Stages of virus reproduction
Paramyxoviruses are adsorbed by glycoprotein receptors on sensitive host cells. Penetration of the virion into cells occurs by receptor endocytosis or by fusion of the viral envelope with the cytoplasmic membrane. Viral RNA replication occurs in the cytoplasm of infected cells. During the formation of virions, certain sections of the cytoplasmic membrane of the host cell are modified due to the incorporation of viral glycoproteins into it from the outside, and the membrane protein from the inside. Viral nucleocapsids are transported to the modified regions of the cell membrane along the actin filaments of the cytoskeleton. The release of viral particles is carried out by budding. In the cytoplasm of infected cells, acidophilic inclusions are formed.
Antigenic structure and antigenic variability
The antigenic structure of the virus is poorly understood. The morphological similarity with the human measles virus made it possible to assume the similarity of their antigenic composition. The main antigens of the measles virus are hemagglutinin, the F protein, and the NP nucleocapsid protein. AT to hemagglutinin and F-protein show a cytotoxic effect directed against infected cells.
antigenic variability. The plague virus is immunobiologically homogeneous, at the same time, according to its origin and some biological features, its strains are divided into two subgroups: classical and variant. Classical strains are highly pathogenic and exhibit strict species specificity.
Hemagglutinating and hemadsorbing properties
The viral envelope consists of three proteins: hemagglutinin (H), fusion protein (F) and matrix (M). Also, in serological reactions, the virus revealed complement-fixing, precipitating, neutralizing and hemagglutinating antigens. In this regard, the virus is able to irregularly agglutinate chicken and guinea pig erythrocytes. The phenomenon of hemagglutination in the virus is considered non-specific.
It is believed that sialic acids present on the membrane of macrophages are receptors for virus adsorption. At the same time, it was found that the canine distemper virus lacked neuraminidase activity. Therefore, the binding of hemagglutinin to the sialic acids of the membrane is rather weak, labile, which reduces the risk for the virus to "get stuck" on the cell surface.
Features of cultivation in various living systems
The first experiments on the cultivation of canine distemper virus in tissue explants were carried out by Mitscheriich in 1938. Later, it was propagated in explants of the spleen, mesenteric lymph nodes, lungs, and testicles of 10-14-day-old puppies. The authors performed 19 passages, while the virus titer in spleen explants reached 2 × 104 ID/g. Canine distemper virus actively multiplies in primary cultures of kidney cells of dogs, ferrets, lungs of dogs and ferrets; in the primary culture of kidney cells of puppies 3-4 days of age. In these cultures on medium 199 supplemented with 20% calf serum, the virus forms plaques under the agar cover. In HeLa cells and a human liver cell line, the virus did not cause CPE.
Various cell cultures are also sensitive to the plague virus after its adaptation by passaging in them. In 1959, the virus was first isolated from distemper dogs by culturing in trypsinized pieces of lungs or kidneys. In subsequent years, it was also isolated in the primary culture of the kidneys of dogs, cattle, sheep, monkeys, fibroblasts of chicken and quail embryos, etc. The transplanted Hela and Vero cell lines are also sensitive to the virus. During reproduction, some strains of the virus cause CPP, which is characterized by granularity and rounding of cells, followed by the destruction of the monolayer and the formation of multinucleated cells and syncytia. Young puppies are used to isolate and maintain the virus in the laboratory. However, it is much more sensitive than thorzofrette. Materials for the isolation of the virus in cell culture are the spleen, liver, kidney.
The virus multiplies in chick embryos when it infects the chorionallantoic membrane (CAO), the allantoic cavity, and the yolk sac. This method is also successfully used to determine the virus titer in 8- to 9-day-old embryos. The virus is titrated for CAO. During the reproduction of the virus in infected embryos, changes appear mainly on the chorion-allantoic membrane in the form of swelling and the formation of light gray nodules the size of a millet grain or light gray strands.
In a comparative study of the reproduction of 3 strains of canine distemper virus on different cell systems, it was found that the 1st of them (Rockborn) did not have a pronounced cytopathic effect, the 2nd strain accumulated in a titer of 3.5-5.0 lg TCDsh /wi and pcs. Akbar-37 accumulated in a titer of 5.0-6.5 lg TCID50/ml (17). The strains adapted to chicken embryos develop well in the culture of chicken embryo fibroblasts, transplanted cell lines HeLa ("immortal" cells that do not have a Hayflick limit), Hep (larynx cancer cells), etc. The maximum accumulation of adapted strains in cell culture was noted at 8 -9th day. The virus reproduces in the culture of alveolar macrophages of the lungs of dogs. After 2-6 days, characteristic round multinucleated giant cells are formed in it, which disappear after 1-2 weeks with the formation of syncytium. Adapted to Vero cells (African green monkey kidney cells) pcs. Canine distemper virus Green is able to form plaques in Hep-2, BS-C-1 and HeLa cells, but not in Vero cells and canine kidney cell culture. Adapted to chick embryos or cell culture, the virus can replicate in many cell systems (dogs, cattle, monkeys, humans). Canine distemper virus causes a cytopathic effect and its titers are higher in roller cultures than in stationary ones.
