Reproduction of viruses in cellular systems. Stages of reproduction
The relationship of the virus with the host cell can develop in different ways. Conventionally, these relationships can be reduced to three types.
Productive infection: the cycle of virus reproduction in the host cell ends with the formation of a new, numerous generation of viruses, usually accompanied by the death of the host cell.
Abortive infection occurs when the reproduction cycle of the virus in the host cell is suddenly interrupted. The host cell retains its vital activity.
Virogenia characterized by the integration (embedding) of the viral nucleic acid into the genome of the host cell, which subsequently leads to synchronous replication of the cell's DNA and the nucleic acid of the virus. The host cell continues to live.
Reproduction of viruses is carried out by their reproduction in the host cell. The reproduction cycle is a process of subordinating cellular mechanisms to foreign viral information.
Functionally, virus enzymes can be divided into 2 groups: enzymes that facilitate the penetration of the viral nucleic acid into the cell and the release of the resulting virions into the environment, and enzymes involved in the processes of transcription and replication of the viral nucleic acid.
reproduction cycle can be divided into separate stages.
Stage 1 - chemisorption of viruses on the surface of the host cell
Chemisorption is possible only if the cell carries on its surface sensitive receptors that are complementary to the receptors of the given virus. In animal and human cells, the function of receptors for picorno- and arboviruses is performed by lipoproteins, for mixo- and paramyxoviruses and adenoviruses - by mucoproteins.
In simply organized viruses, receptors are unique combinations of protein subunits located on the surface of the capsid. In more complexly organized viruses, the function of receptors is performed by outgrowths of the supercapsid in the form of spikes or villi.
Stage 2 - the penetration of the virus into the host cell.
The ways in which viruses enter the cell can be different. It is assumed that many viruses enter the cell by pinocytosis, or viropexis. During pinocytosis in the region of virus chemisorption, the cell membrane forms an invagination and swallows the virus. As part of the pinocytic vacuole, the virus enters the cytoplasm.
Some viruses enter cells by the fusion of cell and viral membranes.
The penetration of phage DNA into a bacterial cell occurs due to the partial destruction of the cell membrane by phage lysozyme and the contractile reaction of the phage residue.
Stage 3 - deproteinization of the virus.
The process of deproteinization of the virus involves the release of its nucleic acid from the proteins of the capsid. As soon as the viral nucleic acid is released from the capsid proteins, the so-called latent period begins - the period eclipse. It is assumed that during the period of eclipse, the viral nucleic acid passes through the cytoplasm of the cell to the region of the nucleus.
Stage 4 - synthesis of virus components.
The totality of the processes of this stage can be divided into three stages:
The first stage is preparatory. It provides for two goals: to suppress the functioning of the genetic apparatus of the cell, to stop the synthesis of cellular proteins and nucleic acids, to transfer the protein-synthesizing apparatus of the cell under the control of the virus genome; prepare conditions for nucleic acid replication and synthesis of viral capsid proteins.
The second stage is the replication of the nucleic acid of the virus. For double-stranded DNA - genomic viruses, the same way of implementing genetic information is typical, as for other living organisms. The process of DNA replication is preceded by transcription of mRNA. The messenger RNA of the virus is translated by the ribosomes of the cell and the synthesis of early virus-specific proteins takes place on the virus-polysome using the mRNA template.
As soon as early virus-specific proteins are synthesized, the process of viral DNA replication begins. Replication of two-strand DNA of the virus follows the principle of DNA replication of cellular organisms in a semi-conservative way.
The process of replication of single-stranded DNA begins with the synthesis of its complementary pair. The result is a double-stranded circular parental DNA.
The study of the replication mechanism of RNA-genomic viruses began in 1961, when RNA-genomic phages were discovered.
In RNA genomic viruses, the RNA molecule is both the genetic material and performs the function of mRNA and DNA.
In 1970, the enzyme RNA-dependent DNA polymerase was found in single-celled RNA viruses, indicating the presence of a reverse transcription process. Later it was proved that in oncogenic RNA viruses, according to their RNA matrix, with the participation of RNA-dependent
The DNA polymerase contained in the virion is transcribed into a DNA copy. The DNA copy from a single-stranded form passes into a replicative double-stranded form, which ensures the replication of the RNA of the virus and the synthesis of the necessary enzymes.
The third stage is the synthesis of capsid proteins.
This process lags behind the viral nucleic acid replication process and begins when replication is in full swing. The synthesis of capsid proteins occurs both in the nucleus and in the cytoplasm of the cell. Virus-specific mRNA is translated by the ribosomes of the cell, and precursor proteins are synthesized on the virus-polysome. From this "fund" of precursor proteins, the proteins of the capsid of the virus are formed.
Stage 5 - assembly of virions, or virus morphogenesis.
In simply organized viruses, the protein subunits of the capsid in a strictly ordered compound are located around the nucleic acid. In complexly organized viruses, cellular structures, such as nuclear and cytoplasmic membranes, also take part in the process of assembling virions.
Stage 6 - the release of the virus from the cell.
This process is different for different viruses. The release of DNA-genomic phages occurs when the cell is completely lysed by phage lysozyme. Complex human and animal viruses exit the cell with a cytoplasmic region by budding through the cytoplasmic membrane and envelope, simultaneously acquiring a supercapsid. Often, the release of viruses from the cell is facilitated by its digestion by blood phagocytes. Plant viruses from cell to cell can pass through intercellular connections - plasmodesmata.
Most often, the virus reproduction cycle ends with a productive infection - the formation of a large population (100-200) of full-fledged virions, which is usually accompanied by the death of the host.
Taxonomy, classification
PARAMIXOVIRUSES
Paramyxoviruses (family Paramyxoviridae from lat. para - about, myxa - slime) is a family of RNA viruses. The family contains respiratory syncytial virus, measles, mumps, parainfluenza viruses transmitted by the respiratory mechanism. Until recently, the family Paramyxoviridae, in accordance with the generally accepted classification of viruses, included three genera: Paramyxovirus, Morbillivirus, Pneumovirus. But recently the classification has been changed.
