Replication of viral RNA molecules. Stages of RNA replication of the viral genome. VIVOS VOCO: A.S. Spirin, “Ribonucleic acids as the central link of living matter” B Replication of the genome of RNA viruses

This is the IV stage of viral reproduction: synthesis of viral proteins and replication of nucleic acids.

1. Picornavirus RNA acts as an mRNA, is translated into ribosomes, and serves as a template for the formation of a single giant polypeptide. The latter is split into several proteins, one of which is a polymerase. Replication of the same RNA, freed from ribosomes, begins.

2. The RNA of other viruses serves as a template on which the mRNA is transcribed. It is translated onto ribosomes, and certain viral proteins are formed, one of which is polymerase. Next, viral RNA replication occurs, and first a form of 2 strands is formed.

3. In oncogenic RNA-containing viruses, synthesis proceeds differently. From the RNA template, a DNA copy is formed, which has 1 strand of DNA. This process involves reverse transcriptase, which is present in the virion. Then this DNA strand is replicated and 2 strands are formed. RNA molecules are synthesized on the template of this DNA copy.

54. What is the difference between (+) and (-) variants of single-stranded RNA genomes?

Viral RNAs are divided into + strands and - RNA strands.

+RNA are presented as single chains with characteristic “caps” at the ends for recognition of ribosomes. This group includes RNAs that are capable of directly translating genetic information on the ribosomes of an infected cell, that is, performing f- and m-RNA. F-i + threads: serve as m-RNA for the synthesis of structural proteins, a template for RNA replication, and are packaged into a capsid to form a daughter population.

-RNA are not able to translate genetic information on ribosomes. Serve as a template for m-RNA synthesis.

The essence of the radioimmune method.

Purified and concentrated Ag and AT labeled with a radioisotope (iodine) are used.

For detection of AT– labeled Ag is added to the test serum. The AT titer in serum is determined by the decrease in free labeled Ag.

For detection of Ag– the test material is mixed with antiserum, then homologous labeled Ag is added. If the labeled Ag remains free, the reaction positive, since the test Ag was bound to the serum. If labeled Ag decreases, this means that it interacts with serum - reaction negative.

Use for the diagnosis of viral hepatitis.

How does HIV become infected?

HIV infection is a typical anthroponosis; the disease cannot be reproduced in animals. The reservoir of the virus is an infected person. Transmission routes:

1. Sexual – through damage to the mucous membranes.

2. Use of the same needles and syringes by drug addicts.

3. Hemotransfusion – transfusion of blood and its preparations.

4. Transfer with donor organs.

HIV is sensitive to high temperatures, ethanol, ether. It is viable in biological material at room temperature for several days.

What cells does HIV affect and what receptor do these cells have? Mech-m of HIV development.

Targets for HIV are T-helper cells, monocytes, macrophages, microglial cells.

Pathogenesis lesions: selective damage to CD4 + cells, since the virus uses CD4 as a receptor. There are 4 stages in pathogenesis:

I. Apoptosis– “programmed” cell death – when viruses interact with the receptor system of macrophages, the “recognition” of the virus as a foreign Ag is disrupted.

II. Syncytia formation– viruses enter the blood and invade new uninfected lymphocytes. Healthy lymphocytes adhere to the affected ones. The activity of lymphocytes decreases under the influence of toxins produced during cell death.

III. Autoimmune reactions– the appearance of viral glycoproteins on the membranes of T-helper cells leads to the activation of T-killer cells. The immune system cannot resist even saprophytic flora. Opportunistic infections occur.

IV. Infection of progenitor cells– with normal immunity, these cells are destroyed, and in conditions of immunodeficiency they actively multiply. Malignant growth diseases occur - Kaposi's sarcoma.

"Opportunistic" infections– diseases caused by a microorganism that can only affect individuals with weakened immune systems.

How does HIV work?

HIV is part of the retroviruses. Characteristic: unique genome structure and the presence of reverse transcriptase. Reverse transcriptase ensures the reverse direction of the flow of genetic information - from RNA to DNA (hence the name).

Genome: 2 identical molecules of 1-stranded non-segmented +RNA.

During reproduction, DNA intermediates are formed - features of retrovirus reproduction. There are HIV-I and HIV-II.

Mature virions: spherical shape, d = 120 nm, in the genome there are 2 strands + RNA, capsid, supercapsid from a lipid bilayer, which is penetrated by glycoprotein spikes. These spikes interact with CD4 molecules on cell membranes.

