Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement
Free PMC article
Item in Clipboard
Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement
Free PMC article
Influenza viruses replicate within the nucleus of the host cell. This uncommon RNA virus trait provides influenza with the advantage of access to the nuclear machinery during replication. However, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. The segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral RNA (vRNA) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. To accomplish this goal, influenza A viruses (IAVs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vRNA gene segments in and out of the cell. The aim of this review is to present the current mechanistic understanding for how IAVs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the IAV infection process.
HA and NA; hemagglutinin; influenza A virus; neuraminidase; viral entry mechanism; viral envelope proteins; viral replication; viral ribonucleoprotein.
Influenza A and B viruses.
(A) Schematic of the eight viral RNA (vRNA) gene segments that comprise the influenza A and B genomes. The 5′ and 3′ untranslated regions (UTRs), which contain the viral promoters, are represented with a line, and the box corresponds to the coding region within each vRNA. (B) Diagram of the viral mRNAs that are transcribed from the IAV (left) and IBV (right) vRNA templates. Boxes indicate the viral gene product encoded by each mRNA and the dashed lines show the alternative splicing of the IAV M and NS transcripts, as well as the IBV NS transcript. Red circles represent the 5′ M 7pppG cap, black lines denote the 10–13 nucleotide, host-derived primers that are obtained by the cap-snatching mechanism of the viral polymerase. A(n) corresponds to the 3′ poly-A tail produced by reiterative stuttering of the viral polymerase. The smaller mRNAs (empty boxes) represent transcripts that encode nonessential accessory proteins found in many strains, whereas those that are less prevalent (PB2-S1, M42, and NS3) are not illustrated (–11). (C) Diagram of an influenza A or B virus. The viral membrane proteins HA, NA, and M2 are shown, along with the eight viral ribonucleoproteins (vRNPs), and the matrix protein M1 that supports the viral envelope. To highlight the vRNP components, the illustration beneath the virus is not to scale. A single vRNA gene segment is shown wrapped around multiple nucleoprotein (NP) copies with the conserved promoter regions in the 5′ and 3′ UTRs forming a helical hairpin, which is bound by a single heterotrimeric viral RNA-dependent RNA polymerase (PB1, PB2, and PA). (D) Top view of an influenza virus cross-section showing the vRNP “1 + 7” configuration. vRNPs are depicted with black circles as it is not known if the positioning of a particular vRNP is conserved or interchangeable.
Receptor-mediated cell entry of IAVs.
(A) Diagram of a bi-antennary N-linked glycan. The terminal sialic acid residues are displayed with an α-2,3 linkage, as well as an α-2,6 linkage, to illustrate the “linear” and “bent” presentations. (B) Illustration of IAV cell entry. (i) IAVs initiate cell entry by using the HA receptor-binding domain (located in the HA1 region) to associate with sialylated glycoconjugates on a host “receptor.” Binding to the “receptor” triggers endocytosis. (ii) The virus then traffics to the endosome where the lower pH facilitates a conformational change in HA, exposing the fusion peptide (located in the HA2 region) for insertion into the endosomal membrane. (iii) The HA pre-hairpin conformation begins to collapse, forming a six-helix bundle that promotes hemifusion of the viral envelop with the endosomal membrane. At some point, the M2 channel opens to release the viral ribonucleoproteins (vRNPs) from M1 by acidifying the viral interior. (iv) HA further collapses into a trimer of hairpins to promote the formation of the fusion pore, which (v) releases the vRNPs into the cytosol. (vi) The exposed nuclear localization signals (NLS) on the vRNPs are recognized by the adaptor protein importin-α, leading to the recruitment of importin-β that (vii) facilitates the transport through the nuclear pore complex (NPC) and into the nucleus.
Transcription of the complimentary RNA (cRNA) and viral RNA (vRNA) by the heterotrimeric viral RNA-dependent RNA polymerase (PB2, PB1, and PA).