A method for large-scale cultivation of canine distemper virus on Gelaspker M microcarriers (Lachema, Bruc) (diameter 150-200 µm) is proposed, for which chick embryo or Vero cells are grown as a pseudo-suspension culture. At the same time, the biological accumulation of the virus was more than 10 times higher than that when using stationary cultures.
A method for differentiation of pathogenic and attenuated canine distemper virus strains in vitro has been developed. MonAbs react with nucleocapsid AG of attenuated pcs. Onderstepoort, which is cultivated in Vero cells and does not react with pathogenic pcs. A75/17 and CH84 cultured in primary canine cell cultures. However, after several passages in Vero cells, the strains acquired an epigone that reacted with mAbs and simultaneously lost their pathogenicity for dogs.
PCS. D84-1 HSF, adapted to the culture of EC fibroblasts, causes pronounced CPI in cell culture and slight plaque formation in CAO. PCS. D84-1 is genetically stable and neurovirulent in mice.
Reproduction viruses are carried out in several stages, successively replacing each other: adsorption of the virus on the cell; penetration of the virus into the cell; "undressing" the virus; biosynthesis of viral components in the cell; the formation of viruses; release of viruses from the cell.
Adsorption . The interaction of a virus with a cell begins with the adsorption process, i.e., the attachment of viruses to the cell surface. This is a highly specific process. The virus is adsorbed on certain parts of the cell membrane - the so-called receptors. Cellular receptors can have a different chemical nature, representing proteins, carbohydrate components of proteins and lipids, lipids. The number of specific receptors on the surface of one cell varies from 104 to 105. Consequently, dozens and even hundreds of viral particles can be adsorbed on the cell. Penetration into the cell. There are two ways for animal viruses to enter a cell: viropexis and fusion of the viral envelope with the cell membrane. With viropexis, after the adsorption of viruses, invagination (invagination) of a section of the cell membrane and the formation of an intracellular vacuole, which contains a viral particle, occur. The vacuole with the virus can be transported in any direction to different parts of the cytoplasm or the cell nucleus. The fusion process is carried out by one of the surface viral proteins of the capsid or supercapsid envelope. Apparently, both mechanisms of virus penetration into the cell do not exclude, but complement each other. "Undressing". The process of "undressing" consists in removing the protective viral membranes and releasing the internal component of the virus that can cause an infectious process. "Undressing" of viruses occurs gradually, in several stages, in certain areas of the cytoplasm or nucleus of the cell, for which the cell uses a set of special enzymes. In the case of virus penetration by fusion of the viral envelope with the cell membrane, the process of virus penetration into the cell is combined with the first stage of its “undressing”. The end products of "undressing" are the core, nucleocapsid or nucleic acid of the virus. Biosynthesis of virus components. The viral nucleic acid that has penetrated into the cell carries genetic information that successfully competes with the genetic information of the cell. It disrupts the work of cellular systems, suppresses the cell's own metabolism and forces it to synthesize new viral proteins and nucleic acids that are used to build viral progeny. The implementation of the genetic information of the virus is carried out in accordance with the processes of transcription, translation and replication. Formation (assembly) of viruses. Synthesized viral nucleic acids and proteins have the ability to specifically “recognize” each other and, if their concentration is sufficient, spontaneously combine as a result of hydrophobic, salt and hydrogen bonds. There are the following general principles for assembling viruses with different structures:
1. The formation of viruses is a multi-stage process with the formation of intermediate forms;
2. The assembly of simply arranged viruses consists in the interaction of viral nucleic acid molecules with capsid proteins and the formation of nucleocapsids (for example, polio viruses). In complex viruses, nucleocapsids are first formed, with which supercapsid envelope proteins interact (for example, influenza viruses);
3. The formation of viruses does not occur in the intracellular fluid, but on the nuclear or cytoplasmic membranes of the cell;
4. Complexly organized viruses in the process of formation include components of the host cell (lipids, carbohydrates).
The release of viruses from the cell. There are two main types of exit of viral progeny from the cell. The first type - explosive - is characterized by the simultaneous release of a large number of viruses. In this case, the cell quickly dies. This exit mode is characteristic of viruses that do not have a supercapsid envelope. The second type is budding. It is inherent in viruses that have a supercapsid shell. At the final stage of assembly, the nucleocapsids of complex viruses are fixed on the cell plasma membrane modified by viral proteins and gradually protrude it. As a result of the protrusion, a "kidney" containing a nucleocapsid is formed. The "kidney" then separates from the cell. Thus, the outer envelope of these viruses is formed during their exit from the cell. With this mechanism, a cell can produce a virus for a long time, retaining to some extent its basic functions.