Below is an article by the laureate of the Great Gold Medal. M.V. Lomonosov 2002, which was written based on a report read on May 15, 2002 at the General Meeting of the Russian Academy of Sciences when presenting this award.

A. S. Spirin
Spirin Alexander Sergeevich- academician, adviser to the Russian Academy of Sciences.

In the 30s of the last century, my teacher Andrei Nikolaevich Belozersky managed to put the last end to the protracted debate about the belonging of two different types of nucleic acids - ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) - to the plant or animal kingdoms of the living world. At that time, nothing was known about the genetic functions of both nucleic acids. For a long time, RNA was considered a component of plants, including fungi, and DNA was considered a typical component of animal cells, as "animal nucleic acid", most often called then "thymonucleic acid" (from thymus- the Latin name of the thymus gland from which it was isolated). Then it turned out that RNA, along with DNA, is widespread in animals. However, there were great doubts about the presence of DNA in plant cells: there was no direct evidence, and the only indirect indication - the positive color cytochemical Feilgen reaction, which was detected by the nuclei of plant cells - could not be considered reliable evidence. A.N. Belozersky first isolated and identified thymine, the most characteristic component of DNA, from pea seeds, and then the DNA itself was preparatively isolated from horse chestnut seeds. Thus, the final understanding was established that both RNA and DNA are two universal types of nucleic acids inherent in all kingdoms of the living world.

In the 40s, based on various cytological and biochemical observations and analyses, the idea began to emerge that DNA, constantly localized in the nuclei of cells, in their chromosomes, is most closely connected with the apparatus of heredity, and RNA is an obligatory component of the cell cytoplasm, responsible for protein biosynthesis [,]. Direct experiments by T. Avery and his colleagues proved that pure, isolated DNA can be a carrier of hereditary characteristics of an organism. (For more details about these experiments, as well as about the composition and structure of DNA, see the review). An increasing number of researchers, primarily biochemists and cytologists, began to lean toward the idea that DNA or its complexes with proteins could be the main carriers of genetic information, and RNA could be an intermediary that receives this information from DNA and implements it in the form of protein biosynthesis.

By the early 50s, E. Chargaff established the fact of species specificity of DNA composition, showing that the ratios of four types of its monomers - guanyl (G), adenyl (A), cytidyl (C) and thymidyl (T) - differ in different species of organisms [ , ]. This fact was directly consistent with the proposed genetic role of DNA. At the same time, amazing patterns were also found in the nucleotide composition of DNA, called “Chargaff’s rules”: regardless of species differences, in all DNA the amount of G was equal to the amount of C, and the amount of A was equal to the amount of T (G = C, A = T). In 1953, J. Watson and F. Crick, using these experimental data on the chemical composition of DNA, as well as the results of X-ray diffraction analyzes of oriented DNA strands, which indicated the helical nature of the stacking of polymer DNA molecules [,], proposed a model of the macromolecular structure of DNA . It was a double helix, where two polymer strands of DNA are twisted relative to each other around a common axis and held together due to pairwise interactions of G with C and A with T. Their guess turned out to be ingenious: a mechanism for its exact reproduction followed directly from the structure, which for the first time provided an explanation reproduction of similar structures in the processes of reproduction and heredity. Thus, half a century ago, a new science was born - molecular biology.

Naturally, molecular biology began with the era of DNA. DNA was proclaimed “the main molecule of life”, “the thread of life”, the beginning of beginnings and the basis of all living things. Proteins, previously considered as the main component of living systems, were now “dismissed” from all leadership positions and “assigned” to secondary roles as catalysts serving the existence of DNA. The role of another type of nucleic acids - RNA - was reduced to the function of intermediaries produced on DNA templates and directing the synthesis of proteins. The “DNA->RNA->protein” scheme with the irreversibility of information transfer processes, indicated by arrows, is called the “central dogma of molecular biology” (for more details, see [,13]).

RNA: DNA REPLICATION AND GENETIC FUNCTION

The reproduction (replication) of the DNA structure is based on the so-called complementarity principle: in a double helix, two polymer chains of DNA are connected side by side by hydrogen bonds due to the formation of pairs G-C, C-G, A-T and T-A (see, Fig. 2 and 3). If two chains of a double helix diverge, then a new one can be built (polymerized) on each of them complementary chain, so that opposite G of the original chain there will be C of the new chain, opposite C of the old chain - G of the new chain, opposite A-T, and opposite T-A; the result will be two daughter double helices, completely identical to the original mother one (see, Fig. 4).

