Did viruses enable sex?

Dengue virus E glycoproteins (monomer at top) lie flat on the virus particle as dimers (middle). At endosomal low pH, three monomers reorient to place the fusion peptide (orange) into the cell membrane. Image credit.

A key step in sexual reproduction is the fusion of haploid cells to form a diploid zygote, yet the molecular mechanism underlying this joining of cells is poorly understood. Two studies reveal amazing similarities between proteins required for fusion of sperm and egg, and virus with host cells.

A screen for genes that cause male sterility in the flowering plant Arabidopsis led to the identification of the HAP2 protein. This protein was later found to be important for sperm-egg fusion in Arabidopsis and in the unicellular algae Chlamyodomonas. 

Homology modeling shows that the HAP2 protein looks very much like a class II viral fusion protein (illustrated). Found in dengue virus and many related viruses, dimers of these viral glycoproteins lie flat on the viral membrane, and are comprised largely of beta-strands. At one end of the protein is a fusion loop which allows the virus and cell membranes to join at the start of infection.

The HAP2 protein also has what looks to be a viral fusion loop. Removal or alteration of this sequence in Tetrahymena prevents fusion of mating cells. The fusion loop of the dengue virus E glycoprotein cannot substitute for the HAP2 sequence. Furthermore, vesicular stomatitis viruses with HAP2 in place of the viral glycoprotein cannot enter cells. However the results of biophysical experiments indicate that the HAP2 fusion loop can interact with membrane lipids in ways reminiscent of viral fusion peptides.

Solution of the atomic structure of HAP2 reveals a trimer with protein folds and an upright ‘hairpin’ configuration (illustrated for dengue virus) typical of class II fusion proteins. While acidification of viral type II fusion proteins is required for rearrangement to the post-fusion form, the trigger for HAP2 is not known.

These results clearly show that HAP2 is a type II fusion protein that mediates the joining of haploid gametes in the first step of sexual reproduction. These viral and cell proteins are so similar that it is highly improbable that they arose by convergent evolution. HAP2 is ancient: besides green algae and plants, it is also found in unicellular protozoa, cnidarians, hemichordates, and arthropods, indicating that it was likely present in the last common ancestor of eukaryotes. But viruses existed before the evolution of eukaryotic sex, raising the scenario that type II fusion proteins first arose in viruses, which provided them to eukaryotic cells for use in gamete cell fusion.

Without viruses, there would be no sex, and therefore no humans, or many other animals on Earth.

We continue to recognize new ways that the evolution of eukaryotic life has depended on viruses. These include a viral gene used to produce the placenta; enhancer elements for innate immunity; prions; and the nucleus. What exactly did eukaryotes invent?

TWiV 431: Niemann-Pick of the weak

The TWiVirions reveal bacteriophage genes that control eukaryotic reproduction, and the biochemical basis for increased Ebolavirus glycoprotein activity during the recent outbreak.

You can find TWiV #431 at microbe.tv/twiv, or listen below.

Click arrow to play
Download TWiV 431 (71 MB .mp3, 118 min)
Subscribe (free): iTunesRSSemail

Become a patron of TWiV!

TWiV 415: Ebola pipettors and the philosopher’s clone

Jeremy Luban, Aaron Lin, and Ted Diehl join the TWiV team to discuss their work on identifying a single amino acid change in the Ebola virus glycoprotein from the West African outbreak that increases infectivity in human cells.

You can find TWiV #415 at microbe.tv/twiv, or listen below.

Click arrow to play
Download TWiV 415 (67 MB .mp3, 110 min)
Subscribe (free): iTunesRSSemail

Become a patron of TWiV!

Increased infectivity of Ebola virus glycoprotein from West Africa

filovirionWhen viruses cross species, serial transmission may lead to the selection for mutations that confer improved replication or transmission in the new host. Identifying such mutations in human viruses is extremely difficult: we cannot conduct the appropriate experiments in humans, and often do not have viral isolates spanning the time from spillover through prolonged circulation. The 2013-2016 outbreak of Ebola virus in West Africa is unique because viral genome sequences were obtained early and throughout the epidemic. The results of two new studies (link to paper one, link to paper two) suggest that some of the observed mutations increase infectivity for human cells. The impact of these mutations on infection of humans, and their role in the West African outbreak, remain unknown.

Many mutations have been identified among the many hundreds of genome sequences obtained during the recent Ebola virus epidemic. One stands out: a mutation that leads to a single amino acid change in the viral glycoprotein, from alanine to valine at position 82 (A82V). This change arose early in the outbreak (it was first observed in Guinea in March 2014) and was subsequently found in most of the isolates. It has never been observed in previous Ebolavirus outbreaks.

The effect of the A82V change on viral infectivity was determined by building pseudotyped viral particles – in this case, HIV particles with the Ebola virus glycoprotein. Human cells in culture were infected with pseudotyped viruses with the Ebola virus glycoprotein with either alanine or valine at position 82. Infectivity was measured by quantifying the production of a protein from the HIV genome. The results show that A82V increases infectivity by twofold. The effect is also observed in cells from non-human primates, but not from rodents, dogs, or cats. However, the A82V change decreased infectivity in bat cells.

