Life Cycle of Influenza - Lecture 7

Learning Objectives

  • By the end of this lecture, students should be able to:

    • Describe the structural features of the influenza virion.
      - The influenza virion has a pleiomorphic (often spherical or filamentous) and enveloped structure, containing helical nucleocapsids. Its genome consists of 8 segments of single-stranded negative-sense RNA.
      Surface viral proteins: Hemagglutinin (HA), Neuraminidase (NA), and M2 ion channel protein.
      Internal viral proteins: Matrix protein M1, Nucleoprotein NP, and Polymerase proteins (PB1, PB2, PA).

    • Explain the molecular interactions involved in viral attachment, focusing on the role of hemagglutinin (HA) and its interaction with host cell sialic acid residues.
      - Hemagglutinin (HA): A trimeric protein on the viral surface; its HA1 subunit binds to sialic acid residues on host cell glycoproteins to initiate infection. The HA2 subunit contains a fusion peptide crucial for membrane merging.
      - Sialic Acid: A sugar molecule on host cell surface glycoproteins that serves as a receptor. Variations in its linkage to galactose influence host specificity and tissue tropism.

    • Illustrate the entry process of influenza virus, including receptor-mediated endocytosis, endosomal acidification, HA conformational changes, and the role of the M2 ion channel in viral uncoating.
      Influenza enters via receptor-mediated endocytosis. Endosomal acidification triggers a conformational change in HA, exposing the HA2 fusion peptide, and activates the M2 ion channel, allowing protons into the virion.
      These changes lead to the fusion of viral and endosomal membranes, disassembly of the M1 matrix protein, and release of viral ribonucleoproteins (vRNPs) into the cytoplasm.

    • Summarize the transcription mechanism of the influenza virus, emphasizing the cap-snatching process and the functions of PB1, PB2, and PA polymerase subunits.
      The virus aims to produce positive-sense RNA from its negative-sense RNA genome, resembling host mRNA. This occurs in the nucleus using an NP-coated vRNA template.
      Polymerase Complex: Includes PB1 (RNA-dependent RNA polymerase activity), PB2 (binds to 5’ cap of cellular mRNAs), and PA (endonuclease activity).
      Cap-snatching: PB2 binds the 5’ cap of host mRNA, PA cleaves it, and PB1 uses the capped fragment as a primer to synthesize viral mRNA. This mRNA is not a full-length copy of vRNA.

    • Compare and contrast the mechanisms and output of transcription and replication of the viral genome.
      Transcription: Produces positive-sense, capped, and polyadenylated mRNA (not full-length copies of the genome) using cap-snatching, primarily for protein synthesis.
      Replication: Produces full-length positive-strand antigenomes (as intermediates) and then full-length negative-strand vRNAs. This process is primer-independent, triggered by high NP levels, and is error-prone (approx. 1 error per 10410^4 nucleotides).

    • Identify the cellular compartments involved in influenza replication.
      Plasma membrane: Attachment, entry via endocytosis, assembly, and release.
      Endosomes: Viral entry and uncoating due to acidification.
      Cytoplasm: Release of vRNPs from endosomes, translation of viral proteins, assembly of new virions.
      Nucleus: Transcription of viral mRNA, replication of the viral genome, assembly of new vRNPs.

    • Describe how viral components are trafficked between the nucleus and the plasma membrane.
      vRNPs: After uncoating in the cytoplasm, vRNPs (containing NP with an NLS) are imported into the nucleus for transcription and replication. Newly replicated vRNPs are then exported from the nucleus to the cytoplasm for assembly.
      Surface Glycoproteins (HA, NA, M2): Synthesized in the ER, processed in the Golgi, and trafficked to the plasma membrane.

    • Evaluate the mechanism of action of antiviral drugs targeting influenza.
      The provided notes do not detail the specific mechanisms of antiviral drugs. Generally, these drugs target various stages, such as HA (attachment inhibitors), NA (neuraminidase inhibitors preventing release), or the M2 ion channel (uncoating inhibitors).

    • Predict how inhibition or mutations of specific viral proteins directly affect the viral life cycle.
      HA inhibition/mutation: Would prevent viral attachment to host cells and membrane fusion during entry, stopping infection.
      NA inhibition/mutation: Would impede the cleavage of sialic acid, preventing the release of new virions from the infected cell surface.
      M2 ion channel inhibition/mutation: Would prevent protons from entering the virion, thus inhibiting the uncoating process and viral genome release.
      PB1, PB2, or PA polymerase subunit inhibition/mutation: Would disrupt viral transcription and genome replication, halting the production of viral mRNA and new genomes.
      NP mutation (especially NLS): Would impair the nuclear import of vRNPs, preventing nuclear-dependent transcription and replication.
      M1 matrix protein inhibition/mutation (disassembly in entry): Would prevent the release of vRNPs from the incoming virion.

Viral Replication Overview

  • Viruses as Parasites:

    • Viruses are obligate intracellular parasites, meaning they require a host cell to replicate.

    • They lack the necessary machinery for replication, relying on host cell interactions for replication.

    • The viral life cycle encompasses several phases: attachment, entry, replication, and spread.

Structural Features of the Influenza Virion

  • Virion Characteristics:

    • Pleiomorphic morphology (often appears spherical or filamentous).

    • Enveloped structure.

    • Contains helical nucleocapsids.

    • Composed of 8 genome segments.

    • Carries a single strand of negative-sense RNA.