Family Paramyxoviridae divided into two subfamilies, increased the number of genera:
1. Subfamily Paramyxovirinae includes childbirth Respirovirus(former name - Paramyxovirus, Morbillivirus And Rubulavirus(new genus);
2. Subfamily Pneumovirinae contains genera Pneumovirus And Metapneumovirus.
2. Morphology, size, features of the genome
The structure of the virion. All representatives of the Paramyxoviridae family have a similar structure. It is a complex RNA genomic virus of large size. The typical representative is the Sendai virus (it is pathogenic for mice), and the ultrastructure of paramyxoviruses is considered in this example (Fig. 5). The virion has a rounded shape, its diameter is 150-300 nm. Outside, there is a lipoprotein supercapsid with many spines of two types on the surface (Fig. 4). From the inside, a layer of matrix M-protein adjoins the supercapsid. In the central part of the virion, there is a nucleocapsid strand (RNP) with a helical symmetry type, folded into a loose ball.
Rice. 4 Scheme of paramyxovirus 5 Sendai virus electronogram
Genome It is represented by a large molecule of linear single-stranded minus RNA encoding 7 proteins. Among them are the main capsid protein NP, proteins of the polymerase complex L and P, non-structural protein C (all of which are part of the nucleocapsid), as well as M-protein and surface glycoproteins. These are attachment proteins and a fusion protein (F-protein). Attachment proteins form one type of spines, while the F-protein forms another type of spines. In different paramyxoviruses, attachment proteins are represented by: HN (hemagglutinin-neuraminidase), H (hemagglutinin) or G-protein.
Parainfluenza. According to the antigens of the viral proteins HN, NP, F, 4 main serotypes of parainfluenza viruses are distinguished. Types 1, 2, 3 cross-react with antibodies to the mumps virus. The type 4 virus is different and has 2 subtypes (thus, 5 types of parainfluenza viruses are assumed). All parainfluenza viruses have HN - protein and therefore exhibit hemagglutinating and neuraminidase activity. Parainfluenza virus 1, type 2 agglutinates chicken erythrocytes, parainfluenza 3 virus agglutinates only guinea pig erythrocytes.
Paramyxovirus (Fig. 5) binds by envelope glycoproteins (HN, H, or G) to the cell surface (1). The F-protein ensures the fusion of the virus envelope with the plasma membrane of the cell, without the formation of endosomes. Genome replication is similar to the replication of minus RNA genomic viruses: RNA polymerase is introduced into the cell with the virus nucleocapsid. The genome is transcribed into separate mRNAs (2) for each protein and a complete plus template (3) for genomic RNA. New genomes interact with L-, P- and NP-proteins, forming nucleocapsids. The synthesized matrix protein moves to the inner layer of the cell membrane. Envelope glycoprotein spike precursors are synthesized on ribosomes associated with endoplasmic reticulum (ER) membranes. They are glycosylated, moving through the ER and the Golgi apparatus (AG), integrating into the cell membrane. The nucleocapsid binds to the matrix protein and the glycoprotein-modified membrane (supercapsid). Virions exit the cell (4) by budding.
Rice. 5 Reproduction of paramyxoviruses
Paramyxoviruses have the ability, with the help of the F-protein, to pass into neighboring cells, causing their fusion. In this case, multinucleated giant cells are formed - syncytia (symplasts). This mechanism allows viruses to spread directly from cell to cell, avoiding the action of virus-neutralizing antibodies. The ability to symplast formation is a characteristic feature of paramyxoviruses.
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Control questions:
1. Reproduction of DNA-genomic viruses: main stages, features of reproduction……………………………………………………..……........……...3
2. Signs of virus reproduction in living systems: laboratory animals, chicken embryos, cell cultures…………………………………………......……………………..… ……16
3. Task ............................................... ................................................. ...20
References………………………………………………………...........25
1. Reproduction of DNA-genomic viruses: main stages, features of reproduction
Virus reproduction
The process of virus reproduction can 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 of the virus 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. Once this goal is achieved, the second phase of reproduction begins, during which the expression of the viral genome occurs. 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.
First phase of reproduction.
I. Adsorption of virions on the cell surface.
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 substances that "recognize" them 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. However, recognition of cellular receptors by viral proteins leading to attachment of the viral particle to the 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 cause 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 picornaviruses 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. They 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. However, such attachment is fragile, 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, which is determined by the mobility, "fluidity" 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 between the virus and the cell surface and stable attachment of the viral particle to the resulting groups.
The number of specific receptors on the cell surface ranges between 104 and 105 per cell. Receptors for a number of viruses can be present only in a limited set of host cells, and this can 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 present on the surface of cells of various types, such as receptors for orthomyxoviruses and paramyxoviruses, which are sialyl-containing compounds. Therefore, 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 exceptionally wide range of hosts: these viruses can adsorb and infect cells of both vertebrates and invertebrates.
II. 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. However, these two mechanisms do not exclude, but complement each other.
The term "viropexis" 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. Thus, 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. However, 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 cell 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 due to the point interaction of the viral fusion protein with cell membrane lipids, as a result of which the viral lipoprotein envelope 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 with cell membrane lipids, causing the internal component to pass through the membrane. Most animal viruses enter the cytosol from the receptorosome.
If during endocytosis the viral particle is a passive passenger, then during 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 (P-protein) is one of two glycoproteins found 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. However, most enveloped and non-enveloped viruses cause membrane fusion only at a low pH of 5.0 to 5.75. If weak bases (ammonium chloride, 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 "proton pump" still 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 virus-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) "fusion from the outside" and 2) "fusion from the inside." "Fusion outside" 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, “fusion from within” 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. "Fusion from the inside" is described for many viruses: herpes viruses, oncoviruses, pathogens of slow infections, etc. This type of fusion is caused by the same viral glycoproteins that ensure the penetration of the virus into the cell.
III. Undressing - virus deproteinization
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 decay 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 undressing of picornaviruses is RNA covalently bound to the VPg protein, the end product of undressing of adenoviruses is DNA covalently bound 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.
Second phase of reproduction .
I. Transcription.
Transcription is carried out with the help of a special enzyme - RNA polymerase, which binds nucleotides by forming 3-5´phosphodiester bridges. Such binding occurs only in the presence of a DNA template.
The products of transcription in cells are mRNAs. Cellular DNA itself, which is the carrier of genetic information, cannot directly program protein synthesis. The transfer of genetic information from DNA to ribosomes is carried out by messenger RNA. This is the basis of the central dogma of molecular biology, which is expressed by the following formula:
DNA - transcription - RNA - translation - protein,
where the arrows show the direction of transfer of genetic information.
