TWiV 282: Tamiflu and tenure too

On episode #282 of the science show This Week in Virology, the TWiV team reviews a meta-analysis of clinical trial reports on using Tamiflu for influenza, and suggestions on how to rescue US biomedical research from its systemic flaws.

You can find TWiV #282 at www.microbe.tv/twiv.

Cutting through mucus with the influenza virus neuraminidase

influenza virusNeuraminidase is one of three different viral proteins embedded in the lipid membrane of influenza virus (NA is blue in the illustration at left). This enzyme has a clear and proven role in virus release from cells. NA is also believed to be important during virus entry, by degrading the mucus barrier of the respiratory tract and allowing virus to reach cells. This role is supported by the finding that treatment of mucus-covered human airway epithelial cells with the NA inhibitor Tamiflu substantially suppresses the initiation of infection.  Further evidence comes from the recent finding that influenza virus binds to sialic acids in mucus and that NA cleaves these sugars to allow infection.

The mucus layer of the respiratory tract has a defensive role because it contains soluble glycoproteins that are rich in sialic acids, which are cell receptors for some viruses. When influenza virions enter the respiratory tract, they are thought to be trapped in mucus where they bind sialic acids, preventing infection of the underlying cells. The role of NA in penetration of the mucus layer was studied in frozen sections of human tracheal and bronchial tissues, in which the mucus layer is preserved. Influenza A virions bind to this mucus layer; the interaction is blocked when the tissues are first treated with a bacterial neuramindase, which removes sialic acids from glycoproteins. These observations indicate that the mucus layer of human airway epithelial cells contain sialic acid-containing decoys that bind influenza A viruses.

Human salivary mucins, which approximate the mucus of the human respiratory tract, can protect cultured cells from influenza virus infection. The protective effect can be achieved by simply adding the mucins to cells. The inhibitory effect is dependent on the sialic acid content of the mucins: fewer cells are infected when higher concentrations are used. In contrast, porcine salivary mucins do not substantially reduce influenza virus infection. The type of sialic acids and the virus strain also determine the extent of protection.

To determine the role of the viral NA in mucin-mediated inhibition, Tamiflu was mixed with virus before infection of mucus-coated cells. The presence of Tamiflu increased the inhibition of infection caused by mucins, indicating that the sialic acid-cleaving (sialidase) activity of NA is needed to overcome inhibition by human salivary mucins. When influenza virions are incubated with human salivary mucins linked to beads, sialic acids are cleaved from mucins, and this enzymatic activity is inhibited by Tamiflu. Human salivary mucins inhibit NA by binding to the active site of the enzyme.

These studies establish a clear role for the influenza viral NA in bypassing the defenses of the mucosal barrier. An important message is that not all strains of influenza virus are equally inhibited by mucus; presumably this property is one of many that determines viral virulence. The balance between how tightly the viral HA binds to sialic acids, and how well the NA cleaves them, is probably one predictor of how well we our protected by our mucus.

 

Changing influenza virus neuraminidase into a receptor binding protein

neuraminidaseThe hemagglutinin (HA) and neuraminidase (NA) glycoproteins of the influenza virus particle serve distinct functions during infection. The HA binds sialic acid-containing cellular receptors and mediates fusion of the viral and cell membranes, while the NA removes sialic acids from glycoproteins. Apparently this division of labor is not absolute: influenza viruses have been identified with NA molecules that serve as receptor binding proteins.

An influenza virus was created that could not bind sialic acid by introducing multiple mutations into the HA gene. This mutant virus was not expected to be infectious, but nevertheless did propagate to moderate titers in cell culture. A single amino acid change was identified in the NA protein of this virus: G147R, which is just above the active site of the enzyme (illustrated; active site marked with green spheres). Passage of the virus in cell culture produced a virus that multiplied to higher titers; improved growth was caused by a K62E change in the HA stalk. The results of site-directed mutagenesis showed that the G147R change allowed the NA protein to serve the receptor binding function normally provided by HA. It is not clear how the HA change leads to improved growth of the G147 virus.

Although the G147R NA can serve as receptor binding protein, the HA is still required for fusion: abolishing this activity by mutation or by treatment with a fusion-blocking antibody did not allow virus growth.

The influenza NA protein is an enzyme (sialidase) that cleaves sialic acids from cellular and viral proteins. The G147R NA is active as a sialidase, and this activity can be blocked by the antiviral compound oseltamivir, which is an NA inhibitor. Treatment of G147R-containing virus with oseltamivir also blocked virus binding to cells. Virus-like particles that contain G147R NA but not HA can attach to sialic acid-containing red blood cells. This attachment can be reversed by oseltamivir. After binding to red blood cells, these virus-like particles slowly fall off, a consequence of NA cleaving sialic acid receptors. These observations indicate that the G147R NA binds to sialic acids at the active site of the enzyme, and cleaves the same receptor that it binds.