(A) The viral polymerase initiates transcription of the positive-sense cRNA upon base-pairing of ATP and GTP with the complimentary nucleotides in the 3′ end of the vRNA. The subsequent formation of the A-G dinucleotide is followed by elongation of the cRNA transcript. Nucleoprotein (NP) molecules successively bind to the cRNA as it exits the polymerase, promoting cRNP assembly. cRNP formation is completed upon the termination of transcription and with the binding of a newly synthesized viral polymerase (yellow outline). (B) vRNA transcription proceeds in a similar manner as cRNA synthesis. Recent structures support a model where (i) ATP and GTP base pair to the nucleotides located 4 and 5 bases from the cRNA 3′ end, and there form a dinucleotide, which then disassociates and reanneals with the bases at positions 1 and 2. (ii) Alternatively, ATP and GTP could bind directly to the terminal nucleotides and form a dinucleotide. Both mechanisms would position the dinucleotide at the cRNA 3′ end, which is necessary to transcribe a full-length vRNA. Similar to cRNP formation, multiple NPs and a viral polymerase bind to the newly transcribed vRNA to produce a new viral ribonucleoprotein (vRNP).
Transcription of IAV mRNAs by the viral polymerase. Viral mRNA transcription occurs when the viral ribonucleoproteins reach the host cell nucleus and is assisted by the association of the viral polymerase (PA subunit) with the cellular RNA polymerase II C-terminal domain (RNA pol II CTD). Transcription initiates by a “cap-snatching” mechanism where the PB2 subunit binds to the 5′ cap of a host mRNA (red). Cap binding positions the region of the mRNA 10–13 nucleotides downstream for cleavage by the endonuclease domain in the PA subunit. Following cleavage, a conformational shift repositions the acquired mRNA capped primer to the PB1 subunit where the 3′ end base-pairs with a complimentary sequence at the vRNA 3′ end. Following the priming event, the viral polymerase extends the mRNA transcript. The transcription is terminated by a “reiterative stuttering” process (depicted in the box), which occurs when the polymerase encounters the 5–7 consecutive uracil bases at the vRNA 5′ end. The “reiterative stuttering” function likely involves multiple cycles of dissociation and reannealing, and effectively polyadenylates [A(n)] the viral mRNA by continuously repositioning the elongating 3′ end on the uracil-rich region of the vRNA template.
Coordination of viral ribonucleoprotein (vRNP) assembly and trafficking to the plasma membrane. Upon entry into the host cell nucleus, (i) the vRNP-associated viral polymerase transcribes the viral mRNAs. (ii) The mRNAs are either directly, or after alternative splicing, exported for translation by cytosolic ribosomes. (iii) Newly synthesized viral polymerase subunits (PA, PB1, and PB2) and nucleoprotein (NP) are imported back into the nucleus. (iv) Due to the inefficient dinucleotide priming, the vRNP-associated viral polymerase also infrequently transcribes complimentary RNA (cRNA) copies that assemble into cRNPs
via (v) binding of a newly synthesized viral polymerase (PA, PB1, and PB2) and NP. (vi) The polymerase transcribes viral RNA (vRNA) copies from the positive strand in the cRNPs and these assemble into vRNPs by (vii) association with a new viral polymerase (PA, PB1, and PB2) and NP. Once assembled, the new vRNPs can (viii) transcribe additional viral mRNAs, (ix) transcribe new cRNA copies, or (x) associate with the newly synthesized viral proteins M1 and NS2 to facilitate the recruitment of CRM1, which (xi) mediates the nuclear export of the vRNP. (xii a) Once exported, the vRNPs then associate with Rab11 that assists in the trafficking of the vRNPs toward the cell surface. The vRNP trafficking either occurs by Rab11-containing vesicles associated with microtubules or (xii b) through Rab11 located in the modified endoplasmic reticulum (ER) membranes. How the vRNPs reach the budding site at the plasma membrane is currently not known.