Implementation of genetic information in viruses. The strategy of the viral genome in relation to the synthesis of mRNA is different for different viruses. In DNA-containing viruses, mRNA is synthesized on the template of one of the DNA strands. The formula for the transfer of genetic information is the same as in the cell:
DNA - transcription - RNA - translation - protein.
DNA-containing viruses that reproduce in the nucleus use a cellular polymerase for transcription. These viruses include papovaviruses, adenoviruses, herpes viruses. DNA-containing viruses that reproduce in the cytoplasm cannot use the cellular enzyme located in the nucleus. Transcription of their genome is carried out by a virus-specific enzyme - DNA polymerase, which enters the cell as part of the virus. These viruses include poxviruses and iridoviruses.
Enzymes that transcribe the viral genome. Transcription of a number of DNA-containing viruses - papovaviruses, adenoviruses, herpes viruses, parvoviruses, hepadnaviruses. It is carried out in the cell nucleus, and in this process the mechanisms of cellular transcription are widely used - transcription enzymes and further modification of transcripts. Transcription of these viruses is carried out by cellular RNA polymerase II, an enzyme that transcribes the cellular genome. However, a special group of adenovirus transcripts is synthesized using another cellular enzyme, RNA polymerase III. In two other families of DNA-containing animal viruses, poxviruses and iridoviruses, transcription occurs in the cytoplasm. Since there are no cellular polymerases in the cytoplasm, the transcription of these viruses requires a special viral enzyme, the viral RNA polymerase. This enzyme is a structural viral protein.
transcription regulation. Transcription of the viral genome is tightly regulated throughout the infectious cycle. Regulation is carried out by both cellular and virus-specific mechanisms. Some viruses, mostly DNA-containing, have three periods of transcription - very early, early and late. These viruses include smallpox, herpes, papovaviruses, adenoviruses. As a result of ultra-early and early transcription, ultra-early and early genes are selectively read with the formation of ultra-early or early mRNAs. During late transcription, another part of the viral genome is read - late genes, with the formation of late mRNAs. The number of late genes usually exceeds the number of early genes. Many superearly genes are genes for non-structural proteins - enzymes and regulators of transcription and replication of the viral genome. In contrast, late genes are usually genes for structural proteins. Usually, during late transcription, the entire genome is read, but with a predominance of transcription of late genes.
Transcription regulation factor in nuclear viruses is the transport of transcripts from the nucleus to the cytoplasm, to the site of mRNA functioning - polysomes.
The product of ultra-early transcription of herpes viruses are A-proteins. The function of one or more of them is necessary for the transcription of the next group of genes encoding P-proteins. In turn, P-proteins include the transcription of the last group of late genes encoding Y-proteins. This type of regulation is called "cascade".
II. Broadcast.
This is the process of translating the genetic information contained in mRNA into a specific amino acid sequence in synthesized virus-specific proteins. Protein synthesis in a cell occurs as a result of mRNA translation on ribosomes. In ribosomes, the flow of information (in mRNA) merges with the flow of amino acids that bring transfer RNA (tRNA). There are a wide variety of tRNAs in the cell. Each amino acid must have its own tRNA.
The tRNA molecule is a single stranded RNA with a complex maple leaf structure.
The binding of a specific tRNA and amino acid is carried out by the enzyme aminoacyl synthetase. One end of the tRNA binds to the amino acid, and the other end to the nucleotides of the mRNA to which they are complementary. Three nucleotides on an mRNA code for one amino acid and are called a "triplet" or "codon", while three nucleotides complementary to a codon on a tRNA are called an "anticodon".
The transcription process consists of three phases: elongation initiation and termination.
Translation initiation is the most critical stage in the translation process, based on the recognition of mRNA by the ribosome and binding to its specific sites. The ribosome recognizes the mRNA due to the "cap" at the 5'-end and slides to the 3'-end until it reaches the initiation codon from which translation begins. In the eukaryotic cell, the initiation codons are the AUG codons (adenine, uracil, guanine), encoding methionine. The synthesis of all polypeptide chains begins with methionine. Specific recognition of viral and RNA by the ribosome is carried out due to virus-specific initiating factors.
The small ribosomal subunit first binds to the mRNA. Other components necessary for the start of translation are attached to the mRNA complex with the small ribosomal subunit. These are several protein molecules, which are called "initiating factors". There are at least three of them in a prokaryotic cell and more than nine in a eukaryotic cell. Initiator factors determine the recognition of specific mRNAs by the ribosome. As a result, a complex is formed that is necessary for the initiation of translation, which is called the "initiation complex". The initiator complex includes: mRNA; small ribosomal subunit; aminoacyl-tRNA carrying the initiator amino acid; initiating factors; several molecules of GTP (guanosine triphosphate).
In the ribosome, the flow of information is merged with the flow of amino acids. The entry of aminoacyl-tRNA into the A-center of the large ribosomal subunit is a consequence of recognition, and its anticodon interacts with the codon of the mRNA located in the small ribosomal subunit. When the mRNA advances by one codon, the tRNA is transferred to the peptidyl center (P-center), and its amino acid is attached to the initiator amino acid to form the first peptide bond. The tRNA free from the amino acid leaves the ribosome and can again function in the transport of specific amino acids. In its place, a new tRNA is transferred from the A-center to the P-center, and a new peptide bond is formed. A vacant mRNA codon appears in the A-center, to which the corresponding tRNA immediately attaches, and new amino acids are added to the growing polypeptide chain.
Translation elongation is a process of lengthening, building up a polypeptide chain, based on the addition of new amino acids using a peptide bond. There is a constant stretching of the mRNA strand through the ribosome and "decoding" of the genetic information embedded in it. Often mRNA functions simultaneously on several ribosomes, each of which synthesizes the same polypeptide strand encoded by this mRNA.
Translation termination occurs at the moment when the ribosome reaches the termination codon in the mRNA (UAA, UGA, UAG). Translation stops and the polypeptide chain is released from the polyribosome. After the end of translation, polyribosomes disintegrate into subunits that can be incorporated into new polyribosomes.
Each RNA functions on several ribosomes. A group of ribosomes working on a single mRNA molecule is called a polyribosome or polysome. Polysomes can consist of 4-6 to 20 or more ribosomes.
