Release of influenza viral RNAs into cells

In “Influenza virus attachment to cells” we left the intact virion on the cell surface. The next step is that the viral genetic information – for influenza virus, the individual RNA segments – must enter the cell so that it can be reproduced. The mechanism for influenza virus, illustrated below, involves a step that is a target of the antiviral adamantanes.


The entry of influenza virus into cells is one of the best undertood of all known viral entry mechanisms. After the virion attaches to sialic-acid containing receptors at the cell surface, the virus-receptor complex is taken into cells by endocytosis, a process by which cells normally take up molecules from the extracellular fluid. As the endosomal vesicles that contain the virus particles move towards the cell nucleus, their pH drops. This change in pH is accomplished by a cellular channel that pumps protons (H+) into the vesicle. When the endosomal pH reaches 5.0, the viral HA protein undergoes a conformational rearrangement. This change exposes a fusion peptide on the HA – a short, hydrophobic sequence that inserts into the endosomal membrane and causes it to fuse with the viral envelope. When this occurs, the viral RNAs are released into the cytoplasm. They are then transported into the cell nucleus where viral RNA replication occurs.

In the influenza virion, the viral RNAs are not naked, but bound to a number of viral proteins, including the M1 protein. This protein forms the shell that underlies the lipid membrane of the virion. Unfortunately, if the viral RNAs are bound to M1 protein when they are released from the virion, they cannot enter the nucleus. To get around this problem, the influenza virion has a few copies in its membrane of a protein called M2. This viral protein forms a channel in the membrane that actively pumps protons from the endosome into the interior of the virion. These protons lower the pH in the interior of the virion, releasing the viral RNAs from M1. In this way the RNAs can enter the nucleus.

The M2 ion channel is the target of the antiviral adamantanes, depicted below. These compounds clog the channel and prevent it from pumping protons into the virion. In the presence of adamantanes, viral RNAs remain bound to M1 and cannot enter the nucleus. Therefore viral replication is inhibited. Resistance to adamantanes occurs by changes in amino acids that line the M2 channel. These changes prevent the drug from plugging the channel.


Influenza vaccine – hold the eggs

2607036664_da729b4bd5_mInfluenza virus undergoes continuous antigenic variation, necessitating production of a new vaccine each year. This is not a trivial task. Six to nine months before the flu season begins, the viral strains for inclusion in the vaccine must be selected. High-yielding recombinant strains are produced and grown in embryonated chicken eggs. This year, approximately 140 million doses of the killed, injected vaccine were produced in the US – from 140 million eggs. The annual formulation of influenza vaccine is a huge burden on both medical care providers and vaccine producers, not to mention the many chickens that must toil to produce millions and millions of eggs. Can we envision an influenza vaccine that provides lifelong protection, like the vaccines against polio and measles?

The protective antibodies induced by the current influenza vaccines are directed against the highly variable viral glycoproteins, HA and NA. Other virion proteins are far more conserved, and have been tested in experimental vaccines. For example, the nucleoprotein (NP), an internal component of the virion, is an attractive immunogen because it induces a cytotoxic T cell response that should provide protection against a broad spectrum of strains. Because the NP protein is not particularly immunogenic, DNA vaccines have been extensively tested. These are effective at inducing cellular immune responses and have met with some success, especially when coupled with adenovirus vectored antigens.

The viral M2 protein has been a focus of recent influenza vaccine efforts. This highly conserved transmembrane protein forms an ion channel in the virion, and is essential for uncoating of the viral genome in endosomes. Immunization with M2 protein has been shown to confer protection in mouse and ferret models of influenza. However, the protein is not sufficiently immunogenic. In the reports cited below, the authors examined the immunogenicity of M2 protein conjugated to carrier proteins, or presented on the surface of hepatitis B virus-like particles. The latter was effective at inducing protective immunity in mice, but not in rhesus monkeys. The authors conclude that data obtained in mice should not be used to select M2 vaccine candidates.

It seems likely that future influenza vaccines will be based on NP and M2 proteins, perhaps in combination. Significantly more work will be required before such vaccines are approved for use in humans – in the meantime, our insatiable thirst for chicken eggs will continue.

DEFILETTE, M., MARTENS, W., SMET, A., SCHOTSAERT, M., BIRKETT, A., LONDONOARCILA, P., FIERS, W., & SAELENS, X. (2008). Universal influenza A M2e-HBc vaccine protects against disease even in the presence of pre-existing anti-HBc antibodies Vaccine, 26 (51), 6503-6507 DOI: 10.1016/j.vaccine.2008.09.038

Fu, T., Grimm, K., Citron, M., Freed, D., Fan, J., Keller, P., Shiver, J., Liang, X., & Joyce, J. (2009). Comparative immunogenicity evaluations of influenza A virus M2 peptide as recombinant virus like particle or conjugate vaccines in mice and monkeys Vaccine, 27 (9), 1440-1447 DOI: 10.1016/j.vaccine.2008.12.034