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 236: Flu gets the VIP treatment

On epside #236 of the science show This Week in Virology, Vincent, Alan and Kathy review novel approaches to preventing influenza virus infection.

You can find TWiV #236 at www.microbe.tv/twiv.

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.

Influenza neuraminidase and H5N1 pathogenicity

influenza_virion_250There are two glycoproteins embedded in the influenza viral membrane: the hemagglutinin (HA) and neuraminidase (NA). The NA, shown in yellow in the illustration, is an enzyme that removes sialic acids from the surface of the cell, so that newly formed virions can be released. The NA protein is composed of a box-like head attached to the viral membrane via a stalk. The length of the stalk may be an important determinant of the virulence of avian influenza H5N1 viruses.

Examination of the sequence of all known influenza N1 NAs reveals that the proteins can be grouped into six classes depending on the length of the stalk region. The stalk regions of some NAs are intact, while others lack from 15 to 22 amino acids. In 2000, influenza H5N1 isolates from humans were identified with a deletion of stalk amino acids 49-68. The percent of human H5N1 isolates with this deletion has steadily increased from 15.8% in 2000 to 100% in 2007. This observation led to the question: does stalk length influence H5N1 pathogenesis in animals?

The authors produced a series of H5N1 reassortant viruses with NA stalks of different lengths. Six of the 8 viral RNAs were derived from the 1933 H1N1 isolate WSN. The pathogenicity of the reassortant viruses was determined in chickens and in mice. Three of the viruses were highly pathogenic in chickens: one with the NA from a 2004 H5N1 isolate containing the deletion of amino acids 49-68, a second with the NA from WSN virus, and a third with the N1 from a 1997 H5N1 isolate. The NA stalks from the latter two viruses have deletions, but they are different from the one in the 2004 H5N1 NA. The other viruses tested were found to be of low pathogenicity in chickens. These included a virus with an N1 from an 1996 H5N1 virus with an intact stalk, as well as viruses with NAs harboring different deletions of various lengths. The findings in mice paralleled those obtained in chickens.

These results show that the NA stalk plays a critical role in virulence of H5N1 avian influenza virus in chickens and in mice. The 20 amino acid deletion from amino acids 49-68 is associated with high virulence, although similar pathogenicity was observed for viruses with different stalk deletions. These results do not answer the important question: why viruses with this particular NA deletion have become prevalent among human H5N1 influenza virus isolates. H5N1 viruses with the same stalk deletion have been isolated from aquatic birds and terrestrial poultry since 2002. It has been suggested that the stalk deletion is associated with adaptation of influenza viruses to land-based poultry, but the advantages conferred by this particular NA are unknown.

Zhou, H., Yu, Z., Hu, Y., Tu, J., Zou, W., Peng, Y., Zhu, J., Li, Y., Zhang, A., Yu, Z., Ye, Z., Chen, H., & Jin, M. (2009). The Special Neuraminidase Stalk-Motif Responsible for Increased Virulence and Pathogenesis of H5N1 Influenza A Virus PLoS ONE, 4 (7) DOI: 10.1371/journal.pone.0006277

Tamiflu in river water

N1-oseltamivirTamiflu (Oseltamivir) is one of the few antiviral drugs available for treatment of influenza. Use of the drug has increased substantially because of the emergence of the 2009 H1N1 pandemic strain, against which no vaccine is yet available. A recent study has shown that low levels of oseltamivir can be detected in the aquatic environment. This finding raises the possibility that aquatic birds which harbor influenza virus could be exposed to the antiviral, leading to selection of drug resistant viruses.

Oseltamivir is an inhibitor of the influenza neuraminidase (NA) glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. Neuraminidase inhibitors such as Tamiflu and Relenza function by preventing cleavage of sialic acid from the cell surface. These inhibitors act by binding to the sialic-acid binding pocket of the NA protein.