Virus-specific polysomes can be either free or membrane-bound. Internal proteins are usually synthesized on free polysomes, glycoproteins are always synthesized on membrane-bound polysomes.
Since the genome of an animal virus is represented by a molecule encoding more than one protein, viruses are faced with the need to synthesize either a long mRNA encoding one giant precursor polypeptide, which must then be cut at specific points into functionally active proteins, or short monocistronic mRNAs, each of which codes for one protein. Thus, there are two ways to form viral proteins:
the first - mRNA is translated into a giant precursor polypeptide, which, after synthesis, is sequentially cut into mature functionally active proteins;
the second - mRNA is translated with the formation of mature proteins or proteins that are only slightly modified after synthesis.
The first mode of translation is characteristic of RNA-containing plus-strand viruses - picornaviruses and togaviruses. Their mRNA is translated into a giant polypeptide chain, the so-called polyprotein, which slides in the form of a continuous ribbon from the ribosomal "conveyor" and is cut into individual proteins of the desired size. The cutting of viral proteins is a multistep process carried out by both virus-specific and cellular proteases.
The second way of forming proteins is characteristic of DNA-containing viruses and most RNA-containing viruses. With this method, short monocistronic mRNAs are synthesized as a result of selective transcription of one region of the genome (gene). However, these viruses make extensive use of the mechanism of post-translational protein cutting.
In a eukaryotic cell, many proteins, including viral ones, undergo post-translational modifications; mature functionally active proteins are often not identical to their newly synthesized precursors. Post-translational covalent modifications such as glycosylation, acylation, methylation, sulfonation (formation of disulfide bonds), proteolytic cutting, and finally phosphorylation are widespread. As a result, instead of 20 genetically encoded amino acids, about 140 amino acid derivatives were isolated from various cells of different organs of eukaryotes.
Glycosylation. The composition of complex RNA- and DNA-containing viruses contains proteins containing covalently attached side chains of carbohydrates - glycoproteins. Glycoproteins are located in the composition of the viral membranes and are located on the surface of the viral particles.
Glycosylation of polypeptides is a complex multistage process, the first stages of which begin already in the process of polypeptide synthesis, and the first carbohydrate residue is attached to the polypeptide chain that has not yet descended from the ribosome. The subsequent stages of glycosylation occur by sequential attachment of carbohydrate residues to the carbohydrate chain during the transport of the polypeptide to the plasma membrane. Carbohydrate residues are attached one at a time, and only when the synthesis of the oligosaccharide chain is initiated, the “block” is transferred. The final formation of the carbohydrate chain may be completed at the plasma membrane prior to assembly of the viral particle.
Glycosylation affects transport; moreover, transport is inextricably linked for glycoproteins with stepwise glycosylation. Convincing proof of this is the effect of glycosylation inhibitors on viral reproduction; they completely suppress the transport of polypeptides without disturbing or inhibiting their synthesis.
When glycosylation is suppressed by appropriate inhibitors (analogues of sugars such as 2-deoxyglycose, antibiotic tunikamycin), the assembly of virions of mixo-, rhabdo-, α-viruses is blocked or non-infectious virions of herpes viruses and oncoviruses are formed.
Sulfonation. Some proteins of complex RNA and DNA viruses are sulfonated after translation. Most often, glycoproteins undergo sulfonation, while the sulfate group binds to the carbohydrate residues of the glycoprotein.
Acylation. A number of glycoproteins of complex RNA-containing viruses (HA2 of the influenza virus, G protein of the vesicular stomatitis virus, HN protein of the Newcastle disease virus, etc.) contain covalently linked 1-2 molecules of fatty acids.
Cutting. Many viral proteins, and primarily glycoproteins, acquire functional activity only after they have been cut at specific points by proteolytic enzymes. Slicing occurs either with the formation of two functional protein subunits (for example, the large and small subunits of hemagglutinin of the influenza virus, two glycoproteins (E2 and E3) of the Semliki forest virus), or with the formation of one functionally active protein and an inactive enzyme, for example, the F and HN proteins of paramyxoviruses. Slicing is usually carried out by cellular enzymes. In many complex animal viruses with glycoproteins, cutting is necessary for the formation of active attachment proteins and fusion proteins and, therefore, for the virus to acquire the ability to infect a cell. Only after cutting these proteins does the viral particle acquire infectious activity. Thus, we can speak about the proteolytic activation of a number of viruses, carried out with the help of cellular enzymes.
Phosphorylation. Phosphoproteins are contained in almost all animal viruses - RNA - and DNA-containing, simple and complex. Most viruses contain protein kinases, but phosphorylation can be carried out by both viral and cellular enzymes. Usually, proteins associated with the viral genome and performing a regulatory role in its expression are phosphorylated. The mechanism of the active action of interferon is associated with the process of phosphorylation.
III. Replication.
Replication is the synthesis of nucleic acid molecules homologous to the genome. DNA replication occurs in the cell, resulting in the formation of daughter double-stranded DNA. Replication occurs on the untwisted regions of DNA and proceeds simultaneously on both strands from the 5'-end to the 3'-end.
Since the two strands of DNA are of opposite polarity, and the replication site (“fork”) moves in the same direction, one strand is built in the opposite direction by separate fragments, which are called Okazaki fragments (after the scientist who first proposed such a model). After synthesis, the Okazaki fragments are “sewn together” by a ligase into a single thread.
DNA replication is carried out by DNA polymerases. To start replication, preliminary synthesis of a short section of RNA on a DNA template, which is called a primer, is necessary. The synthesis of a DNA strand begins with the seed, after which the RNA is quickly removed from the growing site.
Replication of viral DNA. Replication of the genome of DNA-containing viruses is mainly catalyzed by cellular fragments and its mechanism is similar to that of cellular DNA replication.
Each newly synthesized DNA molecule consists of one parent and one newly synthesized strand. Such a replication mechanism is called semi-conservative.
In viruses containing circular double-stranded DNA (papovaviruses), one of the DNA strands is cut, which leads to unwinding and removal of supercoils in a certain part of the molecule.
One can see the lower supercoiled part of the molecule, the untwisted part over a large area, and the newly formed replication loops.
