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Influenza

Influenza properties:
  • Eight RNA segments
  • Antigenic shift and drift
  • Respiratory transmission
  • Frequent childhood and adult disease

It has two binding proteins: a hemagglutinin (H) and a neuramidinase (N). These, combined with the location and strain of the virus, are used in nomenclature:

A/Ann Arbor/6/60 (H2N2)
strain, location, serial #, year, type

HA binds to salicylic acid. These receptors are only in the respiratory tract. There are different types of linkage:

  • α2-3 linkage on avian cells
  • α2-6 linkage on avian cells
  • α2-3 & α2-6 linkage on avian cells

Antigenic Variation

Antigenic variation in influenza virus can occur by antigen shift and antigenic drift. In antigenic drift, small changes accumulate in the epitopes of HA and NA due to RNA replication errors (mutations). This causes the annual variations in influenza. In antigenic shift, a human virus and an animal virus co-infect the same cell, resulting in reassortment of viral RNA segments. Antigenic shift can cause a pandemic if a new virus emerges that:

  • Has mostly human RNA
  • Has human or animal hemagglutinin protein with high affinity for human cells
  • Humans have not evolved antibodies against.

Influenza virus is an avian virus. There are 15 types of HA and 9 types of N. By resassortment, a new HA can come into the human population. BY antigenic drift, the HA protein can adapt to bind better to the human sialic acid receptor α2-6. There are three human A strains (H1N1, H2N2, H3N2) in addition to strain B. They can all adapt easily to bind to the human receptor. The human HA protein is cleaved only in the lung, meaning the humans strains are infectious only in the lung.

Transmission

Influenza has two modes of transmission: person-to-person and by respiratory droplets. Then, influenza viruses enter cells via receptor-mediated endocytosis, a kind of engulfment.

Following internalization, the vesicle is with an endosome. Endosomes are acidic, and this low pH activates the M2 ion channel. This allows ions to enter the virion, leading to a conformational change in the HA protein.

The virus is internalized into clathrin-coated, membrane-bound vesicles.

Amantidine blocks influenza virus replication. Viral mutants resistant to amantidine map to the trans membrane domain of the M2 protein.
First, select for spontaneous AmR mutants
  • Grow viruses in presence of amantidine.
  • After successive passages in presence of amantidine, AmS viruses will be eliminated.
Next, map the gene segment that encodes AmR.
  • Coinfect cells at 20 pfu/cell (a high multiplicity of infection) with WT and AmR viruses.
  • Progeny viruses are a mixture of WT and reassortants.
  • By using several different strains in coinfections, it can be shown that each reassorted AmR virus contained M1 and M2 segments from AmR strain.
Next, obtain direct evidence that M2 is an ion channel influenced by amantidine.
  • Transfect oocytes of Xenopus laevis (or any model organism) with the M2 gene.
  • Observe the function of M2.
  • Observed that at low pH, HA molecule extends ~100 angstroms, causing fusion of viral envelope & endosomal membrane, ∴ releasing viral nucleocapsid into the cytoplasm.
  • Incubation in presence of amantidine blocks M2 protein.

Summary: Upon entry into the cell, the enveloped virus resides in a low pH endosome. The viral M2 protein, part of the envelope, serves as an ion channel to further reduce the pH. This induces a conformational change in the HA protein, causing it to protrude forward and effecting a fusion of the viral envelope with the membrane of the endosome. This releases the nucleocapsid into the cytoplasm.

The flu (disease)

Classic flu-like symptoms include:
  • Fever
  • Malaise
  • Myalgia (muscular pain)
  • Sore throat
  • Nonproductive cough

Influenza vaccines

  • Formalin-inactivated whole virus
  • Chemically disrupted virus (subvirion)
  • Purified antigens

H5N1 virus (Bird flu)

H5N1, an influenza, binds well onto to &alpha2-3 sialic acid. It contains RNA segments that help produce disease. For example, one RNA segment turns off a component of the immune system. H5 is cleaved by the protease furin, which is present in all cells. Therefore, H5N1 is a pantropic virus able to infect all tissues of avians. It is so pathogenic because, although most human lung cells express α2-6 sialic acid, there are a few expressing α2-3 sialic acid. The H5N1 virus infects these cells and causes a potentially fatal very strong immune response. It can infect poultry, and may adapt to be transmissble from human-to-human.

Influenza transcription

Influenza virus uses the caps of eukaryotic mRNAs instead of synthesizing its own.

This was concluded by a series of experiments:

  • Actinomycin D inhibits DNA-dependent RNA transcription. Influenza virus transcription is inhbited by Actinomycin D. As a result, it was proposed than nucleus plays a role in viral replication.
  • α-amantin is an inhibitor of DNA-dependent RNAP II. It was shown that it inhibited transcription of influenza virus in a cell but not in vitro. This inhibition by α-amantin led to experiments regarding the role of cellular RNA transcription in viral RNA synthesis. In vitro transcritpion was performed using:
    • Detergent-disrupted virions, which provide viral RNA templants and polymerases.
    • Labeled nucleotides
  • The dinucleotide ApG, which is complementary to the first 2 nucleotides at 3' end of each viral RNA segment (3'-UpCpG...5') was shown to stimulate transcription. It was also shown that ApG was incorporated directly into the 5' end of the newly synthesized transcript. It is unusual for a dinucleotide to stimulate transcription.
  • Dr. Krug showed that globin mRNA, even more than ApG, greatly stimulates in vitro transcritption. He went on to show that the globin mRNA m7GpppG can structure was transferred to the influenza virus mRNA transcript.

Krug's Cap-Stealing Experiment was as follows:

  • Globin mRNA added to in vitro transcription stimulates that reaction compared to no globin mRNA added.
  • Decapped globin mRNA does not stimulate in vitro transcription, compared to normal globin mRNA.
  • Decapped globin mRNA is used to put a 32P-labeled cap back on, which transfers radioactivity to viral mRNA synthesized in vitro, as shown by gel migration.
m7GpppA˜˜˜˜˜˜˜˜˜˜˜Globin mRNA stimulates in vitro transcription.
˜˜˜˜˜˜˜˜˜˜˜Remove cab with tobacco acid pyrophosphatase. Uncapped mRNA does not stimulate in vitro transcription
32P˜˜˜˜˜˜˜˜˜˜˜Re-cap with 32P-GTP
In vitro transcription without 32P-GTP

Summary: viral polymerase is unable to cap the viral mRNAs. Because uncapped mRNAs are unstable, the virus steals caps from cellular mRNAs. Thus, replication of influenza virus is inhibited by drugs that block DNA-dependent RNA synthesis since these drugs remove a source of cap structures for the virus to steal.

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