  • Surface Viral Proteins:

    • Hemagglutinin (HA)

    • Neuraminidase (NA)

    • M2 ion channel protein

  • Internal Viral Proteins:

    • Matrix protein M1

    • Nucleoprotein NP

    • Polymerase proteins: PB1, PB2, PA

Influenza Life Cycle Stages

  1. Attachment

  2. Entry

  3. Transcription

  4. Translation

  5. Genome replication

  6. Assembly

  7. Release

Mechanism of Attachment

  • Hemagglutinin (HA):

    • HA is a trimeric protein on the influenza virus surface, initially produced as a larger precursor protein HA0.

    • HA1 subunit binds to sialic acid on host cell glycoproteins to initiate infection.

    • HA2 subunit contains the fusion peptide necessary for merging with the host membrane, and cleavage of HA is vital for infectivity.

  • Sialic Acid:

    • A sugar molecule located at the end of glycan chains on host cell surface glycoproteins.

    • Variation in the linkage between sialic acid and galactose by species and tissue type influences host specificity and tropism.

    • Neuraminidase (NA) cleaves sialic acid from host glycoproteins, facilitating viral release from infected cells.

Viral Entry Mechanism

  • Receptor-Mediated Endocytosis:

    • The influenza virus enters the host cell post-attachment through receptor-mediated endocytosis.

    • The endosome undergoes acidification which triggers:

    • Conformational change in HA, exposing the fusion peptide in HA2.

    • Activation of the M2 ion channel that lets protons into the virion.

    • These changes cause:

    • Fusion of viral and endosomal membranes.

    • Disassembly of the M1 matrix protein.

    • Release of viral ribonucleoproteins (vRNPs) into the cytoplasm.

  • Fusion Mechanism:

    • A dramatic shape change in HA triggered by low pH exposes the N-terminus of HA2, allowing it to insert into the host membrane.

    • HA2 folds back, pulling the viral and host membranes together, a mechanism shared with other viruses like HIV and SARS-CoV-2.

Uncoating and Nuclear Import

  • After membrane fusion, the M1 matrix protein disassembles, releasing vRNPs into the cytoplasm.

  • The M2 ion channel assists in acidifying the interior of the virion, promoting disassembly.

  • Each vRNP features a nuclear localization signal (NLS) on the NP protein, guiding it to the nucleus.

Transcription in the Nucleus

  • Transcription Goal:

    • The influenza genome is a single-stranded negative-sense RNA.

    • The aim is to produce single-stranded positive-sense RNA that resembles host mRNA.

    • The template for transcription is the helical NP-coated vRNA, which has 5’ and 3’ ends forming a “panhandle.”

  • Polymerase Complex:

    • The complex includes:

    • PB1: Exhibits RNA-dependent RNA polymerase activity.

    • PB2: Binds to the cap structure at the 5’ end of cellular mRNAs.

    • PA: Displays endonuclease activity involved in cleaving mRNA.

Cap-Snatching Mechanism

  • Process:

    • Influenza employs a cap-snatching mechanism to prime transcription:

    • PB2 binds to the 5’ cap of host mRNA.

    • PA cleaves the 5’ cap a few nucleotides downstream.

    • PB1 utilizes the capped fragment to synthesize viral RNA.

    • This process results in mRNA that is not a full-length copy of the vRNA, missing sequences from the 5’ end but containing extra sequences from cellular mRNA.

Replication of Viral Genomes

  • After transcription, the virus shifts to replicate full-length copies of its genome.

  • Triggered by elevated levels of free NP, the process involves:

    • Primer-independent replication using the same polymerase proteins but in a different conformation.

    • Produces a positive-strand antigenome as an intermediate followed by the synthesis of a negative-strand vRNA to package into new virions.

  • Mutation Rate:

    • Replication is error-prone, contributing to influenza’s high mutation rate—approximately 1 error per 10,00010,000 nucleotides.

Conclusion

  • Assessment of Status:

    • At this stage, glycoproteins reside at the cell surface, and new vRNPs are located in the nucleus, setting the stage to assemble into infectious particles.

Life Cycle Cascade Outline

  • Attachment: Hemagglutinin (HA) on the virion surface binds to sialic acid receptors on the host cell membrane.

  • Entry: The virus enters the host cell via receptor-mediated endocytosis, forming an endosome.

  • Uncoating: Acidification of the endosome triggers HA conformational changes and M2 ion channel activation, leading to membrane fusion and release of viral ribonucleoproteins (vRNPs) into the cytoplasm.

  • Nuclear Import: vRNPs are imported into the nucleus.

  • Transcription: Viral RNA polymerase performs cap-snatching on host mRNAs and synthesizes viral mRNAs (positive-sense) from the negative-sense vRNA genome, primarily in the nucleus.

  • Translation: Viral mRNAs are translated by host ribosomes to produce viral proteins (e.g., HA, NA, M2, M1, NP, polymerase subunits).

  • Genome Replication: The viral polymerase switches to primer-independent replication, producing full-length positive-strand antigenomes, which then serve as templates for new negative-sense vRNAs.

  • Protein Trafficking: Newly synthesized HA, NA, and M2 are trafficked to the plasma membrane. Other viral proteins and new vRNPs are assembled in the cytoplasm (post-nuclear export for vRNPs).

  • Assembly: New vRNPs, matrix proteins, and surface glycoproteins assemble at the plasma membrane.

  • Release: New virions bud from the host cell surface, with Neuraminidase (NA) cleaving sialic acid to facilitate release, preventing self-aggregation.