During the replication of single-stranded DNA (family of parvoviruses), the formation of double-stranded forms occurs, which are intermediate replicative forms.
replication complexes. Since the resulting DNA and RNA strands remain associated with the matrix for some time, replicative complexes are formed in the infected cell, in which the entire process of replication (and in some cases also transcription) of the genome is carried out. The replicative complex contains the genome, replicase, and newly synthesized nucleic acid chains associated with the matrix. Newly synthesized genomic molecules are immediately associated with viral proteins, so antigens are found in replication complexes. In the process of replication, a partially double-stranded structure with single-stranded “tails” arises, the so-called replicative precursor.
Replicative complexes are associated with cellular structures, either pre-existing or virus-induced. For example, replicative complexes of picornaviruses are associated with membranes of the endoplasmic reticulum, poxviruses are associated with the cytoplasmic matrix, replicative complexes of adenoviruses and herpes viruses in the nuclei are associated with newly formed fibrous structures and are associated with nuclear membranes. In infected cells, increased proliferation of cellular structures with which replication complexes are associated, or their formation from pre-existing material, can occur. For example, smooth membrane proliferation occurs in cells infected with picornaviruses. Microtubules accumulate in cells infected with reoviruses; in cells infected with smallpox viruses, the formation of a cytoplasmic matrix occurs.
In replication complexes, simultaneously with the synthesis of genomic molecules, transcription occurs and the assembly of nucleocapsids and cores occurs, and in some infections, viral particles as well.
replication regulation. The newly formed genomic RNA molecule can be used in various ways. It can be associated with capsid proteins and become part of the virion, serve as a template for the synthesis of new genomic molecules, or for the formation of mRNA, and finally, in plus-strand viruses, it can function as mRNA and bind to ribosomes. There are mechanisms in the cell that regulate the use of genomic molecules. Regulation follows the principle of self-regulation and is realized through the interaction of viral RNA and proteins due to the possibility of protein-nucleic acid and protein-protein recognition. For example, the role of the terminal protein of picornaviruses is to inhibit the translation of mRNA and select molecules for the formation of virions. The protein that binds to the 5' end of the genomic RNA is, in turn, recognized by the capsid proteins and serves as a signal for the assembly of the viral particle with the participation of this RNA molecule. By the same principle, genomic RNA molecules are selected from “minus”-stranded viruses. The RNA molecule is part of the virion or serves as a template for replication. To switch it to transcription, a ban on protein-nucleic acid interaction must occur. Adenovirus DNA replication involves a protein molecule that binds to the end of the viral DNA and is required to initiate replication. Thus, the synthesis of viral proteins is necessary for the initiation of replication: in the presence of inhibitors of protein synthesis, there is no switch from transcription to replication.
IV. assembly of viral particles.
The synthesis of the components of viral particles in the cell is disconnected and can proceed in different structures of the nucleus and cytoplasm. Viruses that replicate in the nucleus are called nuclear viruses. These are mainly DNA-containing viruses: adenoviruses, papovaviruses, parvoviruses, herpes viruses.
Viruses that replicate in the cytoplasm are called cytoplasmic. These include the DNA-containing variola virus and most RNA-containing viruses, with the exception of orthomyxoviruses and retroviruses. However, this division is very relative, because in the reproduction of both viruses there are stages that occur, respectively, in the cytoplasm and nucleus.
Within the nucleus and cytoplasm, the synthesis of virus-specific molecules can also be uncoupled. So, for example, the synthesis of some proteins is carried out on free polysomes, and others - on polysomes associated with membranes. Viral nucleic acids are synthesized in association with cellular structures away from polysomes that synthesize viral proteins. With such a disjunctive method of reproduction, the formation of a viral particle is possible only if the viral nucleic acids and proteins are able, at sufficient concentration, to recognize each other in the variety of cellular proteins and nucleic acids and spontaneously combine with each other, i.e., are capable of self-assembly.
Self-assembly is based on specific protein-nucleic and protein-protein recognition, which can occur as a result of hydrophobic, salt and hydrogen bonds, as well as steric conformity. Protein-nucleic acid recognition is limited to a small region of the nucleic acid molecule and is determined by unique nucleotide sequences in the non-coding part of the viral genome. With this recognition of the genome region by viral capsid proteins, the process of assembly of the viral particle begins. The attachment of the remaining protein molecules is carried out due to specific protein-protein interactions or non-specific protein-nucleic acid interactions.
Due to the diversity of the structure of animal viruses, the ways of forming virions are also diverse, however, the following general assembly principles can be formulated:
In simply arranged viruses, provirions are formed, which then, as a result of protein modifications, turn into virions. In complex viruses, the assembly is carried out in many stages. First, nucleocapsids or cores are formed, with which the proteins of the outer shells interact.
The assembly of complex viruses (with the exception of the assembly of smallpox viruses and reoviruses) is carried out on cell membranes. The assembly of nuclear viruses occurs with the participation of nuclear membranes, the assembly of cytoplasmic viruses - with the participation of the membranes of the endoplasmic reticulum or plasma membrane, where all components of the viral particle arrive independently of each other.
A number of complex viruses have special hydrophobic proteins that act as intermediaries between the formed nucleocapsids and viral envelopes. Such proteins are matrix proteins in a number of "minus"-stranded viruses (orthomyxoviruses, paramyxoviruses, rhabdoviruses).
The assembly of nucleocapsids, cores, provirions and virions does not occur in the intracellular fluid, but in pre-existing in the cell or induced by the virus (“factories”).
Complicated viruses use a number of elements of the host cell to build their particles, for example, lipids, some enzymes, in DNA-genomic 5V40 - histones, in enveloped RNA-genomic viruses - actin, and even ribosomes were found in arenoviruses. Cellular molecules have certain functions in the viral particle, but their inclusion in the virion may also be the result of accidental contamination, such as the inclusion of a number of cell membrane enzymes or cell nucleic acids.
Assembly of DNA-containing viruses. There are some differences in the assembly of DNA-containing viruses from the assembly of RNA-containing viruses. As with RNA-containing viruses, the assembly of DNA-containing viruses is a multi-step process with the formation of intermediate forms that differ from mature virions in the composition of polypeptides. The first step in assembly is the association of DNA with internal proteins and the formation of cores or nucleocapsids. In this case, DNA is connected to pre-formed "empty" capsids.