NA contributions to viral release and intercellular movement. (i) Viral mRNAs encoding the membrane proteins NA, HA, and M2 are exported for translation by cytosolic ribosomes. (ii) Exposure of the N-terminal signal sequence (HA) or transmembrane domains (NA and M2) recruits the signal recognition particle (SRP), which (iii) targets the ribosome–nascent chain complex for synthesis at the endoplasmic reticulum (ER). Following synthesis, the proteins oligomerize and are trafficked through the
Golgi to the plasma membrane. (iv) Late in replication, the viral ribonucleoproteins (vRNPs) are exported from the nucleus and (v a) trafficked to the budding regions in the plasma membrane, where (v b) HA and NA have co-localized, with M2 at the budding boundary. (vi) Following budding, progeny virus can remain associated with the infected cell’s surface through HA binding to sialic acid (SA). (Box A) The envelope protein NA promotes release of the virus from the infected cell surface by hydrolyzing the glycosidic bond attaching the SAs. (vii) SAs present on the glycans of HA and NA can result in HA-mediated virus–virus association. (Box B) NA can separate the viruses by removing these SAs. (viii) In the respiratory tract, the epithelium is protected by mucus, rich in sialylated glycoproteins such as mucin, which can associate with HA and slow viral movement. (Box C) NA can cleave off the SAs from the glycoproteins within the mucus to facilitate movement of the virus to neighboring cells.
Intracellular Colocalization of Influenza Viral RNA and Rab11A Is Dependent upon Microtubule Filaments.
J Virol. 2017 Sep 12;91(19):e01179-17. doi: 10.1128/JVI.01179-17. Print 2017 Oct 1.
J Virol. 2017.
28724771 Free PMC article.
Heterologous Packaging Signals on Segment 4, but Not Segment 6 or Segment 8, Limit Influenza A Virus Reassortment.
J Virol. 2017 May 12;91(11):e00195-17. doi: 10.1128/JVI.00195-17. Print 2017 Jun 1.
J Virol. 2017.
28331085 Free PMC article.
Transport of the influenza virus genome from nucleus to nucleus.
Viruses. 2013 Oct 2;5(10):2424-46. doi: 10.3390/v5102424.
24104053 Free PMC article.
The Feat of Packaging Eight Unique Genome Segments.
Viruses. 2016 Jun 17;8(6):165. doi: 10.3390/v8060165.
27322310 Free PMC article.
Comparative review of respiratory diseases caused by coronaviruses and influenza A viruses during epidemic season.
Microbes Infect. 2020 May 13:S1286-4579(20)30083-6. doi: 10.1016/j.micinf.2020.05.005. Online ahead of print.
Microbes Infect. 2020.
32405236 Free PMC article.
Antiviral Activity of Chicken Cathelicidin B1 Against Influenza A Virus.
Front Microbiol. 2020 Mar 19;11:426. doi: 10.3389/fmicb.2020.00426. eCollection 2020.
Front Microbiol. 2020.
32265870 Free PMC article.
Interaction of influenza A virus NS2/NEP protein with the amino-terminal part of Nup214.
Turk J Biol. 2020 Apr 2;44(2):82-92. doi: 10.3906/biy-1909-49. eCollection 2020.
Turk J Biol. 2020.
32256144 Free PMC article.
Silent Infection of B and CD8
+ T Lymphocytes by Influenza A Virus in Children with Tonsillar Hypertrophy.
J Virol. 2020 Apr 16;94(9):e01969-19. doi: 10.1128/JVI.01969-19. Print 2020 Apr 16.
J Virol. 2020.
Hause BM, Collin EA, Liu R, Huang B, Sheng Z, Lu W, et al. Characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the Orthomyxoviridae family. MBio (2014) 5:e31–14.10.1128/mBio.00031-14
Palese P, Shaw ML. Fields Virology. Philadelphia: Lippincott Williams & Wilkins; (2007).
Palese P, Schulman JL. Mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes. Proc Natl Acad Sci U S A (1976) 73:2142–6.10.1073/pnas.73.6.2142
McGeoch D, Fellner P, Newton C. Influenza virus genome consists of eight distinct RNA species. Proc Natl Acad Sci U S A (1976) 73:3045–9.10.1073/pnas.73.9.3045
Houser K, Subbarao K. Influenza vaccines: challenges and solutions. Cell Host Microbe (2015) 17:295–300.10.1016/j.chom.2015.02.012