The reproduction of the virus in the cell occurs in several phases:
The first phase is the adsorption of the virus on the cell surface that is sensitive to this virus.
The second phase is the penetration of the virus into the host cell by viropexis.
The third phase is the "undressing" of virions, the release of the nucleic acid of the virus from the supercapsid and capsid. In a number of viruses, the penetration of the nucleic acid into the cell occurs by the fusion of the virion envelope and the host cell. In this case, the second and third phases are combined into one.
Depending on the type of nucleic acid, this process occurs as follows.
Reproduction occurs in the nucleus: adenoviruses, herpes, papovaviruses. Use DNA-dependent RNA - cell polymerase.
Reproduction occurs in the cytoplasm: viruses have their own DNA-dependent RNA polymerase.
Riboviruses with a positive genome (plus-nitium): picorna-, toga-, coronaviruses. There is no transcription.
RNA -> protein
Riboviruses with a negative genome (minus-strand): influenza, measles, parotitis, ortho-, paramyxoviruses.
(-)RNA -> mRNA -> protein (mRNA is complementary to (-)RNA). This process takes place with the participation of a special viral enzyme - virion RNA-dependent RNA polymerase (there cannot be such an enzyme in a cell).
Retroviruses
(-)RNA -> DNA -> mRNA -> protein (and RNA is homologous to RNA). In this case, the process of DNA formation on the basis of (-) RNA is possible with the participation of the enzyme - RNA-dependent DNA polymerase (reverse transcriptase or reverse transcriptase)
The fourth phase is the synthesis of virion components. The nucleic acid of the virus is produced by replication. The information of the viral mRNA is translated to the ribosomes of the cell, and a virus-specific protein is synthesized in them.
The fifth phase is the assembly of the virion. Nucleocapsids are formed by self-assembly.
The sixth phase is the release of virions from the cell. Simple viruses, such as the polio virus, destroy the cell when they exit. Complex viruses, such as the influenza virus, exit the cell by budding. The outer shell of the virus (supercapsid) is formed during the release of the virus from the cell. The cell during this process remains alive for some time.
The described types of interaction of the virus with the cell are called productive, as they lead to the production of mature virions.
Another way - integrative - is that after the penetration of the virus into the cell and "undressing" the viral nucleic acid integrates into the cell genome, that is, it is integrated into the cell chromosome in a certain place and then replicated along with it in the form of the so-called provirus. For DNA- and RNA-containing viruses, this process takes place in different ways. In the first case, viral DNA integrates into the cellular genome. In the case of RNA-containing viruses, reverse transcription occurs first: DNA is formed on the viral RNA template with the participation of the "reverse transcriptase" enzyme, which is integrated into the cellular genome. The provirus carries additional genetic information, so the cell acquires new properties. Viruses capable of this type of interaction with a cell are called integrative. Integrative viruses include some oncogenic viruses, hepatitis B virus, herpes virus, human immunodeficiency virus, temperate bacteriophages.
In addition to ordinary viruses, there are prions - protein infectious particles that do not contain nucleic acid. They look like fibrils, up to 200 nm in size. They cause slow infections in humans and animals with brain damage: Creutzfeldt-Jakob disease, kuru, scrapie and others.
Methods for the indication of viruses in the test material.
The reproduction of viruses in cell cultures is judged by their cytopathic action (CPE), which has a different character depending on the type of virus, by plaque formation on a cell monolayer covered with a thin agar layer, erythrocyte hemadsorption, and other tests.
Thus, the indication of viruses is performed microscopically by the presence of CPP, plaque formation on the cell monolayer, hemadsorption of erythrocytes added to the cell culture of the virus, as well as in the hemagglutination reaction with the tested virus-containing material. The hemagglutination reaction is caused by viruses containing hemagglutinin in their capsid or supercapsid.
Virus classification principles:
Viruses make up the kingdom Vira, which is subdivided by nucleic acid type into two sub-kingdoms - riboviruses and deoxyriboviruses. Sub-kingdoms are divided into families, which in turn are subdivided into genera. The concept of the type of viruses has not yet been clearly formulated, as well as the designation of different types.
As taxonomic characteristics, paramount importance is attached to the type of nucleic acid and its molecular biological features: double-stranded, single-stranded, segmented, non-segmented, with repeating and inverted sequences, etc. However, in practical work, the characteristics of viruses obtained as a result of electron microscopy and immunological studies: morphology, structure and size of the virion, the presence or absence of an outer shell (supercapsid), antigens, resistance to high temperature, pH, detergents, etc.
