Influenza Virus Assembly and More - Lecture 8

Influenza Virus Assembly, Pandemic Emergence, and Vaccine Development Study Notes

Course Objectives

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

    • Describe the process of influenza virus assembly

    • Nuclear export of viral ribonucleoproteins (vRNPs)

    • Targeting of HA, NA, and M2 to lipid rafts on the apical membrane

    • Interaction of vRNPs with surface proteins during virion formation

    • Explain the role of NEP, M1, and NP

    • In facilitating nuclear export and genome packaging

    • Discuss the function of neuraminidase (NA)

    • In viral release and how its inhibition affects viral spread

    • Define antigenic shift and antigenic drift

    • Compare their mechanisms

    • Explain their roles in seasonal flu and pandemics

    • Describe the concept of viral reassortment

    • Contribution to the emergence of pandemic influenza strains

    • Explain host specificity of influenza viruses

    • Differences in sialic acid linkages (α2-3 vs. α2-6)

    • How HA mutations, e.g., Q226L, G228S, enable cross-species transmission

    • Discuss the role of pigs as mixing vessels

    • In influenza evolution and interspecies transmission

    • Compare and contrast vaccine types

    • Inactivated influenza vaccines

    • Live attenuated influenza vaccines

Assembly of Influenza Virus

Step 1: Nuclear Export of vRNPs

  • Objective: Get the vRNPs out of the nucleus.

    • Components involved:

    • Polymerase proteins: PB1, PB2, PA

    • Nucleoprotein (NP)

    • Viral components include HA (Hemagglutinin) and M2 proteins.

    • Transport:

    • NP contains a nuclear localization signal (NLS) to retain vRNPs in the nucleus.

    • M1 and NEP (NS2) bind to vRNPs, masking NP’s NLS and exposing nuclear export signals (NES).

    • NES are recognized by exportins for transport through the nuclear pore into the cytoplasm.

Step 2: Targeting Envelope Proteins

  • Objective: Concentrate HA, NA, and M2 at the apical surface of epithelial cells.

    • The proteins contain transmembrane and cytoplasmic sequences directing them to lipid rafts.

    • Lipid Rafts: Cholesterol-rich microdomains that serve as platforms for viral budding.

    • Targeting ensures release of new virions into the airway lumen.

Step 3: Packaging vRNPs into Budding Virions

  • Interaction: vRNPs must interact with surface proteins at the plasma membrane for incorporation into budding virions.

    • NP targets vRNPs to lipid rafts facilitating this integration.

    • M1 acts as a bridge, binding to cytoplasmic tails of HA and NA and vRNPs, ensuring correct genome packaging.

    • Ensures that each virion contains the correct set of 8 genome segments.

Viral Release

  • Neuraminidase (NA): A tetrameric enzyme on the viral surface responsible for viral release.

    • Cleaves sialic acid residues from host cell glycoproteins and glycolipids.

    • Prevents newly formed virions from re-binding to the host cell surface.

    • Inhibition Example:

    • Oseltamivir (Tamiflu) blocks this cleavage, leading to virions remaining attached to the host cell, thereby reducing viral spread.

Host Receptors and Specificity

  • Sialic Acid:

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

    • HA1 binds to sialic acid to initiate infection.

    • Variance in linkage between sialic acid and galactose influences host specificity and tissue tropism.

    • Neuraminidase assists in cleaving sialic acid, enabling viral release.

Antigenic Shift vs. Drift

Antigenic Shift

  • Occurs when a new HA or NA subtype emerges in humans due to two different influenza viruses infecting the same cell.

  • Gene segments mix during viral assembly, creating reassortant viruses, which may evade immune detection due to a lack of pre-existing immunity, leading to pandemic potential.

Antigenic Drift

  • Involves small, gradual changes in HA and NA due to point mutations resulting in antibody escape.

  • Responsible for seasonal flu and annual vaccine updates, approximately 1% drift/year corresponding to about 3 amino acid changes in HA1.

Host Adaptation and Cross-Species Transmission

  • HA mutations such as Q226L and G228S shift receptor specificity, enabling avian viruses to bind to human-type receptors and facilitating infection of human respiratory cells.

  • These mutations are critical in host adaptation and zoonotic transmission.

Vaccine Insights

Inactivated Influenza Vaccine (Standard Flu Shot)

  • Production Process:

    • Trivalent vaccine protecting against 2 A and 1 B strain; strain selection based on global surveillance.

    • HA and NA genes integrated into a vaccine backbone, virus grown in embryonated chicken eggs, and subsequently inactivated using formalin or beta-propiolactone.

  • Immune Response:

    • The inactivated virus is non-infectious but remains antigenic; antigen-presenting cells (APCs) recognize HA and NA, stimulating B cells for antibody production.

    • Provides strain-specific immunity.

Live Attenuated Influenza Vaccine

  • Design and Delivery:

    • Contains live but weakened virus delivered via nasal spray (FluMist®); licensed for ages 5 to 49.

    • HA and NA from circulating strains are inserted into an attenuated backbone.

  • Attenuation Strategies:

    • Passaged in non-human cells to reduce virulence and utilize temperature-sensitive mutations to restrict replication to cooler nasal passages.

  • Immune Response:

    • Mimics natural infection, stimulating mucosal immunity and a systemic antibody response, potentially providing broader protection than inactivated vaccines.