Influenza Virus: Intimate Life Details

Electron microscopy is the only method for a direct visualization of viruses – “nanoorganisms” ranging in size from 20 to 250—300 nm. Naturally, such minute size essentially restricts the set of structural components forming an organism: in fact, viruses are mere hereditary material (DNA or RNA) packed into a protein “case” of various degrees of complexity. Such a particle (virion) in water, air, or on the surface of various objects behaves as an inanimate matter; this is why debates on whether viruses are “living substance” continue.

As soon as a virion encounters an appropriate cell, the intricate program of virus reproduction – the program of genetic parasitism – is launched. Having entered the cell, a virus “switches over” the work of cell macromolecular systems to the synthesis of virus molecules. Viruses exploit all the cell structures without any exclusion, which provides not only for the synthesis of virus proteins and nucleic acids, but also for the formation of virus progeny. In addition to virus reproduction, infected cells give the parasite a reliable protection.

The only chance to stop infection progress in the body is to destroy infected cells; on the one hand, the body itself can cope with this task and, on the other, this is a real challenge for the developers of antivirals. It is impossible to solve this problem without going into the fine details of the virus-cell interaction. During their reproduction, viruses utilize different cell structures and mechanisms of various degrees of complexity. In particular, adenoviruses self-assemble in the cell nucleus into hexagonal particles, while measles viruses “dress” their nucleic acid and proteins with the cell plasma membrane.

Similar to many other viruses, the influenza virus may enter the cell by various routes, whose mechanisms are still vague. Nonetheless, electron microscopy allows us to see with our own eyes many “intimate” details of the virus life in the infected cell.

“Full face and side face”

Outwardly, an influenza virus is a bubble or an elongated rod. The membrane envelope conceals an unusual RNA genome composed of eight separate segments or fragments. The surface bristles with spikes formed of the protruding components of the membrane proteins – hemagglutinin and neuraminidase. These particular glycoprotein molecules are responsible for the binding of a virus particle to host cell receptors.

According to the classical concept, the upper part of a virus hemagglutinin molecule binds to glycoproteins and glycolipids of the cell plasma membrane, namely, to sialic acid residues, which usually end the side chains of these molecules.

Interestingly, human influenza viruses bind to the α2,6-galactose–linked sialic acids, whereas the avian influenza viruses bind to the α2,3-galactose–linked acids. (A high binding specificity is determined by the presence of the amino acids leucine of glutamine at a certain position in the virus hemagglutinin molecule.) Swine tracheal cells contain sialic acids of both types, making these animals susceptible to both avian and human influenza viruses. This fact allows swine to be regarded as a sort of “Pandora’s vessel”, in which new variants of influenza virus, dangerous for humans, can form.

Dangerous liaisons

So the foot is in the door – a virus has bound to cell receptors. A pocket in the plasma membrane is formed at the binding site, and the virus finds itself within the so-called endocytic vesicle. In general, endocytosis is a common process for the cells of higher organisms, which they use to take in large molecules. Thus, the virus takes advantage of the host cell’s transport system, doing this “on legal grounds” – its “ticket” is the binding to cell receptors.

The first sorting station in the endocytic traffic flow is an endosome – a membrane vesicle with protrusions and small vesicles inside. Despite its apparent simplicity, the endosome is involved in complex logistic functions in the cell, namely, it “recognizes” and assorts the macromolecules that have entered the cell directing them to the appropriate metabolic pathways. However, this is absolutely unnecessary for a virus, as it knows itself what to do next. Taking the advantage of an acid medium inside the endosome, the virus membrane “squeezes up” against the endosome membrane to fuse with it. Thus, virus RNA enters the cytoplasm, as quick as half hour after the virus has bound to the cell surface. Note that the rimantadine drugs act on this particular stage of influenza virus “uncoating”, thereby blocking the fusion between the virus envelope and endosome membrane.

In addition, influenza virus utilizes various endocytic pathways at the stage of entering the host cell, which increases infection efficiency and provides a better chance to escape an attack of the immune system.

With a solid “backing”

Once the virus genome enters the cytoplasm, it has to reach the site of its replication (reproduction), that is, the cell nucleus. Replication in the cell nucleus is a rare event among the RNA viruses. Although the nucleus is the most protected part of the cell, this virus has somehow learned to use the most solid backing.

It is anything but easy to get there: the cell nucleus is safely isolated from the surrounding cytoplasm, and all the transported molecules are subject to an “ID check” at the gate to nuclear pores. The ID of this virus is a special signaling nucleotide sequence identical to the corresponding cellular sequence, which is encoded in its genome.

In this way, the virus RNA gets into the nucleus, obtaining a secure protection. Note another interesting specific feature of our “hero”: its RNA is of the so-called negative polarity, i.e., it cannot serve as a template for synthesizing its daughter virus RNA (future virus genome) and mRNA (the template for synthesizing virus proteins).

Therefore, two species of positive-polarity (+)RNA are first formed in the nucleus of an infected host cell on the template of (–)RNA. The first species is a complementary virus RNA, c(+)RNA, which is then used for synthesizing daughter (–)RNA. The second species is a messenger virus RNA, mv(+)RNA, which after an intricate chain of transformations by cell enzymes is transported into the cell cytoplasm to serve in future synthesis of virus proteins. Certainly, all these migrations are also provided by the cell transport systems.