Tamiflu is s prodrug, oseltamivir phosphate, which is converted to the active form, oseltamivir carboxylate (OC) in the liver. The active form of oseltamivir is excreted in the urine, and is not removed by sewage treatment plants. It has therefore been suggested that OC may present in the aquatic environment, and could expose natural reservoirs of influenza virus to low levels of the antiviral.

Because Japan is the largest consumer of Tamiflu, the levels of OC were determined in the Yodo River system in the Kyoto and Osaka prefectures. This river was selected because it is distant from the sea and located in a densely populated area. Surface water was collected before (June 2007) and during (December 2007 and February 2008) the flu season. No OC was detected in water samples from June 2007. At the onset of the flu season, December 2007, the antiviral was found at levels between 2 and 7 nanograms per liter (ng/L). At the peak of the flu season, in February, levels increased to 12 – 58 ng/L. Levels of OC were higher in water samples taken near sewage treatment plants, compared with those obtained farther away. The amounts detected are close to the concentration of drug that causes 50% inhibition of virus replication (the IC50) in cell cultures, reported to be between 80–230 ng/L.

The authors suggest that dabbling ducks, a natural reservoir of influenza virus, could ingest OC. As influenza in dabbling ducks is a gastrointestinal infection, the virus would encounter oseltamivir in the gut, which could promote selection of viruses resistant to the drug.

It is not known whether OC in aquatic environments leads to influenza virus resistance to Tamiflu. Clearly additional studies must be done to determine whether the antiviral drug can be found in other waters around the world. Influenza viruses should be isolated from aquatic birds living in OC contaminated environments to monitor resistance to Tamiflu.

Söderström, H., Järhult, J., Olsen, B., Lindberg, R., Tanaka, H., & Fick, J. (2009). Detection of the Antiviral Drug Oseltamivir in Aquatic Environments PLoS ONE, 4 (6) DOI: 10.1371/journal.pone.0006064

Assembly of influenza virus

Our discussion of influenza virus replication has so far brought us to the stage of viral RNA synthesis. Last time we discussed the formation of viral RNAs, an event which takes place in the cell nucleus. Now we’ll consider how these RNAs participate in the assembly of new infectious viral particles, as illustrated in the following figure.

influenza-assembly

For simplicity, the nucleus is not shown. But remember that the viral RNAs have to be exported from the nucleus to the cytoplasm, where viral assembly occurs. First, the viral mRNAs are translated to produce all the proteins needed to synthesize a new virus particle. The mRNAs encoding the HA and NA glycoproteins are translated by ribosomes that are bound the the endoplasmic reticulum – the membranous organelle that assists in transporting certain proteins to the plasma membrane. As the HA and NA proteins are produced, they are inserted into the membrane of the endoplasmic reticulum as shown. These proteins are then transported to the cell surface via small vesicles that eventually fuse with the plasma membrane. As a result, the HA and NA are inserted in the correct direction in the lipid membrane of the cell. The M2 protein is sent to this location in a similar way.

The (-) strand viral RNAs that will be packaged into new virus particles are produced in the cell nucleus, then exported to the cytoplasm. These RNAs are joined with the viral proteins PA, PB1, PB2, and NP. Viral proteins other than HA, NA, and M2 are produced by translation on free ribosomes, as shown for M1. The latter protein binds to the membrane where HA, NA, and M2 have been inserted. The assembly consisting of viral RNAs and viral proteins – called a ribonucleoprotein complex or RNP –  travels to the site of assembly. The virion then forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.

As new virions are produced by budding, they would immediately bind to sialic acid receptors on the cell surface, were it not for the action of the viral NA glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. This requirement explains how the neuraminidase inhibitors Tamiflu and Relenza function: they prevent cleavage of sialic acid from the cell surface. In the presence of these inhibitors, virions bud from the cell surface, but they remain firmly attached. Therefore Tamiflu and Relenza block infection by preventing the spread of newly synthesized virus particles to other cells.