segments, facilitating bundling; while on the left, the compartmentalised model sees the formation of bundling hot spots (for example by phase separation) in which several vesicles can exchange segments, then the bundles would be able to progress to the membrane. Graphical representations of cell membrane, nucleus, virion, microtubules, host proteins, NPC, vesicles, and arrows are adapted from Servier medical art repository (https://smart.servier.com).