The A82V change is located at the binding site of the Ebola virus glycoprotein with the cell fusion receptor, NPC1. It appears to increase the fusion activity of the viral glycoprotein.

Other amino acid changes in the Ebola virus glycoprotein were also observed to increase infectivity in human cells, and decrease infectivity in bat cells.

The pattern of increased infectivity in primate cells, and decreased infectivity in bats, is consistent with the hypothesis that the outbreak virus came from bats, and after circulation of the virus in humans, it lost some ability to infect bat cells while becoming better at infecting human cells. However there is still no solid proof that bats are a reservoir of Ebolaviruses.

What does increased infectivity have to do with infection of humans? The idea is that the mutation increases the efficiency of virus entry into cells, and hence increased viral gene expression is observed. Fewer viruses needed to infect a cell, the better chance of initiating an infection. But is the two-fold increase observed in cells enough to impact infection in humans?

The assays used in these papers measure protein production from an HIV genome. The experiments need to be repeated using bona fide Ebola virus, to make sure that the mutations have the same effect. The changes might have impacts on other stages of viral replication. Furthermore, the impact of the changes in the viral glycoprotein should be assessed in animal models, to determine if improved infectivity has any impact on pathogenesis and transmission. Ultimately, we can’t prove that these mutations have any effect in humans – the needed experiments cannot be done.

I’m curious about why the A82V change was not seen in previous Ebola virus outbreaks. Those were in different parts of Africa – could the changes be driven by population genetics, ecology, or other factors? It will be important to determine if the same change is selected in future outbreaks.

The authors are sufficiently cautious in their conclusions. From paper #2:

Despite the experimental data provided here, it is impossible to clearly establish whether the adaptive mutations observed were in part responsible for the extended duration of the 2013–2016 epidemic. Indeed, it seems likely that the prolonged nature of the outbreak in West Africa was primarily due to epide- miological factors, such as an increased circulation in urban areas that in turn led to larger chains of transmission.

From paper #1:

Our findings raise the possibility that this mutation contributed directly to greater transmission and thus to the severity of the outbreak. It is difficult to draw any conclusions from this hypothesis, though…

As I feared, press coverage of these findings has been inaccurate. For example, a BBC headline proclaims “Ebola adapted to easily infect people”. Even the journal Cell, which published both papers, made an incorrect conlcusion: see the screen capture below from the journal website.key mutations ebola virusBoth Cell and the BBC might have taken too literally the unfortunate title of one of the papers,  “Human adaptation of Ebola virus during the West African Outbreak.” The results suggest adaptation to human cells, not to humans. The title of the second paper is sufficiently careful: “Ebola virus glycoprotein with increased infectivity dominated the 2013-2016 epidemic”. But that’s not a BBC headline.

Structure of Zika virus

Zika virus reconstructionSix months after Zika virus became a household word, we now know the three-dimensional structure of the virus particle. And it looks like very much like other flaviviruses, such as West Nile and dengue viruses.

In the old days, solving a virus structure was a big deal. A virus is, after all, a very large assembly of many proteins. To solve the structure of a virus – which will tell us the location of the amino acid chains in three dimensional space – was a technical tour de force. It was necessary to purify large amounts of virus particles, and then find the conditions to produce crystals, a hit and miss affair. If you were lucky to grow virus crystals – which could take a year or more – you then crossed your fingers to see if they diffracted in an X-ray beam. When X-rays are aimed at a crystal, the beams bounce off atoms in the crystals, and their reflections provide information on where the atoms are located. Finally you could collect the diffraction data, do a lot of math on a computer, and determine the three dimensional structure.

The first virus structure to be solved by X-ray crystallography was of a plant virus, tomato bushy stunt virus in 1976, followed by poliovirus and rhinovirus in 1985. Many X-ray structures of viruses have been solved, with resolutions less than 2 Angstroms that allow us to see not only the amino acid chain, but all the atoms in the side chains.

The Zika virus structure was not solved by X-ray crystallography. It was done by cryo-electron microscopy (cryo-EM) and image reconstruction. It’s easier and faster than X-ray crystallography, and can achieve comparable resolutions.

It is not necessary to produce crystals to determine structures by cryo-EM. Instead, samples of purified viruses are rapidly frozen and photographed with an electron microscope at very low temperatures. This procedure preserves native structure, and allows visualization of the contrast inherent in the virus particle. Photographs of thousands of virus particles – each in a slightly different orientation – are taken and processing computationally to create the final three-dimensional image.

The cryo-EM structure of Zika virus tells us how the virus particle is put together. It looks very much like other flaviviruses, which consist of a membrane surrounding the capsid, which in turn carries the viral RNA genome. Inserted into the membrane are 180 copies of the viral proteins E and M. Although inserted in a fluid lipid bilayer, they are arranged with a symmetry that reflects their contacts with the underlying icosahedral capsid. In the illustration, which I produced from the freely available cryo-EM data, you can clearly see five copies of the E glycoprotein (red) at one five-fold axis of symmetry.