Currently, human and animal viruses are included in 18 families. The belonging of viruses to certain families is determined by the type of nucleic acid, structure, integrity or fragmentation of the genome, as well as the presence or absence of an outer shell. When determining belonging to the family of retroviruses, the presence of reverse transcriptase is necessarily taken into account.
Virus reproduction
The reproduction of the virus in the cell occurs in several phases:
The first phase is the adsorption of the virus on the cell surface that is sensitive to this virus.
The second phase is the penetration of the virus into the host cell by viropexis.
The third phase is the "undressing" of virions, the release of the nucleic acid of the virus from the supercapsid and capsid. In a number of viruses, the penetration of the nucleic acid into the cell occurs by the fusion of the virion envelope and the host cell. In this case, the second and third phases are combined into one.
Depending on the type of nucleic acid, this process occurs as follows.
1. Reproduction occurs in the nucleus: adenoviruses, herpes, papovaviruses. Use DNA-dependent RNA - cell polymerase.
2. Reproduction occurs in the cytoplasm: viruses have their own DNA-dependent RNA polymerase.
1. Riboviruses with a positive genome (plus-nitium): picorna-, toga-, coronaviruses. There is no transcription.
RNA -> protein
2. Riboviruses with a negative genome (minus-strand): influenza, measles, parotitis, ortho-, paramyxoviruses.
(-)RNA - > mRNA - > protein (mRNA complementary (-)RNA ). This process takes place with the participation of a special viral enzyme - virion RNA-dependent RNA polymerase (there cannot be such an enzyme in a cell).
3. Retroviruses
(-)RNA -> DNA -> mRNA -> protein (and RNA is homologous to RNA ). In this case, the process of DNA formation on the basis of (-) RNA is possible with the participation of the enzyme - RNA-dependent DNA polymerase (reverse transcriptase or reverse transcriptase)
The fourth phase is the synthesis of virion components. The nucleic acid of the virus is produced by replication. The information of the viral mRNA is translated to the ribosomes of the cell, and a virus-specific protein is synthesized in them.
The fifth phase is the assembly of the virion. Nucleocapsids are formed by self-assembly.
The sixth phase is the release of virions from the cell. Simple viruses, such as the polio virus, destroy the cell when they exit. Complex viruses, such as the influenza virus, exit the cell by budding. The outer shell of the virus (supercapsid) is formed during the release of the virus from the cell. The cell during this process remains alive for some time.
The described types of interaction of the virus with the cell are called productive, as they lead to the production of mature virions.
Another way - integrative - is that after the penetration of the virus into the cell and "undressing" the viral nucleic acid integrates into the cell genome, that is, it is integrated into the cell chromosome in a certain place and then replicated along with it in the form of the so-called provirus. For DNA- and RNA-containing viruses, this process takes place in different ways. In the first case, viral DNA integrates into the cellular genome. In the case of RNA-containing viruses, reverse transcription occurs first: DNA is formed on the viral RNA template with the participation of the "reverse transcriptase" enzyme, which is integrated into the cellular genome. The provirus carries additional genetic information, so the cell acquires new properties. Viruses capable of this type of interaction with a cell are called integrative. Integrative viruses include some oncogenic viruses, hepatitis B virus, herpes virus, human immunodeficiency virus, temperate bacteriophages.
In addition to ordinary viruses, there are prions - protein infectious particles that do not contain nucleic acid. They look like fibrils, up to 200 nm in size. They cause slow infections in humans and animals with brain damage: Creutzfeldt-Jakob disease, kuru, scrapie and others.
20. Phages (viruses of microbes): morphology and ultrastructure. Phases of interaction of virulent and temperate phages with a bacterial cell. Determination of activity (titer) of a bacterial cell. Prophage. Phage typing of microorganisms, significance. Practical use of phages.
The phenomenon of bacteriophagy was discovered and studied by the French microbiologist d'Errel. In 1917, he observed the lysis of a culture of dysentery bacteria after introducing into it the feces filtrate of a patient recovering from dysentery. activity and even increased it. The scientist made the correct conclusion from this that the lysing agent is alive and multiplies in bacteria during passages. D "Errell called this agent a bacteriophage (lat. phagos - devouring), and the very phenomenon of lysis - bacteriophage.
Later it was confirmed that the bacteriophage is alive. This is a bacterial virus, it multiplies in bacteria, causing their lysis. The addition of a bacteriophage to a bacterial culture on a liquid nutrient medium causes the medium to become clear. On dense nutrient media, when a mixture of bacteria and a bacteriophage is inoculated, sterile spots or negative colonies of phages appear against the background of a continuous growth of bacteria.
Bacteriophages are specific, that is, they lyse certain types of bacteria. Hence their names: dysenteric bacteriophage, staphylococcal bacteriophage. Phages of not only bacteria, but also actinomycetes were found.