RNA is chemically similar to DNA. In both cases, they are linear, unbranched polymers of nucleotides with a pentose phosphate backbone and four types of nitrogenous bases (purine and pyrimidine) as side groups (Figure 1). There are only two small differences between an RNA strand and a single strand of DNA:

1) five-carbon sugar (pentose) in RNA is represented by ribose, and in DNA - by its derivative 2"-deoxyribose;

2) one of the two pyrimidine nucleotides in RNA is represented by a uridyl residue (U) instead of its methylated derivative T in DNA.

Being copies of certain functional segments of the DNA chain - genes, RNA chains are designed to serve as templates for the synthesis of another type of polymer - polypeptide chains of proteins. Since proteins consist of twenty different types of monomers (amino acids), and RNA - of only four types of monomers (nucleotides), the determination of the amino acid sequence of a polypeptide chain by the nucleotide sequence of RNA requires that each amino acid be encoded by a combination of several - at least three - nucleotides. It was the triplet code that was first postulated on the basis of theoretical considerations, and then proven experimentally. Firmly attached to RNA genetic role an intermediary between genes and proteins: on the one hand, RNA was represented as a set of gene copies, that is, copies of DNA segments, and on the other hand, as direct matrices, the sequences of nucleotide triplets of which are decoded into amino acid sequences of polypeptide chains in the process of protein synthesis.

Based on these ideas, in 1956 I, then a graduate student A.N. Belozersky at the Institute of Biochemistry of the USSR Academy of Sciences, work began on experimental verification of the correspondence between DNA and RNA. First, we showed that the nucleotide composition (the ratio of four types of nucleotides) of DNA can vary greatly among different species of organisms, in particular among bacteria of different taxonomic groups. We further proceeded from the fact that if RNA is a copy of DNA, then the nucleotide composition of these two types of nucleic acids must coincide or at least be similar. Our analyzes yielded a completely unexpected result: despite strong variations in DNA composition, the ratios of nucleotides in the total RNA of different species turned out to be surprisingly conservative (Table). At the same time, statistical analysis of these data showed that there is a reliable positive correlation between the composition of RNA and the composition of DNA, although with a small regression value (Fig. 3). The data were interpreted in such a way that, against the background of the bulk of evolutionarily conservative RNA, similar in different species, there is a relatively small fraction of species-specific RNA that copies DNA.

In 1959, F. Crick described this early period in the history of molecular biology:

"The coding problem has now gone through three phases. In the first, the wandering phase, various assumptions were made, but none were accurate enough to be disproved. The second phase, the optimistic one, was initiated in 1954 by Gamow, who was bold enough to to propose a fairly accurate code. This stimulated a number of researchers who sought to show that his assumptions were incorrect, and thereby somewhat raised the accuracy of thinking in this area. The third phase - the phase of confusion - was initiated by the paper of Belozersky and Spirin in 1958... The data presented there showed that our understanding was overly simplistic in many important respects." .

Somewhat earlier, it was established that DNA-like RNA is formed when bacteria are infected with a virus (bacteriophage): the introduction of new genetic material - viral DNA - into the cell induced the synthesis of RNA, similar in composition to viral DNA and, obviously, determining the synthesis of viral proteins. Our work was the first to show that a fraction of DNA-like RNA is a normal component of normal, uninfected cells, where it apparently functions to carry genetic information from its DNA to determine the synthesis of its proteins. This RNA fraction was later named messenger RNA, messenger RNA (mRNA), or messenger RNA.

Further research on mRNA in my group (laboratory since 1960) at the Institute of Biochemistry of the USSR Academy of Sciences and the transition from the study of microorganisms to higher organisms led to another discovery. It turned out that in the cells of higher organisms - animals and higher plants - mRNA does not exist in free form; it is presented in the form of ribonucleoprotein particles (mRNP particles), which we called infosomes[ , ].

This new type of intracellular particles was characterized by a number of unique physicochemical properties, and in particular, a constant ratio of structural protein and mRNA with a significant predominance of the protein component. The role of the nucleoprotein form of existence of mRNA in the cells of higher organisms was subsequently studied by many researchers in connection with the mechanisms of regulation of protein synthesis at the translation level. These works provided the key to understanding a number of molecular mechanisms of oogenesis, spermatogenesis and early embryogenesis, cell differentiation and morphogenesis, erythropoiesis and other processes in the life of multicellular creatures, including humans (see reviews [, ]).