As a result of DNA binding to capsids, a new class of intermediate forms appears, which are called incomplete forms. In addition to incomplete forms with different DNA content, there is another intermediate form in morphogenesis - immature virions, which differ from mature ones in that they contain uncut polypeptide precursors. Thus, the morphogenesis of viruses is closely related to the modification (processing) of proteins.
Assembly of nuclear viruses begins in the nucleus, usually by association with the nuclear membrane. Formed in the nucleus, intermediate forms of the herpes virus bud into the perinuclear space through the inner nuclear membrane, and the virus acquires in this way an envelope that is a derivative of the nuclear membrane. Further completion and maturation of virions occurs in the membranes of the endoplasmic reticulum and in the Golgi apparatus, from where the virus is transported to the cell surface as part of cytoplasmic vesicles.
In non-budding lipid-containing viruses - smallpox viruses, the assembly of virions occurs in the already described cytoplasmic viral "factories". The lipid envelope of viruses in "factories" is formed from cellular lipids by autonomous self-assembly, so the lipid composition of the membranes differs significantly from the composition of lipids in cell membranes.
V. Release of viral particles from the cell.
There are two ways for the viral progeny to leave the cell:
1) by "explosion";
2) by budding.
The exit from the cell by explosion is associated with the destruction of the cell, the violation of its integrity, as a result of which the mature viral particles inside the cell end up in the environment. This way of leaving the cell is inherent in viruses that do not contain a lipoprotein membrane (picorna-, reo-, parvo-, papova-, adenoviruses). However, some of these viruses can be transported to the cell surface before cell death. Exit from cells by budding is inherent in viruses containing a lipoprotein membrane, which is a derivative of cell membranes. With this method, the cell can remain viable for a long time and produce viral offspring until its resources are completely depleted.
The process of reproduction of viruses can be conditionally divided into 2 phases . The first phase includes 3 stages: 1) virus adsorption on sensitive cells; 2) penetration of the virus into the cell; 3) virus deproteinization . The second phase includes the stages of realization of the viral genome: 1) transcription, 2) translation, 3) replication, 4) assembly, maturation of viral particles and 5) release of the virus from the cell.
The interaction of a virus with a cell begins with the process of adsorption, i.e., with the attachment of the virus to the surface of the cell.
Adsorption is a specific binding of a virion protein (antireceptor) to a complementary cell surface structure - a cell receptor. By chemical nature, the receptors on which viruses are fixed belong to two groups: mucoprotein and lipoprotein. Influenza viruses, parainfluenza, adenoviruses are fixed on mucoprotein receptors. Enteroviruses, herpes viruses, arboviruses are adsorbed on lipoprotein receptors of the cell. Adsorption occurs only in the presence of certain electrolytes, in particular Ca2+ ions, which neutralize excess anionic charges of the virus and the cell surface and reduce electrostatic repulsion. virus and cell, and then comes the specific interaction of the attachment protein of the virion with specific groups on the plasma membrane of the cell. Simple human and animal viruses contain attachment proteins in the capsid. In complexly organized viruses, attachment proteins are part of the supercapsid. They can take the form of threads (fibers in adenoviruses), or spikes, mushroom-like structures in mixo-, retro-, rhabdo- and other viruses. Initially, a single bond of the virion with the receptor occurs - such attachment is fragile - adsorption is reversible. For irreversible adsorption to occur, multiple bonds must appear between the virus receptor and the cell receptor, i.e., stable multivalent attachment. The number of specific receptors on the surface of one cell is 10 4 -10 5 . Receptors for some viruses, such as arboviruses. are found on cells of both vertebrates and invertebrates; for other viruses, only on cells of one or more species.
The penetration of human and animal viruses into the cell occurs in two ways: 1) viropexis (pinocytosis); 2) fusion of the viral supercapsid envelope with the cell membrane. Bacteriophages have their own penetration mechanism, the so-called syringe, when, as a result of the contraction of the protein outgrowth of the phage, the nucleic acid is, as it were, injected into the cell.
Deproteinization of the virus release of the virus hemiome from viral protective shells occurs either with the help of viral enzymes or with the help of cellular enzymes. The end products of deproteinization are nucleic acids or nucleic acids associated with an internal viral protein. Then the second phase of viral reproduction takes place, leading to the synthesis of viral components.
Transcription is the rewriting of information from the DNA or RNA of a virus to mRNA according to the laws of the genetic code.
Translation is the process of translating the genetic information contained in mRNA into a specific sequence of amino acids.
Replication is the process of synthesis of nucleic acid molecules homologous to the viral genome.
The implementation of genetic information in DNA-containing viruses proceeds in the same way as in cells:
DNA transcription and RNA translation protein
RNA transcription and RNA translation protein
In viruses with a positive RNA genome (togaviruses, picornaviruses), transcription is absent:
RNA translation protein
Retroviruses have a unique way of transferring genetic information:
RNA reverse transcription DNA transcription i-RNA translation protein
The DNA integrates with the genome of the host cell (provirus).
After the cell has produced viral components, the last stage of viral reproduction begins, the assembly of viral particles and the release of virions from the cell. The release of virions from the cell is carried out in two ways: 1) by "explosion" of the cell, as a result of which the cell is destroyed. This path is inherent in simple viruses (picorna-, reo-, papova- and adenoviruses), 2) exit from cells by budding. Inherent in viruses containing supercapsid. With this method, the cell does not die immediately, it can give multiple viral offspring until its resources are depleted.
Virus culture methods
For the cultivation of viruses in laboratory conditions, the following living objects are used: 1) cell cultures (tissues, organs); 2) chicken embryos; 3) laboratory animals.
Cell cultures
The most common are single-layer cell cultures, which can be divided into 1) primary (primarily trypsinized), 2) semi-transplantable (diploid) and 3) transplantable.
Origin they are classified into embryonic, neoplastic and from adult organisms; by morphogenesis- on fibroblast, epithelial, etc.
