TWiV 267: Snow in the headlights

On episode #267 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy review a protease essential for influenza pathogenesis in mice, and directionality of rhinovirus RNA exit from the capsid.

You can find TWiV #267 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.

 

TWiV 263: Game of clones

On episode #263 of the science show This Week in Virology, Ben tenOever joins the TWiV team to reveal the winner of his contest in which influenza viruses carrying different interferon-stimulated genes vie against one another in mice.

You can find TWiV #263 at www.microbe.tv/twiv.

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 257: Caveat mTOR

On episode #257 of the science show This Week in Virology, the TWiV team consider how the kinase mTOR modulates the antibody response to provide broad protection against influenza virus, and explore the problems with scientific research.

You can find TWiV #257 at www.microbe.tv/twiv.

Virus-induced fever might change bacteria from commensal to pathogen

Stem-loopNeisseria meningitidis may cause septicemia (bacteria in the blood) and meningitis (infection of the membrane surrounding the brain), but the bacterium colonizes the nasopharynx in 10-20% of the human population without causing disease. Although understanding how the bacterium changes from a commensal to a pathogen has been elusive, an important property is believed to be the ability to resist destruction by the immune response. Fever caused by a viral infection might be the trigger that makes N. meningitidis evade immunity.

A property of N. meningitidis that makes it cause disease is resistance to complement, a collection of proteins in the blood that help clear pathogens. N. meningitidis has evolved several mechanisms to avoid destruction by complement, including the production of a polysaccharide capsule, addition of sialic acid to a component of the bacterial outer membrane, and the production of a protein that binds one of the complement proteins. It is not clear why a commensal organism would have evolved such evasion mechansims – invading the blood and the brain are dead ends, as they do not lead to transmission to a new host.

An answer to this question comes from the finding that N. meningitidis proteins essential for resistance to complement are under the control of an RNA thermosensor. This control element is an RNA stem loop structure (pictured) formed by base pairing of local sequences within the RNA. Buried in the base-paired stem is a short sequence called the ribosome binding site that is essential for translation of the mRNAs into protein. At 30°C, the stem loop structure is intact, preventing binding of ribosomes to the mRNA; protein synthesis is blocked. At elevated temperatures – 37 or 40°C – abundant protein synthesis takes place, because the RNA secondary structure is denatured, allowing ribosomes to more efficiently access the ribosome binding site on the mRNA.

How do these findings explain why N. meningitidis becomes a pathogen? The temperature of the upper respiratory tract, where the bacterium normally colonizes, is low, so the RNA sensor is intact, preventing production of proteins needed for resistance to complement. If the respiratory tract is infected with a virus, a local immune response occurs which is accompanied by high temperatures – a fever. The RNA thermosensors of N. meningitidis have evolved to sense elevated temperature and turn on the synthesis of proteins that help it to avoid destruction by the immune response. Unfortunately, inflammation also damages the mucosal barriers that normally prevent microbes from invading the underlying tissues, where they have access to the bloodstream. N. meningitidis enters the blood stream, and because it is resistant to complement, it is not cleared. The result may be septicemia and infection of the brain. Moving away from its niche in the respiratory tract is probably not part of the microbe’s plan, but rather a consequence of the fact that it has evolved to survive immune responses to other pathogens in the respiratory tract.

There is some epidemiological support for this scenario: peaks of Neisseria meningitidis disease may follow outbreaks of influenza.

The conversion of commensal bacteria into pathogens by a second infection may be more common than we know. Streptococcus pneumoniae is a human nasopharyngeal commensal that colonizes 10 to 40% of healthy individuals. The bacterium is also a leading cause of respiratory disease. There is evidence that infection with influenza virus releases S. pneumoniae bacteria from biofilms; the free-living bacteria are then able to cause respiratory disease. One of the influenza virus-induced host signals responsible for changing S. pneumoniae from a commensal to a pathogen is fever.  For more on this story, listen to This Week in Microbiology #62.

TWiV 255: Longhorns go viral

On episode #255 of the science show This Week in Virology, Vincent and Rich visit the University of Texas at Austin and meet up with Bob and Chris to talk about their work on influenza virus and microRNAs.

You can find TWiV #255 at www.microbe.tv/twiv.

Jeffrey Almond on vaccine development

Dr. Jeffrey Almond began his career as an academic virologist studying influenza virus, then moved to poliovirus. He made major contributions to our understanding of the molecular basis of poliovirus attenuation and reversion to virulence. After 20 years in academics he moved to Sanofi Pasteur, where he is currently Vice President, discovery research and external R&D.

I interviewed Jeffrey Almond, Ph.D., in Manchester UK at the 2013 meeting of the Society for General Microbiology. We spoke about the eradication of poliovirus, challenges in making a universal influenza vaccine, a dengue virus vaccine developed by Sanofi Pasteur, and moving from academia to industry.

 

Influenza H7N9 gain of function experiments on Dispatch Radio

I spoke with Robert Herriman, executive editor of The Global Dispatch, about the proposed avian influenza H7N9 virus gain of function experiments on Dispatch Radio.