Treatment of cells with a bacterial sialidase that removes a broad range of sialic acids only partially inhibits G147R NA-mediated binding to cells. In contrast, growth of wild type influenza virus is completely blocked by this treatment. Therefore the receptor recognized by G147R NA is not the same as that bound by wild type virus.

Changing the influenza virus NA to a receptor binding protein is not simply a laboratory curiosity: the G147R NA change was found in 31 of 19,528 NA protein sequences in the Influenza Virus Resource. They occur in seasonal H1N1 viruses that circulated before 2009, in the 2009 swine-origin pandemic H1N1 virus, and in avian H5N1 viruses. The presence of this change in phylogenetic clusters of seasonal H1N1 and chicken H5N1 sequences suggests that they are also found in circulating viruses, and are not simply sequence errors or the product of passage in the laboratory.

These observations emphasize the remarkable flexibility of the influenza viral glycoproteins in their ability to switch receptor binding function from HA to NA. They might also have implications for vaccines, whose effectiveness are thought to depend largely on the induction of antibodies that block the function of HA protein. The work underscores the importance of serendipity in science: the HA receptor binding mutant virus was originally produced as a negative control for a different experiment.

The neuraminidase of influenza virus

influenza virusThe influenza virus particle is made up of the viral RNA genome wrapped in a lipid membrane (illustrated). The membrane, or envelope, contains three different kinds of viral proteins. The hemagglutinin molecule (HA, blue) attaches to cell receptors and initiates the process of virus entry into cells. I have written about the HA and its function during infection (article one and two) but not about the neuraminidase (NA, red) or M2 (purple) proteins. Let’s first tackle NA.

An important function of the NA protein is to remove sialic acid from glycoproteins. Sialic acid is present on many cell surface proteins as well as on the viral glycoproteins; it is the cell receptor to which influenza virus attaches via the HA protein. The sialic acids on the HA and NA are removed as the proteins move to the cell surface through the secretory pathway. Newly released virus particles can still potentially aggregate by binding of an HA to sialic acid present on the cell surface. Years ago Peter Palese showed that influenza virus forms aggregates at the cell surface when the viral neuraminidase is inactivated. The NA is therefore an enzyme that is essential for release of progeny virus particles from the surface of an infected cell.

The NA protein also functions during entry of virus into the respiratory tract. The epithelial cells of the respiratory tract are bathed in mucus, a complex protective coating that contains many sialic acid-containing glycoproteins. When influenza virions enter the respiratory tract, they are trapped in mucus where they bind sialic acids. This interaction would prevent the viruses from binding to a susceptible cell were it not for the action of the NA protein which cleaves sialic acids from glycoproteins. When the virus particle encounters a cell, it binds the sialic acid-containing receptor and is rapidly taken into the cell before the NA protein can cleave the carbohydrate from the cell surface.

The essential nature of the NA for virus production has been exploited to develop new drugs designed to inhibit viral release. Both Tamiflu (Oseltamivir) and Relenza (Zanamivir) are structural mimics of sialic acid that bind tightly in the active site of the NA enzyme. When bound to drug, the NA cannot remove sialic acids from the cell surface, and consequently newly synthesized virus remains immobilized. The result is an inhibition of virus infection because virions cannot spread from one cell to another.

This article is part of Influenza 101, a series of posts about influenza virus biology and pathogenesis.

TWiV 223: EEEV and the serpent

On episode #223 of the science show This Week in Virology, Vincent, Alan, and Kathy discuss new influenza virus NA inhibitors, detection of EEEV antibody and RNA in snakes, and replication of the coronavirus EMC in human airway epithelial cells.

You can find TWiV #223 at www.microbe.tv/twiv.

Frederick Hayden on influenza antivirals

Frederick Hayden, Professor of Medicine and Pathology, University of Virginia School of Medicine, U.K., has focused on the use of antiviral agents to prevent and treat respiratory viral infections. His interests range from the use of in vitro assays to study viral susceptibility and antiviral mechanisms of action, to clinical trials utilizing experimentally induced and naturally occurring infections. Work from his laboratory includes the demonstration that intranasal administration of interferons can prevent transmission of rhinovirus colds, studies of transmission of drug-resistant influenza A viruses in families, and the antiviral activity and clinical use of influenza neuraminidase inhibitors. His laboratory currently focuses on the application of nucleic acid hybridization to study rhinovirus pathogenesis, elucidating the phenotypic and genotypic basis of antiviral drug resistance in rhinovirus and influenza viruses, and clinical testing candidate antiviral agents for influenza and rhinovirus infections.

