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.

Virologists plan influenza H7N9 gain of function experiments

A group of virologists lead by Yoshihiro Kawaoka and Ron Fouchier have sent a letter to Nature and Science outlining the experiments they propose to carry out with influenza H7N9 virus.

Avian influenza H7N9 virus has caused over 130 human infections in China with 43 fatalities. The source of the virus is not known but is suspected to be wet market poultry. No human to human transmission have been detected, and the outbreak seems to be under control. According to the authors of the letter, the virus could re-emerge this winter, and therefore additional work is needed to assess the risk of human infection.

The research that the virologists propose involve gain-of-function experiments which provide the H7N9 virus with new properties. The isolation of avian influenza H5N1 viruses that can transmit by aerosol among ferrets is an example of a gain-of-function experiment.

The proposed gain-of-function experiments fall into five general categories:

  • Determine whether viruses with altered virulence, host range, or transmissibility have changes in antigenicity, or the ability of the virus to react with antibodies. The results of these studies would suggest whether, for example, acquisition of human to human transmissibility would have an impact on protection conferred by a vaccine produced with the current H7N9 virus strain.
  • Determine if the H7N9 virus could be adapted to mammals and whether it could produce reassortants with other influenza viruses. The results of this work would provide information on how likely it is that the H7N9 virus would become better adapted to infect humans.
  • Isolate mutants of H7N9 virus that are resistant to antiviral drugs. The purpose of these experiments is to identify how drug resistance arises (the mutations can then be monitored in clinical isolates), determine the stability of drug resistant mutants, and whether they confer other properties to the virus.
  • Determine the genetic changes that accompany selection of H7N9 viruses that can transmit by aerosol among mammals such as guinea pigs and ferrets. As I have written before, the point of these experiments, in my view, is not to simply identify specific changes that lead to aerosol transmission. Such work provides information on the mechanisms by which viruses can become adapted to aerosol transmission, still an elusive goal.
  • Identify changes in H7N9 virus that allow it to become more pathogenic. The results of these experiments provide information on the mechanism of increased pathogenicity and whether it is accompanied by other changes in properties of the virus.

I believe that the proposed gain-of-function experiments are all worth doing. I do not share the concerns of others about the potential dangers associated with gain-of-function experiments: for example the possibility that a virus selected for higher virulence could escape the laboratory and cause a lethal pandemic. Gain-of-function is almost always accompanied by a loss-of-function. For example, the H5N1 viruses that gained the ability to transmit by aerosol among ferrets lost their virulence by this route of infection. When these experiments are done under the proper containment, the likelihood that accidents will happen is extremely small.

All the proposed experiments that would use US funds will have to be reviewed and approved by the Department of Health and Human Services:

The HHS review will consider the acceptability of these experiments in light of potential scientific and public-health benefits as well as biosafety and biosecurity risks, and will identify any additional risk-mitigation measures needed.

While I understand that the authors wish to promote a dialogue on laboratory safety and dual-use research, I question the ultimate value of the communication. Because the letter has been published in two scientific journals, I assume that the target audience of the letter is the scientific community. However, the letter will clearly have coverage in the popular press and I am certain that it will be misunderstood by the general public. I can see the headlines now: “Scientists inform the public that they will continue to make deadly flu viruses”. The controversy about the H5N1 influenza virus transmission studies in ferrets all began with a discussion of the results before the scientific papers had been published. I wonder if the publication of these letters will spark another controversy about gain-of-function research.

In my view, science is best served by the traditional process known to be highly productive: a grant is written to secure funding for proposes experiments, the grant proposal is subject to scientific review by peers, and based on the review the work may or may not be supported. The experiments are done and the results are published. I do not understand why it is necessary to trigger outrage and debate by announcing the intent to do certain types of experiments.

I am curious to know what the many readers of virology blog – scientists and non-scientists – feel about the publication of this letter. Please use the comment field below to express your views on this topic.

TWiV 241: The ferret looks ill

On episode #241 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy review how human placental trophoblasts confer viral resistance via exosome-mediated delivery of microRNAs, and isolation of the first human influenza virus in 1933.

You can find TWiV #241 at www.microbe.tv/twiv.

TWiV 240: Virology in Vermont

On episode #240 of the science show This Week in Virology,  Vincent travels to the University of Vermont to talk with Markus and Jason about their work on HIV, influenza virus, arenaviruses and hantaviruses.

You can find TWiV #240 at www.microbe.tv/twiv.

Influenza vaccines for individuals with egg allergy

baculovirusA CDC (US) advisory committee has recommended the use of FluBlok for individuals with egg allergy:

The Advisory Committee on Immunization Practices (ACIP) voted today, 13 to 0, in favor of recommending FluBlok during the 2013-2014 influenza season for vaccination of persons 18 through 49 years of age with egg allergy of any severity.

FluBlok is an influenza virus vaccine that is produced by expressing the influenza virus hemagglutinin (HA) protein in insect cells using a baculovirus vector. Baculoviruses are rod-shaped viruses (see photograph) that infect insects and other arthropods. The baculovirus virion contains a double-stranded DNA genome. To express the influenza virus HA proteins, recombinant baculoviruses are produced in which DNA encoding the HA protein is inserted into the baculovirus DNA genome. When insect cells are infected with the recombinant baculoviruses, the influenza HA protein is produced.

To manufacture FluBlok, three recombinant baculoviruses were isolated  that contain the HA gene from A/Panama/2007/99 (H3N2), A/New Caledonia/20/99 (H1N1), and B/Hong Kong/330/2001. After infection of insect cells with these recombinant baculoviruses, the HA proteins were purified to greater than 95% purity. In a randomized, placebo-controlled clinical trial, FluBlok was shown to be 44.6% effective in preventing culture-confirmed influenza. The low efficacy might in part be due to antigenic mismatch between the HA proteins used in the vaccine and circulating viruses.

Because FluBlok is not produced in eggs it may be used in individuals with egg allergies. Another alternative for such individuals is Flucelvax, an influenza vaccine produced in cell culture, which was approved by the Food and Drug Administration in November 2012.