In practical medicine, bacteriophages have found application as therapeutic and prophylactic agents,
It is important that many problems of general virology and molecular genetics were discovered and studied using the example of bacteriophage.
Structure of bacteriophages
The sizes of bacteriophages range from 20 nm to 200 nm. Like all viruses, they contain DNA, or RNA, and a protein capsid. The most common and best studied are bacteriophages that have the shape of a spermatozoon or a tadpole. They consist of a head, a caudal process, a battle plate with short tires and tail filaments. Inside the head is a spirally twisted drink of DNA, covered with a protein capsid. The caudal process is a hollow cylindrical rod surrounded by a contractile sheath. The basal plate and filaments carry out the process of adsorption of the bacteriophage on the bacterial cell. There are bacteriophages that have a different structure: with a short process, with a process without a contractile sheath, without a process, filamentous.
Interaction of a bacteriophage with a bacterial cell
Like all viruses, bacteriophages do not reproduce on nutrient media. Their reproduction occurs only in bacterial cells sensitive to them, in the process of interaction, in which the same phases are observed as in the interaction of other viruses with the cell.
Bacteriophage adsorption. Like all viruses, phages are immobile, and the collision with a bacterium occurs by chance, then adsorption becomes strong if the cell has phage-specific receptors on the surface. Phages with a contractile sheath are adsorbed by the tail process.
Entry of phage into the cell. Under the action of the enzyme lysozyme, which is located in the tail segment, a hole is formed in the cell wall of the bacterium. Through this hole, as a result of contraction of the tail sheath, the phage DNA passes into the bacterial cell. The protein capsid remains outside.
Synthesis of DNA and protein of bacteriophage. The synthesis of bacterial proteins stops in the cell. Phage DNA is formed, and phage protein molecules are synthesized on bacterial ribosomes.
Phage formation. The assembly of mature phages from DNA and capsid occurs in the cytoplasm of the cell. The release of mature phages from the cell occurs when bacteria are destroyed by lysozyme, and then mature phages are introduced into new cells.
The "harvest" of a phage, depending on its type, ranges from 20 to 200 particles. The entire cycle of interaction, which takes from 10 minutes to several hours, is called the lytic cycle, and the phage during this interaction - virulent .
Unlike virulent, temperate phages do not lyse bacteria. Their genome, having penetrated into the cell, is integrated into the bacterial chromosome and subsequently remains in the chromosome in the form prophage and replicated along with it. Prophage-bearing bacteria are called lysogenic, and the phenomenon itself is called lysogeny. Lysogenic bacteria are very common. The prophage, being in the genome of the bacterium, gives it some new properties. For example, the production of exotoxin in diphtheria and botulism bacilli is associated with the presence of a prophage.
Under certain conditions (exposure to temperature, chemicals, etc.), prophages can turn into virulent bacteriophages. Reproducing, they lyse bacteria and can pass into other bacterial cells. When leaving the chromosome, the prophage can capture neighboring genes of the bacterial chromosome and, when it infects another bacterium, integrates into its chromosome and transfers these genes. The transfer of genetic material from one bacterium to another by a temperate bacteriophage is called transduction. Thus, traits such as resistance to antibiotics, the ability to produce any enzymes can be transmitted. Temperate bacteriophages are used in genetic engineering as a gene carrier vector.
The practical importance of bacteriophages
Bacteriophage preparations are used for diagnosis, prevention and treatment. Phage diagnostics is based on the specificity of bacteriophages: species-specific bacteriophages lyse only certain types of bacteria. Moreover, bacteria of the same species differ in sensitivity to different typical bacteriophages. Thus, using a set of typical bacteriophages, it is possible to determine the fagovars of staphylococci, salmonella, and vibrios. Phage typing helps to establish the source of infection and the route of transmission.
The therapeutic and prophylactic effect of phages is based on their lytic activity.
To obtain a bacteriophage preparation, a culture of bacteria is infected with a bacteriophage. The next day, the deprived culture is filtered through a bacterial filter. Quinosol is added to the filtrate as a preservative.
For the quantitative characterization of bacteriophages, such a criterion as the bacteriophage titer is used. Phage titer can be expressed in two terms:
1) the highest dilution of the drug, at which the bacteriophage lyses the corresponding bacteria:
2) the number of active bacteriophage corpuscles in 1 ml of the preparation. Bacteriophage titration methods:
1) the method of serial dilution in test tubes with liquid nutrient medium according to Appslman;
2) two-layer agar method, in which the number of negative phage colonies is counted against the background of a continuous growth of bacteria - the Gracia method.