One structural difference between Zika virus and other flaviviruses is a loop of amino acids exposed on the surface of the particle. This sequence of the E glycoprotein, and a sugar molecule attached to it, might be involved in regulating Zika virus tropism and pathogenesis. The ability of West Nile virus to enter the central nervous system of mice has been linked to glycosylation at a similar position, while cell receptors are thought to attach to sugars on the dengue virus capsid.

The authors of the Zika virus cryp-EM structure have produced an animation which illustrates aspects of the structure (below). Watch my lecture on virus structure for more information how viruses are put together.

Updated 7 April 2016 to provide an explanation of how the sugar attached to the E glycoprotein of Zika virus might regulate tropism and pathogenesis.

Covering up a naked virus

Sabin type 2 poliovirusViruses can be broadly classified according to whether or not the particle is enveloped – surrounded by a membrane taken from the host cell – or naked. Some naked viruses apparently are more modest than we believed.

Members of the family Picornaviridae, which include Hepatitis A virus, poliovirus, and Coxsackieviruses, have non-enveloped particles that consist of a protein shell surrounding the viral RNA genome (poliovirus is illustrated). Examples of viruses that are enveloped include dengue virus, influenza virus, and measles virus.

Recently it was discovered that hepatitis A virus (HAV) particles are released from cells in membrane vesicles containing 1-4 virus particles. These membranous structures resemble exosomes, which are also released from uninfected cells and play roles in various biological processes. Enveloped hepatitis A virus particles are present in the blood of infected humans. However virus in the feces, which is transmitted to other hosts, is not enveloped.

Viral envelopes typically contain viral glycoproteins, such as the HA protein of influenza viruses, which serve important functions during replication, such as attachment to cell receptors. Envelope glycoproteins are also the target of antibodies that block viral infection. The presence of an envelope makes HAV resistant to neutralization with antibodies, because the membrane contains no viral proteins that can be blocked by antibodies.

Two other non-enveloped picornaviruses, Coxsackievirus B and poliovirus, are also released from cells within membrane vesicles. These virus particles are in vesicles derived from the autophagy pathway, which captures and recycles cytoplasmic contents by ejecting them from the cell.

What is the function of the membrane acquired by these naked viruses? Perhaps immune evasion: the presence of the cell membrane makes HAV and Coxsackievirus B virus particles resistant to neutralization with antibody. The ability to deliver multiple virus particles to a single cell might help to overcome genetic defects in the viral genome that are a consequence of the high mutation rates of these viruses.

An interesting problem is how these cloaked viruses enter cells, because there is no evidence that the membranes contain any viral proteins that could interact with a cell receptor. Nevertheless, entry of enveloped HAV and poliovirus into cells requires the known viral receptor. Perhaps the vesicles are taken into the cell by endocytosis, where viral particles are released from the vesicles, and then bind receptors to initiate escape of the genome.

Should HAV, poliovirus, and Coxsackievirus B be reclassified as enveloped viruses? Probably not, in part because the membranes surrounding these virus particles are not needed for infectivity. In contrast, removal of the membrane from influenza virus, dengue virus, or measles virus destroys their infectivity. Enveloped viruses acquire a membrane after the internal components have been assembled, whether they are helical or icosahedral nucleocapsids. In contrast, HAV, poliovirus, and Coxsackievirus B become fully infectious particles before they acquire an envelope.

Another argument against calling picornaviruses enveloped is that viral membranes contain viral glycoproteins that allow attachment to cell receptors and release of the viral genome into the cell. There is no evidence that the membranes of picornaviruses contain viral proteins.

The acquisition of a membrane may have taken place later in the evolution of picornaviruses, to allow more efficient infection or evasion of host responses. Alternatively, the membrane may simply be a by-product acquired when these viruses exit the cell by a non-lytic mechanism.

While the finding of membranes around picornavirus particles is intriguing, I am not yet convinced that these viruses should be considered to be enveloped. I would like to know if other non-enveloped viruses are similarly released from cells in membranous cloaks, and the function of this addition for viral replication in the host.

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.

Virology lecture #5: Attachment and entry

Download: .wmv (386 MB) | .mp4 (131 MB)

There are some errors in this lecture – I’ll correct them during the next session.

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

TWiV 33: Live in Philly

twiv-200Episode 33 of the podcast “This Week in Virology” was recorded before an audience at the ASM General Meeting in Philadelphia. Vincent, Alan, Dick, and Raul Andino discussed increased arterial blood pressure caused by cytomegalovirus infection, restriction of influenza replication at low temperature by the avian viral glycoproteins, first isolation of West Nile virus in Pennsylvania, and current status of influenza.

Click the arrow above to play, or right-click to download TWiV #33 or subscribe in iTunes or by email.