In the year 1918 the «Spanish flu» took away several dozens of millions of lives. At the time the cause of the disease was unknown: Influenza viruses were first isolated only 15 years after the pandemic. Now scientists have discovered hundreds of influenza strains, developed drugs that are specific to individual viral proteins and carefully described the structure of the virus particle. Based on the data accumulated over the course of almost 100 years since the deadly flu hit Europe we have created a full 3D model of influenza virus H1N1. Our model allows to see the smallest details of the virion — from the loops of RNA to oligosaccharides attached to the surface proteins.
Strains and epidemics
Influenza viruses infect humans, several other mammals and birds. Water birds harbor the widest diversity of flu strains, however the symptoms of the disease are usually not detectable in them (1). Before infecting humans individual influenza strains usually spread among cattle and fowl (2). Seasonal flu pandemics, which cause from 250 to 500 thousands of deaths every year are sometimes accompanied by non-seasonal strains that may cause more severe symptoms and higher mortality level (3). Aside from the Spanish flu, two larger pandemics took place in the 20th century: Asian and Hong-Kong flu in 1957 and 1968, respectively. Together they killed 2-3 million of people (4, 5). The most recent remarkable pandemic — so called swine influenza — occurred in the 2009 and had a much smaller fatality rate compared to its predecessors. Still, 17-18 thousands of people died from the infection worldwide (6). This pandemic began not in Asia, as it is common to influenza, but in California and Mexico (7).
Influenza viruses are transmitted through the air and can stay infectious for up to several hours on clothes or paper and even for a day on metallic surfaces (8, 9). The infection spreads more easily during winter season. Some researchers think that this might be due to better preservation of the viral particles in the cold and dry air (10). Usually the complications caused by influenza are most dangerous to people older than 65 or kids under 2 years of age. People who suffer from chronic diseases are also at a risk. However,there was an unusual amount of young victims in case of swine flu in 2009. This may be explained by the immunity to the similar flu strains from the past that remained in older people but was non-existent among the young (11, 12).
There are three types of influenza viruses: A,B and C. Each of them has many different strains. Type A is the most widespread (13, 14). All types of the influenza viruses are part of the Orthomyxoviridaefamily which also include Isavirusescausing infectious salmon anemia (15) and Thogotoviruses, which may be among the causative agents of encephalitis in humans (16).
The classification of the influenza strains is based on the diversity of their surface proteins and their combinations. There are 16 types of hemagglutinins (H) and 9 types of neuraminidases (N) but only some of their combinations are harmful to humans (17). Both Spanish and swine flu were caused by the A/H1N1 type of virus, the Hong-Kong pandemics strain was H3N2.
Virulence and mortality rate of the influenza are highly diverse among strains. For example, the avian influenza H5N1, that caused the epidemic in Eastern Asia in 1997, was characterized by very high mortality (50%), but was transmitted very ineffectively and the number of infected people did not exceed several hundred (18).
Morphology and proteins
Orthomyxoviruses have lipid membrane taken from the host cell during the budding of the virus (19, 20). There are two types of surface membrane proteins: Hemagglutinin which mediates the virus entry to the host cell, and neuraminidase, that allows the new particle to leave the cell surface by cutting the bonds with the cell receptors. The protein membrane channels in the viral envelope participate in the process of the uncoating of the virus. The matrix layer located under the envelope is composed of the M1 protein. M1 plays a structural role and is a crucial element in virus budding. The genetic material of the influenza is located in the very center of the virion and consists of 8 different RNA molecules connected with the nucleoprotein (NP) and polymerase complexes. NP and polymerase complexes are responsible for the replication and transcription of the genome (5, 21, 22).
Influenza viruses may be spherical or filamentous (23). The shape of the particle depends on the structures of the matrix protein. Diameter of the spherical particles is around 80-120 nm, which is comparable to the size of the HIV particle (24, 25).
Individual influenza proteins are potential targets for the antiviral therapy. For example, the neuraminidase inhibitors — used in Tamiflu ® and Relensa ® — block the enzyme and don’t let the new particle to leave the host cell and infect another one (26, 27). There are also drugs that block the uncoating of the virus in the cytoplasm and antibodies to different types of hemagglutinins, which inhibit the virus attachment to the cell or the fusion of the particle and cell membrane (28).
Influenza fusion protein — hemagglutinin resembles the fusion protein of Ebola virus (29, 30). It is a homotrimer that has a transmembrane and a surface part connected by disulfide bonds. The viral entry into the cell happens after hemagglutinin connects to the sialic acid receptors located on the cell surface (31). Subsequently the virus travels to the cytoplasm inside a vesicle. When the pH inside the vesicle decreases hemagglitinins change their conformation and the fusion of the viral and vesicle membranes happen (32). The channels in the viral membrane formed by M2 protein let the protons enter the inner space of the virioncausing dissociation of the ribonucleoprotein complex (RNP) from the matrix layer (33). There are several compounds that can inhibit the process of viral uncoating by blocking the M2 protein, rimantadine and amantadine as an example, but theyare less effective than neuraminidase inhibitors (27).