Primary cell cultures are cells of any human or animal tissue that have the ability to grow as a monolayer on a plastic or glass surface coated with a special nutrient medium. The life span of such crops is limited. In each case, they are obtained from the tissue after mechanical grinding, treatment with proteolytic enzymes and standardization of the number of cells. Primary cultures derived from monkey kidneys, human embryonic kidneys, human amnion, chicken embryos are widely used for the isolation and accumulation of viruses, as well as for the production of viral vaccines.
semi-transplantable (or diploid ) cell cultures - cells of the same type, capable of withstanding up to 50-100 passages in vitro, while maintaining their original diploid set of chromosomes. Diploid strains of human embryonic fibroblasts are used both for the diagnosis of viral infections and in the production of viral vaccines.
transplanted cell lines are characterized by potential immortality and heteroploid karyotype.
The source of transplanted lines can be primary cell cultures (for example, SOC, PES, VNK-21 - from the kidneys of day-old Syrian hamsters; PMS - from the kidney of a guinea pig, etc.), individual cells of which show a tendency to endless reproduction in vitro. The set of changes leading to the appearance of such features from cells is called transformation, and the cells of transplanted tissue cultures are called transformed.
Another source of transplanted cell lines are malignant neoplasms. In this case, cell transformation occurs in vivo. The following lines of transplanted cells are most often used in virological practice: HeLa - obtained from cervical carcinoma; Ner-2 - from carcinoma of the larynx; Detroit-6 - from lung cancer metastasis to the bone marrow; RH is from the human kidney.
For cell cultivation, nutrient media are needed, which, according to their purpose, are divided into growth and supporting ones. The composition of growth media should contain more nutrients in order to ensure active reproduction of cells to form a monolayer. Supporting media should only ensure the survival of cells in an already formed monolayer during reproduction of viruses in the cell.
Standard synthetic media, such as Synthetic 199 media and Needle media, are widely used. Regardless of the purpose, all nutrient media for cell cultures are designed on the basis of a balanced salt solution. Most often it is Hank's solution. An integral component of most growth media is the blood serum of animals (calf, bull, horse), without the presence of 5-10% of which, cell reproduction and the formation of a monolayer do not occur. Serum is not included in maintenance media.
Isolation of viruses in cell cultures and methods for their indication.
When isolating viruses from various infectious materials from a patient (blood, urine, feces, mucous discharge, swabs from organs), cell cultures are used that are most sensitive to the alleged virus. For infection, cultures in test tubes with a well-developed monolayer of cells are used. Before infecting the cells, the nutrient medium is removed and 0.1-0.2 ml of a suspension of the test material, previously treated with antibiotics to kill bacteria and fungi, is added to each test tube. After 30-60 min. contact of the virus with the cells, remove the excess material, add a supporting medium to the test tube and leave it in a thermostat until signs of virus reproduction are detected.
An indicator of the presence of the virus in infected cell cultures can be:
1) the development of specific cell degeneration - the cytopathic effect of the virus (CPE), which has three main types: round or small cell degeneration; the formation of multinucleated giant cells - symplasts; development of foci of cell proliferation, consisting of several layers of cells;
2) detection of intracellular inclusions located in the cytoplasm and nuclei of affected cells;
3) positive hamagglutination test (RGA);
4) positive hemadsorption reaction (RGAds);
5) the phenomenon of plaque formation: a monolayer of virus-infected cells is covered with a thin layer of agar with the addition of a neutral red indicator (pink background). In the presence of the virus in the cells, colorless zones ("plaques") are formed on a pink agar background.
6) in the absence of CPE or GA, an interference reaction can be set: the culture under study is re-infected with the virus that causes CPE. In a positive case, there will be no CPP (the interference reaction is positive). If there was no virus in the test material, CPE is observed.
Isolation of viruses in chicken embryos.
For virological studies, chicken embryos of 7-12 days of age are used.
Before infection determine the viability of the embryo. When ovoscoping, live embryos are mobile, the vascular pattern is clearly visible. With a simple pencil, mark the boundaries of the air sac. Chicken embryos are infected under aseptic conditions with sterile instruments, having previously treated the shell above the air space with iodine and alcohol.
The methods of infection of chicken embryos can be different: applying the virus to the chorion-allantoic membrane, to the amniotic and allantoic cavities, to the yolk sac. The choice of infection method depends on the biological properties of the virus under study.
Indication of the virus in the chicken embryo is made by the death of the embryo, a positive hemagglutination test on glass with allantoic or amniotic fluid, by focal lesions ("plaques") on the chorion-allantoic membrane.
III. Isolation of viruses in laboratory animals.
Laboratory animals can be used to isolate viruses from infectious material when more convenient systems (cell cultures or chick embryos) cannot be used. They take mainly newborn white mice, hamsters, guinea pigs, rats. Infect animals according to the principle of cytotropism of the virus: pneumotropic viruses are injected intranasally, neurotropic - intracerebral, dermatotropic - on the skin.
The indication of the virus is based on the appearance of signs of disease in animals, their death, pathomorphological and pathohistological changes in tissues and organs, as well as a positive hemagglutination reaction with extracts from organs.
Not done by binary fission. Back in the 50s of the last century, it was established that reproduction is carried out by the reproduction method (translated from English reproduce - make a copy, reproduce), that is, by reproducing nucleic acids, as well as protein synthesis, followed by the collection of virions. These processes occur in different parts of the cell of the so-called host (for example, in the nucleus or cytoplasm). This disjointed method of virus reproduction is called disjunctive. This is what we will focus on in more detail in our article.
reproduction process
This process has its own characteristics of virus reproduction and is distinguished by a successive change of some stages. Let's consider them separately.
Phases
Viral reproduction in a cell occurs in several phases, which are described below:
- The first phase is the adsorption of the virus, which was discussed above, on the surface of the cell, which is sensitive to this virus.
- The second is the penetration of the virus into the host cells by the viropexis method.
- The third is a kind of "undressing" of virions, the release of nucleic acid from the capsid and supercapsid. In a number of viruses, the nucleic acid enters cells by the fusion of the virion envelope and the host cell. In this case, the third and second phases are combined into a single one.
Adsorption
This stage of viral reproduction refers to the penetration of a viral particle into cells. Adsorption begins on the cell surface through the interaction of cellular as well as viral receptors. Translated from Latin, the word "receptors" means "receiving". They are special sensitive formations that perceive irritations. Receptors are molecules or molecular complexes located on the surface of cells, and are also capable of recognizing specific chemical groups, molecules or other cells, and binding them. In the most complex virions, such receptors are located on the outer shell in the form of a spike-like outgrowth or villus; in simple virions, they are usually located on the surface of the capsid.
