Treacherous genes

Molecular double-dealing: Human genes work for the flu virus
Anton Chugunov, "Biomolecule"

The genome of the influenza A virus (including of swine origin) encodes no more than 11 proteins, as a result of which the virus actively uses the cellular mechanisms of the infected organism for its own purposes. As a result of genome-wide scanning using RNA interference, a list of almost 300 human genes that the virus needs for the early stages of its life cycle has been established. Among the "traitor" proteins are vacuolar ATPase, Golgi complex coatomers, fibroblast growth factor receptor, calmodulin-dependent protein kinase and many others. This information will be used to create new generations of antiviral drugs – inhibitors of certain human proteins.

Influenza viruses belong to the Orthomyxoviridae family. They are pathogenic to humans, some mammals and many birds. In humans and mammals, the virus affects the upper respiratory tract, and in birds, the infection mainly affects the intestines. These viruses are characterized by high variability and a wide range of hosts. The flu disease periodically spreads in the form of epidemics and pandemics. The severity and nature of the course of the disease varies depending on the strain of the virus.

The device of the viral particle is extremely simple compared to the cell of the infected organism
it is more than compensated by the ability of viruses to exploit cellular mechanisms for their own purposes.
At the same time, many "host" proteins unwittingly turn out to be accomplices in the spread of viral infection.
In the picture: swine flu virus and fluorescently colored cells

Influenza viruses annually cause waves of morbidity, and in some cases cause pandemics with a high mortality rate. Among such incidents are the pandemics of 1918, 1957 and 1968, as well as the rumored outbreak of swine flu in 2009. The genome of the influenza virus encodes no more than eleven (!) proteins, of which only two are targets of modern antiviral agents approved by the US FDA – neuraminidase and the M2 ion channel. The main of these drugs are oseltamivir (tamiflu), zanamivir (relenza), amantadine and rimantadine, and modern strains of the virus have already developed resistance to each of them.

Taking into account the fundamentally small number of possible viral target proteins, the phenomenal variability of viruses (and, consequently, the ability to develop resistance) and their deep introduction into many cellular processes during infection, the idea of antiviral agents directed not directly at the virus proteins, but at the cellular mechanisms of the "host" involved in the vital process becomes increasingly attractive. the aggressor's cycle. However, this pathway requires a thorough study of the molecular mechanisms of reproduction of the virus inside the host cell, as well as compiling a complete list of "host" proteins that are involved in the process of infection and replication of the virus.

Some such proteins have been known for a long time: for example, sialoglycoreceptors of the cell membrane, recognized by viral hemagglutinin before "hacking" the cell. (By the way, it is the variability and diversity of forms of hemagglutinin of various strains of the virus that determines the range of potential "hosts" (humans, other mammals, birds) and the ability to jump from one carrier to another – see, for example, "The different virulence of influenza viruses – pathogens of the "Spanish flu" is explained" [1].)

But this is a separate, most well–studied path. And how many are there in total? The answer to this question began to emerge about two years ago (in 2008), when in a systematic genome-wide scan with the sequential "shutdown" of almost every gene in human cell culture, a list of "traitor" genes working for the AIDS virus was compiled ("Footboard for the AIDS virus" [2]). The staggering size of this list is more than 250 genes! – almost exceeded the assumptions by an order of magnitude, showing how deeply viruses are embedded in the biochemistry of the carrier, in our case, a person.

This work marked the beginning of a number of similar projects – in addition to HIV, hepatitis C viruses, tropical dengue fever and West Nile encephalitis have been studied in a similar way. Now this list has been supplemented by the influenza A virus, and large-scale scanning based on RNA interference was performed by two teams of scientists who published their reports in the same issue of Nature [3, 4].

The tasks, the formulation of the experiment and, consequently, the results of the German [4] and American [3] researchers were somewhat different, but the main principle was the same: based on a library of several tens of thousands of short interference RNAs (KIRNAS), the development of the influenza virus in the culture of human epithelial cells with the "switched off" gene – in In each case, one of about 20,000. (RNA interference is a phenomenon by which it is possible to selectively and reversibly suppress the activity of genes.) If the virus did not develop in such cells or developed slowly (but the cells themselves felt normal at the same time), it was believed that the gene was associated with virus replication. By the way, given the huge number of studied genes and, therefore, the necessary experiments, it should be noted that such a scan was carried out in the format of high-density micromatrix (see Fig. 1b). The number of identified genes – potential "traitors" – in both cases approaches 300, which practically coincides with a similar figure for HIV [2].

 
Figure 1. Large-scale scanning of genes "aiding" the reproduction of the influenza virus.