Neuraminidase plays a crucial role during the final stages of the virus life cycle. Neuraminidase is composed of four uniform subunits (34). Neuraminidase cleaves the bonds between terminal sialic (neuraminic) acid residues of cell surface receptors and viral hemagglutinins to allow the newly formed particles to leave the cell. The activity of the neuraminidase also prevents the new virions from entering already infected cells (35). The active ingredients of Tamiflu and Relenza — oseltamivir and zanamivir, respectively— are neuraminidase inhibitors that slow down the propagation of the virus inside the organism (36). Oseltamivir is very effective but it loses its function if the histidine at the 275 position of the neuraminidase is substituted to tyrosine. Zanamivir is active irrespective of the amino acid substitution at 275 but has some side effects that limit its usage to cases when the oseltamivir is ineffective (27, 37).
Matrix proteins M1 play a crucial role during the assembly and budding of the viral particles (38, 39). They bind to the host cell membrane at specific spot and interact with other components of the virus — surface proteins and RNP complexes. The activity of M1 protein allows all the parts of the virionto concentrate in one area and to start budding. Viruses usually use the lipid rafts areas on the host membrane to initiate budding. These parts of membranes have slightly different lipid composition that may help to make virions more stable. Matrix proteins of HIV, Ebola and some other enveloped viruses use similar mechanisms (40 — 42).
Inside the virus: how the genetic material is packed
The genome of the influenza is divided into 8 RNA molecules. Each molecule encodes different proteins. This makes flu capable of producing new strains by means of rearrangement between two initial strains if they enter the same cell. This feature enhances the evolution of influenza and makes the combat with virus more difficult (43).
RNA segments of influenza are different and they need to combine in a specific pattern to make the fully functional virion with all 8 molecules in the set. It is not absolutely clear now how the problem is solved in nature. Many researchers think that there are specific interactions between RNA molecules that allow them to aggregate in the appropriate manner (44). Dr. Jaime Martin-Benito makes comments about the specific of his work on this topic (45):
When we started the structural determination of the complete native influenza virus RNP at the Centro Nacional de Biotecnología in Madrid the information available of the complex was limited to the X-ray structure of the RNA free nucleoprotein(46, 47) and fragments of the polymerase (48, 49). RNPs are heterogeneous in size, flexible and supercoiled helical particles (50), all of these features hamper any structural study. In this scenario, electron microscopy appears as the unique technique able to produce an acceptable result. The main technical difficulties to analyse the structure of RNPs arise from their length heterogeneity and the high flexibility. To overcome these problems we carried out a separate analysis of the central sections and the termini of RNPs (isolated from purified virions), followed by techniques of three-dimensional reconstruction (45). In this project the most challenging step was the classification of the images in order to obtain homogeneous groups of images that allowed the final reconstruction. More than 90.000 images were used and several millions of hours of computation were spent to generate the final structure. The RNP arrangement is based on a double helix showing two opposite-polarity NP strands, defining a major and a minor groove and a dimeric inter-strand NP-NP interaction; in one of the ends of the double helix the polymerase is located, whereas on the other end short terminal loop containing only three unpaired NP monomers closes the structure.
Influenza genome is composed of the segmented negative-stranded RNAs of different length. The number of 8 segments is common for all Orthomyxoviruses except Thogotoviruses which have only 6 RNAs. RNAs are associated with the polymerase complexes (48) and nucleoproteins (51), forming ribonucleoproteins (RNPs) (44). RNPs have a double-helical conformation in which two NP strands of opposite polarity are connected with each other along the helix (45).
RNPs are different in length corresponding to the differences between RNA molecules. The longest molecules are 2500 nucleotides long and the shortest one is only about 800 nucleotides. RNPs are usually grouped around one of the longest molecules. The ends of the RNPs that are associated with polymerase complexes are interconnected and arranged in the one pole of the virion. The nuclear export protein (NEP) that mediates the transfer of RNPs from the host cell nucleus to the site of budding is also locatedon this side of the virion.In our model we demonstrate aplausibleway of interactions between viral RNAs — the connection of two partially complementary RNA loops. However, the exact mechanism of such interaction still needs further investigation (44, 45, 52).
Many enveloped viruses incorporate some host proteins into the particle or the membrane. For example, HIV carries human actin and cyclophilin that help to uncoat the virus. Inside the HIV membrane there are several surface proteins of main histocompatibility complex that may increase the viral infectivity (53, 54, 55). Apparently no such mechanisms are used by influenza: there are very few works that demonstrate the presence of human proteins inside the influenza particles (56).
How to avoid new «Spanish flu»
The World Health Organization (WHO) and its regional departments in different countries constantly monitor the influenza activity and distribution. The set of flu strains changes each year and a new deadly strain can potentially emerge at anytime. The high rate of influenza variability is a challenge for pharmacological companies and medical organizations: the number of new oseltamivir-resistant strains is growing, and new vaccines against influenza need to be developed each year (27, 57). Flu vaccines are based on full inactivated virions, fragments of the virion or even individual surface proteins. The vaccines are usually made against the prevailing strains of the A and B types of the influenza (58). The symptoms of flu — fever, head and muscle ache, sore throat —are frequently caused by other viruses such as coronaviruses, picornaviruses and adenoviruses (59).