The finished liquid bacteriophage preparation must be completely transparent. For intestinal infections, the drug is used together with a solution of baking soda, since the acidic contents of the stomach destroy the bacteriophage. Preparations of some bacteriophages for injection and topical application are produced in ampoules. For oral administration, bacteriophage preparations are also available in the form of tablets with an acid-resistant coating, which dissolves in the alkaline environment of the small intestine. Pectin or acetylphthalylcellulose (LPP) is used as a coating.
Preparations of dysentery, salmonella, coli-proteus, staphylococcal and other bacteriophages, as well as sets of typical phages for phage typing of staphylococci, typhoid and other bacteria are produced in our country.
Preparatory phase of viral reproduction
Lecture 4
Expression of the viral genome. Virus genetics
Integrated goal of the module
The complex goal of the module is to combine lecture material relating to all possible ways to realize the genetic potential of viruses, to give students an idea of the basic stages of virus reproduction, the biological essence of all phases and stages of their reproduction. The task of the lecture material combined in this module is extremely important to generalize information about the reproduction of various viruses with their genetic potential, to show the essence of the processes. controlling the heredity and variability of viruses.
The module consists of four lectures, the material of which allows to solve the set goal.
The process of virus reproduction should be conditionally divided into two phases. The first phase covers the events that lead to the adsorption and entry of the virus into the cell, the release of its internal component and its modification in such a way that it is able to cause infection. Accordingly, the first phase includes three stages: 1) virus adsorption on cells; 2) penetration into cells; 3) undressing of the virus in the cell. These stages are aimed at ensuring that the virus is delivered to the appropriate cellular structures, and its internal component is released from the protective shells. As soon as this goal is achieved, the second phase of reproduction begins, during which the expression of the viral genome takes place. This phase includes the following steps: 1) transcription, 2) translation of messenger RNA, 3) genome replication, 4) assembly of viral components. The final stage of reproduction is the release of the virus from the cell.
ADSORPTION
The interaction of a virus with a cell begins with the process of adsorption, i.e., the attachment of viral particles to the cell surface. The process of adsorption is possible in the presence of appropriate receptors on the surface of the cell and ʼʼʼʼʼ recognizing their substances on the surface of the virus. The very initial adsorption processes are nonspecific and may be based on the electrostatic interaction of positively and negatively charged groups on the surface of the virus and the cell. In this case, the recognition of cell receptors by viral proteins, leading to the attachment of a viral particle to a cell, is a highly specific process. Proteins on the surface of the virus that recognize specific groups on the plasma membrane of the cell and determine the attachment of a viral particle to them are called attachment proteins.
Viruses use receptors designed to pass into the cell the substances necessary for its vital activity: nutrients, hormones, growth factors, etc. Receptors can have a different chemical nature and represent proteins, the carbohydrate component of proteins and lipids, lipids. Receptors for influenza viruses and paramyxoviruses are sialic acid in the composition of glycoproteins and glycolipids (gangliosides), for rhabdoviruses and reoviruses - also a carbohydrate component in the composition of proteins and lipids, for picorn and adenoviruses - proteins, for some viruses - lipids. Specific receptors play a role not only in the attachment of a viral particle to the cell surface. Οʜᴎ determine the further fate of the viral particle, its intracellular transport and delivery to certain areas of the cytoplasm and nucleus, where the virus is able to initiate the infectious process. The virus can attach to non-specific receptors and even enter the cell, but only attachment to a specific receptor will lead to infection.
Attachment of a viral particle to the cell surface first occurs through the formation of a single bond between the viral particle and the receptor. Moreover, such attachment is unstable, and the viral particle can easily come off the cell surface (reversible adsorption). In order for irreversible adsorption to occur, multiple bonds must appear between the viral particle and many receptor molecules, i.e., stable multivalent attachment must occur. The number of cell receptor molecules in adsorption sites can reach up to 3000. Stable binding of a viral particle to the cell surface as a result of multivalent attachment occurs due to the possibility of free movement of receptor molecules in the lipid bilayer of the plasma membrane, ĸᴏᴛᴏᴩᴏᴇ is determined by the mobility, ʼʼfluidityʼʼ of the protein-lipid layer. An increase in lipid fluidity is one of the earliest events in the interaction of a virus with a cell, which results in the formation of receptor fields at the site of contact of the virus with the cell surface and the stable attachment of the viral particle to the resulting groups - irreversible adsorption.
The number of specific receptors on the cell surface varies between 10 4 and 10 5 per cell. Receptors for a number of viruses are present only in a limited set of host cells, and this may determine the sensitivity of an organism to a given virus. For example, picornaviruses are adsorbed only on primate cells. Receptors for other viruses, on the contrary, are widely represented on the surface of cells of various types, such as, for example, receptors for orthomyxoviruses and paramyxoviruses, which are sialyl-containing compounds. For this reason, these viruses have a relatively wide range of cells on which adsorption of viral particles can occur. Receptors for a number of togaviruses are possessed by cells of an extremely wide range of hosts: these viruses can adsorb and infect cells of both vertebrates and invertebrates.