A. Scheme of a recombinant virus based on strain A/WSN/33, in which the hemagglutinin gene is replaced by a luciferase gene to facilitate the "reading" of infection data by a fluorescent signal.
B. The kiRNA library, prepared in the format of a micromatrix of wells, is used to "turn off" one of the genes in the cells of the pulmonary epithelium A549 on the principle of RNA interference. (Inactivation of ~19,000 genes has been investigated.) Transfected cells in the wells were infected with a virus and the intensity of fluorescence controlled the development of the infectious process.
B. Network of interactions of ten viral proteins and cellular factors.
G. Predicted interactions with human proteins of influenza viruses, AIDS, hepatitis C, dengue fever and West Nile fever (data for all viruses except the first one are taken from the literature). The intensity of the red color shows the significance of the prediction.

Let's take a closer look at the work of Americans from the University of California, the California Salk Institute and several other scientific institutions [3]. In their experiment, the kiRNA library was previously applied to the matrix of wells of a laboratory die, in which a culture of human lung epithelial cells A549 infected with a modified influenza virus strain A/WSN/33 was to develop. In fact, in each well there were cells in which, according to the principle of RNA interference, one or another gene was "turned off" (there are about 19,000 in total – almost every gene in the human genome). For convenient observation of the infection process in the micromatrix format, the hemagglutinin gene was replaced with a luciferase gene, which makes it easy to read information about the progress of infection by a fluorescent signal (Fig. 1). However, such a recombinant virus is unable to completely go through its entire cell cycle, so the main attention was paid to the penetration of particles into the cell, the release of the viral genome and genetic processes, but not the assembly or exit of virus particles from the cell.

Analysis of the rate of development of the virus in cells with one or another "switched off" gene made it possible to identify 295 human genes involved in the development of the virus: each of them, being inactivated, slowed down the process by 35% or more or stopped the infection altogether. Many of these genes are "old acquaintances" in the history of the flu virus, but most of them "have not been seen in the discrediting connections before."

Among these genes, groups of signaling molecules (participants of the PI3K/AKT pathway), regulators of cytoskeleton dynamics, proteins involved in phosphorylation and ubiquitylation [5] of other protein molecules, coatomer proteins of the Golgi complex vesicles (COPI) and others are especially richly represented (Fig. 1d). 53 genes from this list are "double-dealing" and in favor of other RNA-containing viruses, which indicates a certain generality of the mechanisms of their life cycle. The found genes were also tested on a wild-type virus with normal hemagglutinin: 219 genes from the list confirmed that they were "not clean on hand." An additional check showed that the complicity of 23 human genes is required by the virus at the stage of penetration into the cell. The swine flu virus (A/Netherlands/602/2009 H1N1) was also not ignored, for which a list of 12 genes was compiled, the inhibition of the activity of which suppressed the development of infection.

Scientists have hypothesized that inhibitors of certain human proteins, such as fibroblast growth factor receptor FGFR4, vacuolar ATPase, glycogen synthase kinase GSK3ß and some others, may be useful to combat the influenza virus, including the swine virus.

Mapping the interaction of viruses with human cells reveals a picture of tremendous complexity in a place where a few years ago no one would have imagined anything like this. On the one hand, this complicates the understanding of the whole process, but on the other hand, it indicates new opportunities to make the virus – the named guest in our organisms – a "bandwagon". After all, in order to "dodge" today's antiviral drugs, he only needs to accidentally change himself (more precisely, change only one or two proteins), but tomorrow's antiviral strategies can "block oxygen" much more thoroughly, making it impossible to interact with intracellular "traitors", without which the invader will become completely helpless.

LiteratureBiomolecule: "Different virulence of influenza viruses – pathogens of the "Spanish flu" is explained";

2. biomolecule: "A footboard for the AIDS virus";
3. König R., Stertz S., Zhou Y., Inoue A., Hoffmann H.H., Bhattacharyya S., Alamares J.G., Tscherne D.M., Ortigoza M.B., Liang Y., Gao Q., Andrews S.E., Bandyopadhyay S., De Jesus P., Tu B.P., Pache L., Shih C., Orth A., Bonamy G., Miraglia L., Ideker T., García-Sastre A., Young J.A., Palese P., Shaw M.L., Chanda S.K. (2010). Human host factors required for influenza virus replication. Nature 463, 813–817;
4. Karlas A., Machuy N., Shin Y., Pleissner K.P., Artarini A., Heuer D., Becker D., Khalil H., Ogilvie L.A., Hess S., Mäurer A.P., Müller E., Wolff T., Rudel T., Meyer T.F. (2010). Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463, 818–822;
5. Biomolecule: "Ubiquitous ubiquitin is coming back."

Portal "Eternal youth" http://vechnayamolodost.ru16.02.2010