The mechanism of adsorption on the surface of a receptive cell is based on the interaction of receptors with the so-called complementary receptors of the "host" cell. Virion receptors and cells are some specific structures that are located on the surface.
Adenoviruses and myxoviruses adsorb directly on mucoprotein receptors, while arboviruses and picornaviruses adsorb on lipoprotein receptors.
In the myxovirus virion, neuraminidase destroys the mucogphotein receptor and cleaves N-acetylneuraminic acids from the oligosaccharide, which contains galactose and galactosamine. Their interactions at this stage are reversible, because they are significantly affected by temperature, the reaction of the medium and salt components. Adsorption of the virion is prevented by heparin and sulfated polysaccharides, which carry a negative charge, but their inhibitory effect is removed by some polykaryons (ecmolin, DEAE-dextran, protamine sulfate), which neutralize the negative charge from sulfated polysaccharides.
Entry of the virion into the host cell
The route of introduction of a virus into a cell sensitive to it will not always be the same. Many virions are able to enter cells by pinocytosis, which in Greek means "drink", "drink". With this method, the pinocytic vacuole seems to draw the virion directly into the cell. The remaining virions can enter the cell directly through its membrane.
The contact of the enzyme neuraminidase with cellular mucoproteins promotes the entry of virions into the cell among myxoviruses. The results of recent studies prove that the DNA and RNA of virions are not separated from the outer shell, i.e., the virions penetrate entirely into sensitive cells by pinocytosis or viropexis. To date, this has been confirmed in relation to the smallpox virus, vaccinia, as well as other viruses that choose animal organisms as their habitat. Speaking of phages, they infect cells with nucleic acid. The infection mechanism is based on the fact that those virions that are contained in cell vacuoles are hydrolyzed by enzymes (lipases, proteases), during which DNA is released from the phage membrane and enters the cell.
For the experiment, a cell was infected with a nucleic acid that was isolated from some viruses, and one complete cycle of virion reproduction was induced. However, under natural conditions, infection with such an acid does not occur.
Disintegration
The next stage of virus reproduction is disintegration, which is the release of NK from the capsid and outer shell. After the virion enters the cells, the capsid undergoes some changes, acquiring sensitivity to the cellular protease, then it is destroyed, simultaneously releasing NK. In some bacteriophages, free NA enters the cells. The phytopathogenic virus enters through the damage in the cell wall, and then it is adsorbed on the internal cell receptor with the simultaneous release of NA.
RNA replication and viral protein synthesis
The next stage of viral reproduction is the synthesis of a virus-specific protein, which occurs with the participation of the so-called messenger RNA (in some viruses they are part of virions, and in some they are synthesized only in infected cells directly on the virion DNA or RNA template). Viral NK replication occurs.
The process of reproduction of RNA viruses begins after the entry of nucleoproteins into the cell, where viral polysomes are formed by complexing RNA with ribosomes. After that, early proteins are also synthesized, which should include repressors from cellular metabolism, as well as RNA polymerases that are translated with the parent RNA molecule. In the cytoplasm of the smallest viruses, or in the nucleus, viral double-stranded RNA is formed by complexing the parent plus chain (“+” - RNA chain) with the newly synthesized, as well as complementary with it minus chain (“-” - RNA chain) . The connection of these strands of nucleic acid provokes the formation of only a single-stranded RNA structure, which is called the replicative form. Syntheses of viral RNA are carried out by replication complexes, in which the replicative form of RNA, the RNA polymerase enzyme, and polysomes take part.
There are 2 types of RNA polymerases. These include: RNA polymerase I, which catalyzes the formation of the replicative form directly on the plus-strand template, as well as RNA polymerase II, which takes part in the synthesis of single-stranded viral RNA on the replicative-type template. Synthesis of nucleic acids in small viruses occurs in the cytoplasm. As for the influenza virus, internal protein and RNA are synthesized in the nucleus. RNA is then released from the nucleus and penetrates into the cytoplasm, in which, together with ribosomes, it begins to synthesize the viral protein.
After the virions enter the cells, they suppress the synthesis of nucleic acids, as well as cellular proteins. During reproduction on a matrix, mRNA is also synthesized in the nucleus, which carries information for protein synthesis. The mechanism of viral protein synthesis is carried out at the level of the cellular ribosome, and the source of construction will be the amino acid fund. Activation of amino acids is carried out by enzymes, with the help of mRNA they are transferred directly to ribosomes (polysomes), in which they are already located in the synthesized protein molecule.
Thus, in infected cells, the synthesis of nucleic acids and virion proteins is carried out as part of a replicative-transcriptive complex, which is regulated by a certain system of mechanisms.
Virion morphogenesis
The formation of virions can occur only in the case of a strictly ordered connection of structural viral polypeptides, as well as their NA. And this is ensured by the so-called self-assembly of protein molecules near the NC.
Virion formation
The formation of the virion occurs with the participation of some structural components that make up the cell. Herpes, polio, and vaccinia viruses are produced in the cytoplasm, while adenoviruses are produced in the nucleus. The synthesis of viral RNA, as well as the formation of the nucleocapsid, occurs directly in the nucleus, and hemagglutinin is formed in the cytoplasm. After that, the nucleocapsid moves from the nucleus to the cytoplasm, in which the formation of the virion envelope takes place. The nucleocapsid is covered on the outside with viral proteins, and the hemagglutinins and neuraminidase are included in the virion. This is how the formation of offspring, for example, the influenza virus.
Release of the virion from the host cell
Virus particles are released from the "host" cell simultaneously (during cell destruction) or gradually (without any cell destruction).
It is in this form that the reproduction of viruses occurs. Virions are released from cells, usually in two ways.
First method
The first method implies the following: after the absolute maturation of virions directly inside the cell, they are rounded, vacuoles are formed there, and then the cell membrane is also destroyed. Upon completion of these processes, the virions are released all at the same time and completely from the cells (picornaviruses). This method is called lytic.
Second method
The second method involves the release of virions as they mature for 2–6 hours on the cytoplasmic membrane (myxoviruses and arboviruses). The secretion of myxoviruses from the cell is facilitated by neuraminidase, which destroys the cell membrane. During this method, 75-90% of the virions are released spontaneously into the culture medium, and the cells gradually die.