The presence of specific receptors on the cell surface in some cases causes the phenomenon of host-dependent restriction, i.e., the ability of the virus to infect only certain types of animals. In general, restrictions on the interaction of the receptor systems of the virus and the cell are biologically justified and expedient, although in some cases they are "reinsurance". Thus, many cell lines resistant to polio and Coxsackie viruses can be infected with deproteinized RNA preparations isolated from these viruses. Such infection of cells bypasses the natural entry routes of infection through interaction with cellular receptors. Known potential ability of animal viruses to replicate in the protoplasts of yeast, fungi and bacteria, and bacteriophages - in animal cells. Τᴀᴋᴎᴍ ᴏϬᴩᴀᴈᴏᴍ, viral DNA and RNA have the ability to infect a wider range of hosts than viruses.
Viral attachment proteins. Attachment proteins can be found in unique organelles, such as outgrowth structures in T-bacteriophages or fibers in adenoviruses, which are clearly visible under an electron microscope; can form morphologically less pronounced, but no less unique arrangements of protein subunits on the surface of viral membranes, such as spikes in enveloped viruses, ʼʼʼʼʼ in coronaviruses.
Simply organized animal viruses contain attachment proteins in the capsid. In complexly organized viruses, these proteins are part of the supercapsid and are represented by multiple molecules. For example, the Semliki forest virus (alpha virus) has 240 glycoprotein molecules in one virion, the influenza virus has 300-450 hemagglutinating subunits, the reovirus has 24 protein molecules, and the adenovirus has 12 fibers.
VIRUS ENTRY INTO THE CELL
Historically, there has been an idea of two alternative mechanisms for the penetration of animal viruses into the cell - by viropexis (endocytosis) and by fusion of the viral and cellular membranes. Moreover, these two mechanisms do not exclude, but complement each other.
The term ʼʼviropexisʼʼ, proposed in 1948 ᴦ. Fazecas de San Gro means that the viral particle enters the cytoplasm as a result of invagination of a section of the plasma membrane and the formation of a vacuole that contains the viral particle.
receptor endocytosis. Viropexis is a special case of receptor or adsorption endocytosis. This process is the usual mechanism by which nutritional and regulatory proteins, hormones, lipoproteins, and other substances enter the cell from the extracellular fluid. Receptor endocytosis occurs in specialized areas of the plasma membrane, where there are special pits covered from the side of the cytoplasm with a special protein with a large molecular weight - clathrin. Specific receptors are located at the bottom of the fossa. The pits provide rapid invagination and the formation of clathrin-coated intracellular vacuoles. The half-life of penetration of a substance into the cell by this mechanism does not exceed 10 min from the moment of adsorption. The number of vacuoles formed in one minute reaches more than 2000. Τᴀᴋᴎᴍ ᴏϬᴩᴀᴈᴏᴍ, receptor endocytosis is a well-coordinated mechanism that ensures the rapid penetration of foreign substances into the cell.
Coated vacuoles fuse with other, larger cytoplasmic vacuoles to form receptor-containing receptors but no clathrin, which in turn fuse with lysosomes. In this way, proteins that have entered the cell are usually transported to lysosomes, where they are broken down into amino acids; they can both bypass lysosomes and accumulate in other parts of the cell in an undegraded form. An alternative to receptor endocytosis is liquid endocytosis, when invagination does not occur in specialized areas of the membrane.
Most enveloped and non-enveloped animal viruses enter the cell by the mechanism of receptor endocytosis. Endocytosis provides intracellular transport of the viral particle within the endocytic vacuole, since the vacuole can move in any direction and fuse with cell membranes (including the nuclear membrane), releasing the viral particle in the appropriate intracellular sites. In this way, for example, nuclear viruses enter the nucleus, and reoviruses enter the lysosomes. At the same time, the viral particles that have entered the cell are part of the vacuole and are separated from the cytoplasm by its walls. They have to go through a number of stages before they can cause an infectious process.
Fusion of viral and cellular membranes. In order for the internal component of the virus to pass through the cell membrane, the virus uses the mechanism of membrane fusion. In enveloped viruses, fusion is mediated by a point interaction of the viral fusion protein from lipids of the cell membrane, as a result of which the viral lipoprotein membrane integrates with the cell membrane, and the internal component of the virus is on its other side. In non-enveloped viruses, one of the surface proteins also interacts from lipids of cell membranes, due to which the internal component passes through the membrane. Most animal viruses enter the cytosol from the receptorosome. If during endocytosis the viral particle is a passive passenger, then during the fusion it becomes an active participant in the process. The fusion protein is one of its surface proteins. To date, this protein has been identified only in paramyxoviruses and orthomyxoviruses. In paramyxoviruses, this protein (F-protein) is one of two glycoproteins located on the surface of the viral particle.