I discussed the use of antiviral drugs to treat influenza with Dr. Hayden during ICAAC Boston 2010, as part of TWiV 99. View the video below, or at YouTube.

TWiV 99: ICAAC Boston 2010

Host: Vincent Racaniello

Vincent tours the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in Boston, speaking with exhibitors and visitors, including Professors Derek Smith, Michael Schmidt, Frederick Hayden, and Myra McClure.

Many thanks to Chris Condayan and Ray Ortega of the American Society for Microbiology for recording and editing this episode.

Click the arrow above to play, or right-click to download TWiV #99 (45 MB .mp3, 62 minutes)

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Secondary changes allow spread of oseltamivir resistant influenza virus

The influenza virus neuraminidase (NA) protein is required for virus release from the cell, a property exploited by the antiviral drugs oseltamivir (Tamiflu) and zanamavir (Relenza). During clinical testing of oseltamivir in 2001, some individuals shed drug-resistant viruses with an amino acid change from histidine to tyrosine (H274Y) in NA. Such viruses are not inhibited by oseltamivir because the amino acid change leads to  decreased binding of the drug. But these viruses replicated less well in cell culture, and had reduced infectivity in ferrets. It was concluded that oseltamivir resistant influenza virus mutants would not spread in the population. Why was this conclusion wrong?

During the 2008-09 flu season oseltamivir resistant influenza H1N1 viruses with the H274Y change began to spread, and within a year they were found in most seasonal isolates. It was hypothesized that these viruses contained other amino acid changes that masked the deleterious effect of H274Y. The H274Y mutation does not affect the catalytic activity of the NA: the ability to cleave sialic acid from glycoproteins. However it does lead to a decease in the amount of NA protein that is transported to the surface of infected cells.

Computational methods were used to identify amino acids in NA that could potentially compensate for the effect of H274Y. A single amino acid change at position 194 of NA, when present with H274Y, restored NA on the cell surface to normal levels.

Did a similar amino acid change in seasonal H1N1 strains allow the spread of oseltamivir resistant viruses with H274Y? Introduction of this amino acid change into the seasonal H1N1 strains A/Texas/91 and A/New Caledonia/99 causes a decrease in surface NA. However the same change has a lesser effect on surface NA in cells infected with A/Solomon Islands/2006. Two amino acid changes were identified in the NA protein of recent oseltamivir-resistant seasonal H1N1 viruses that restore surface levels of NA in the presence of H274Y: V234M and R222Q.

It seems likely that the amino acid changes V234M and R222Q emerged first in the NA of seasonal H1N1 viruses. Why these changes appeared is unknown, but they could be a consequence of random drift, antigenic selection, or a need to balance HA and NA activities. Once these changes were in place, oseltamivir resistant viruses with the H274Y could be selected, and because they had no defect in fitness, they spread globally.

The conclusion is that H274Y in NA attenuates the fitness of influenza virus by reducing the amount of NA on the cell surface. Spread of such viruses in the population is impossible without secondary amino acid changes that restore adequate levels of surface NA. H274Y probably causes a defect in NA folding or transport that is balanced by the secondary mutations.

These findings are another example of how drug resistance frequently comes with a cost to protein stability or folding, and prevents evolution unless compensated by secondary mutations.

There have been scattered isolations of oseltamivir-resistant, pandemic 2009 H1N1 influenza virus with the H274Y change. Will these viruses spread globally, or are they less fit, evolutionary dead ends? Introduction of the H274Y change into the NA of 2009 pandemic H1N1 virus leads to a large decrease in surface NA. Unless the 2009 swine-origin viruses already produce excess NA, viruses with the H274Y change are not likely to spread without secondary mutations that rescue NA surface expression.

Bloom JD, Gong LI, & Baltimore D (2010). Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science (New York, N.Y.), 328 (5983), 1272-5 PMID: 20522774

Virology lecture #21: Antivirals

Download: .wmv (349 MB) | .mp4 (90 MB)

Visit the virology W3310 home page for a complete list of course resources.

TWiV 71: Please Mr. Postman

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Rich Condit

Vincent, Dickson, Alan, and Rich answer listener questions about maternal infection and fetal injury, viral gene therapy, eyeglasses and influenza, filtering prions from blood, eradication of rinderpest, Tamiflu resistance of H1N1 influenza, bacteriophages and the human microbiome, H1N1 vaccine recalls, human tumor viruses, RNA interference, and junk DNA.

This episode is sponsored by Data Robotics Inc. Use the promotion code VINCENT to receive $50 off a Drobo or $100 off a Drobo S.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #71 (63 MB .mp3, 88 minutes)

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Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.