Now all the main components for the assembly line aimed at production of millions of virus clones are ready.

A cell factory

According to the cell laws, the membrane proteins of future viruses are synthesized on chains of ribosomes (cell’s protein “factories”), attached to the rough endoplasmic reticulum – intracellular transport system composed of vesicles and tubules. The virus protein blocks are then transported to another cell organelle, Golgi apparatus, where they, together with the own cell proteins, are subject to glycosylation (attachment of specific carbohydrate residues).

Ready molecules of the virus membrane proteins hemagglutinin and neuraminidase are joined together and transported in this form to the cell perimeter by specialized transport vesicles, which provide for inclusion of the virus molecules into special domains in the plasma membrane, the so-called lipid rafts (caveolae).

The remaining virus proteins necessary for complexes with the hereditary material of future virus progeny, (–)RNA, are synthesized on free cell polyribosomes (polysomes), as is usual for all non-membrane proteins. Note here that the influenza virus even “roughs it” in a sense, since its proteins accumulate in the cytoplasm, while the (–)RNA accumulates in the nucleus. This is why the virus proteins once again go to the cell transport system to reach the nucleus, where they join the virus (–)RNA to form ribonucleoprotein (RNP) particles. These particles can be seen with the help of an electron microscope: they look as rods with “loops” at their ends.

Thus, a multitude of copies of the virus genome has formed in the host cell nucleus; and the future spikes of virus membrane proteins are “stored up” on the host cell membrane – now it is only necessary to assemble these pieces of our Meccano set. The next stage is to export the RNP particles from the nucleus to the cytoplasm and farther, to the cell plasma membrane (the corresponding mechanisms still require further studies).

On the assembly line

Now we reach the final stage in virus reproduction – assembly of new virions. To this end, the entire virus genome – all eight RNP particles and the remaining virus proteins – should meet at a strictly determined place.

The meeting place has already been “appointed” – it is the lipid rafts, the regions of membrane containing incorporated hemagglutinin and neuraminidase molecules, which will serve for the virus as platforms for its assembly and budding. It is amazing how RNP particles find their way to the budding site of the future virus: recent years have produced ample evidence that each segment of the virus genome has specific “packaging signals”.

IS VACCINATION AGAINST INFLUENZA NECESSARY?

Influenza viruses have a “special relationship” with the human immune system, reflected in the so-called “original antigenic sin” phenomenon, or Burkitt’s paradox (Francis et al., 1953), discovered over 50 years ago and reconfirmed recently. This phenomenon is analogous to imprinting, known from ethology (the science dealing with animal behavior), that is, establishing of a long-lasting response in psyche to a single experience (for example, imprinting of the mother’s image in kids).
The fact is that in response to infection with any variant of influenza virus (or to vaccination) our immune system will always produce protective antibodies to the first virus encountered rather than to this particular virus. Thus, if a newly met virus is not identical to the “imprinted” variant, the induced antibodies rather than providing protection against infection, can complicate its course dramatically. This puts the fundamental question on the advisability of vaccination against influenza

Thus, RNP particles, the main component of new virions, are delivered to the plasma membrane ready for packaging. A set of factors warps the plasma membrane at the assembly site for a virus particle, and virion budding commences. The cell surface extrudes, the bulges detach from cells, and here are the “newly minted” virus particles that pass into the intercellular space. A special mechanism is provided to prevent virus particles from binding again back to “parental” cell – neuraminidase of the newly formed virus cleaves receptors from the cell surface, as though with a wisp. The drugs Tamiflu and Relenza block neuraminidase, thereby inhibiting separation of the virus progeny from the host cell and, correspondingly, decrease the number of infected cells.

Each infected cell produces a tremendous number of virus particles; however, not all of them are viable because of regular standstills on the “assembly line” and imbalanced production of the “component parts”. Moreover, the cell itself is not always able to provide proper operation of its synthetic and transport systems. This is why influenza virus, similarly to all other viruses, reproduces most efficiently in “healthy” cells, the fact known well to the virologists working with cell cultures.

In a body, the virus progeny finds itself in the layer of mucus covering the inner surface of the nasopharynx, and an infected person spreads the virus with drops of mucus when sneezing and coughing. Naturally, cells try to defend themselves from the aggressor by switching on the mechanisms of interference and apoptosis; however, this defense usually comes late, and the parasite succeeds in reproducing and infecting new cells. This is why it is so important to administer interferon preparations during the first day (better, first hours after the disease onset) to prevent a mass cell infection and to stop disease development.

Having acquainted ourselves with the intricate interaction between influenza viruses and individual host cells, we cannot but wonder at the virtuosity of this parasite in exploiting the cell systems.

However, at the body level, the interaction of a virus with the host is determined by a multitude of additional factors, which may or may not lead to disease development. The major factor here is the response of the immune system, and the debates on whether it is necessary to stimulate this response by vaccination have not only calmed down, but are becoming ever more heated.

Currently, much is known about influenza viruses and their biological properties; however, influenza is still among the diseases that “you get over in a week if you treat it and in seven days if you don’t”. Presumably, such stability of this disease is determined by an almost complete inability to interfere therapeutically with the reproduction cycle of influenza virus, so safely hidden in the “heart” of an infected cell.

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