The function of the fusion protein in the influenza virus is performed by a small hemagglutinating subunit.
Paramyxoviruses cause membrane fusion at neutral pH, and the internal component of these viruses can enter the cell directly through the plasma membrane. Moreover, most enveloped and non-enveloped viruses cause membrane fusion only at a low pH value - from 5.0 to 5.75. If weak bases (ammonium chloride, f chloroquine, etc.) are added to the cells, which increase the pH to 6.0 in endocytic vacuoles, membrane fusion does not occur, viral particles remain in the vacuoles, and the infectious process does not occur. The strict dependence of membrane fusion on pH values is due to conformational changes in viral fusion proteins.
The lysosome constantly has a low pH value (4.9). In the endocytic vacuole (receptosome), acidification is created by an ATP-dependent ʼʼproton pumpʼʼ even on the cell surface during the formation of a coated vacuole. Acidification of the endocytic vacuole is of great importance for physiological ligands penetrating the cell, since a low pH promotes the dissociation of the ligand from the receptor and the recycling of receptors.
The same mechanism that underlies the fusion of viral and cellular membranes causes viral-induced hemolysis and fusion of plasma membranes of adjacent cells to form multinucleated cells, symplasts and syncytia. Viruses cause two types of cell fusion: 1) ʼʼfusion from the outsideʼʼ and 2) ʼʼfusion from the insideʼʼ. ʼʼFusion outsideʼʼ occurs at high multiplicity of infection and is detected within the first hours after infection. This type of fusion, described for paramyxoviruses, is mediated by the proteins of the infecting virus and does not require intracellular synthesis of viral components. On the contrary, ʼʼfusion from withinʼʼ occurs at a low multiplicity of infection, is found at relatively late stages of the infectious process, and is due to newly synthesized viral proteins. ʼʼFusion from withinʼʼ is described for many viruses: herpes viruses, oncoviruses, pathogens of slow infections, etc.
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This type of fusion is caused by the same viral glycoproteins that ensure the penetration of the virus into the cell.
STRIP
Virus particles that have entered the cell must undress in order to cause an infectious process. The meaning of undressing is to remove the viral protective shells that prevent the expression of the viral genome. As a result of undressing, the internal component of the virus is released, which can cause an infectious process. Undressing is accompanied by a number of characteristic features: as a result of the breakdown of the viral particle, infectious activity disappears, in some cases sensitivity to nucleases appears, resistance to the neutralizing effect of antibodies arises, photosensitivity is lost when using a number of drugs.
The end products of undressing are cores, nucleocapsids, or nucleic acids. For a number of viruses, it has been shown that the product of stripping is not naked nucleic acids, but nucleic acids associated with an internal viral protein. For example, the end product of the undressing of picornaviruses is RNA covalently linked to the VP g protein, the end product of the undressing of adenoviruses, polyoma virus, and SV40 is DNA covalently linked to one of the internal viral proteins.
In some cases, the ability of viruses to cause an infectious process is determined by the possibility of their undressing in the cell of this system. Thus, this stage is one of the stages limiting the infection.
The undressing of a number of viruses occurs in specialized areas inside the cell (lysosomes, structures of the Golgi apparatus, perinuclear space, nuclear pores on the nuclear membrane). When the viral and cellular membranes merge, penetration into the cell is combined with undressing.
Undressing and intracellular transport are interrelated processes: if proper intracellular transport to the undressing sites is disturbed, the viral particle enters the lysosome and is destroyed by lysosomal enzymes.
Intermediate forms when undressing. The undressing of the virus particle is carried out gradually as a result of a series of successive reactions. For example, during the undressing process, picornaviruses go through a series of stages with the formation of intermediate subviral particles with sizes from 156 S to 12 S. The undressing of ECHO viruses has the following stages: virions (156 S) - A-particles (130S), RNP and empty capsids (80S) -RNA with a terminal protein (12S). The undressing of adenoviruses occurs in the cytoplasm and nuclear pores and has at least 3 stages: 1) the formation of subviral particles with a higher density than virions; 2) the formation of cores in which 3 viral proteins are absent; 3) the formation of a DNA-protein complex, in which DNA is covalently linked to a terminal protein. In the process of undressing, the polyoma virus loses its outer proteins and turns into a subviral particle with a sedimentation coefficient of 48 S. Then the particles bind to nuclear proteins (histones) and a 190 S complex is formed (with a sedimentation coefficient of 190 S) that can cause an infectious process. The influenza virus first loses its lipoprotein envelope and turns into a subviral particle, from which, after removal of the M-protein, the nucleocapsid is released.
The preparatory phase of the reproduction of viruses - the concept and types. Classification and features of the category "Preparatory phase of virus reproduction" 2017, 2018.