STRUCTURAL RNA

After 1958, the main efforts of my group were aimed at studying the conserved, non-genetic RNA that makes up the bulk of total cellular RNA. It quickly became clear that the predominant part (about 90%) is a component of ribosomes - intracellular ribonucleoprotein particles that are molecular “factories” for the production of proteins. In a series of brilliant works by English, French and American researchers [ - ] it was proven that ribosomes and ribosomal RNA themselves do not carry genetic information for the synthesis of proteins, but serve as a universal, non-specific apparatus that must be programmed by messenger RNA (mRNA) in order to synthesize specific , proteins determined by the corresponding genes.

The first studies of ribosomal RNA in our laboratory showed that these are large macromolecules (molecular weight of the order of 10 6), each of which represents one covalently continuous polynucleotide chain [, ], in contrast to the previously put forward idea about the subunit nature of the structure of ribosomal RNA molecules. Somewhat earlier, when studying high-polymer biologically active (infectious) RNA from the tobacco mosaic virus, we were able to discover its ability to form secondary and tertiary structures, that is, to fold and fold its polynucleotide chain into structures with short- and long-range intrachain interactions [,30]. It was possible to demonstrate a similar behavior in the case of ribosomal RNA in solution [,]. Taken together, studies of the physicochemical properties and structural characteristics of isolated high-polymer RNA in solution, carried out in 1958-1962, led to the formulation of the following general principles of their spatial organization:

RNA, unlike DNA, is single chain polymer"

RNA forms secondary structure- a set of short helical sections - mainly due to antiparallel complementary pairing of adjacent chain segments;

RNA is capable of forming tertiary structure due to long-range complementary interactions within the chain and interhelical interactions;

High-polymer RNA is capable of folding into compact particles;

RNA has significant conformational mobility(Fig. 4).

These observations were preceded by our experiments on induced structural transformations of ribosomes, such as the unfolding of ribosomal particles into ribonucleoprotein strands without loss of ribosomal proteins [, ], as well as disassembly and reverse self-assembly of ribosomal proteins on the RNA nucleus (ribosome reconstruction), which proved that RNA serves in the formation of ribosomal particles frame. Study of neutron scattering of ribosomes together with the group of I.N. Serdyuk at the Institute of Protein of the USSR Academy of Sciences (see) provided additional physical evidence of the relative position of RNA and proteins in ribosomal particles. In general, all this made it possible to formulate three general principles of ribosome structure:

the ribosome is built from two unequal divided subunits - the small and large ribosomal subunits;

Two specifically self-folding high-polymer RNAs - ribosomal RNAs - form compact structural cores of two ribosomal subunits;

A variety of ribosomal proteins and their groups are specifically assembled on the ribosomal RNA framework, mainly at the periphery of these compact cores.

In addition to information about the detailed structure of the ribosome, the results obtained fully confirmed the above general principles of its structure, and primarily the fact that the shape of the subunits is determined by their compactly folded RNA, and the ribosomal proteins are located on the periphery of these nuclei. A complete ribosome is an associate of two different, compactly and specifically folded RNAs, only on part of the surface “decorated” with proteins. It is important that it is ribosomal RNA, as it turns out, that forms the main functional centers of the ribosome and determines the fundamental structure of the ribosome as a molecular machine that carries out protein synthesis according to a program written in mRNA. It is likely that at the dawn of life the ribosome consisted only of RNA, and ribosomal proteins are a later evolutionary acquisition to stabilize ribosomal RNA or improve its function.

ALMIGHTY RNA

The ability of RNA to form compact three-dimensional structures, as in the case of proteins, provides the basis for specific interactions with other molecules - macromolecules and small ligands. For RNA molecules folded into a specific globule, due to which a unique spatial pattern is created on its surface, it is necessary to assume the possibility of a molecular recognition function, as in proteins. In turn, highly selective recognition leads to the possibility of specific catalysis of chemical reactions in the manner of enzymatic catalysis of reactions by proteins.

Perhaps the first known “recognizing” RNAs can be considered the so-called “transfer” RNAs, or tRNAs, which play an adapter role in protein biosynthesis (see, Fig. 1). These relatively small RNAs (molecular weight about 30,000) are compactly folded molecules with the same type of spatial structure (see, Fig. 3). Their purpose is to transfer amino acids from a free state into the protein polypeptide chain synthesized by the ribosome. To perform this task, tRNA must alternately and very selectively interact with a number of macromolecular structures in the cell: first with an enzyme protein (aminoacyl-tRNA synthetase), carrying a specific activated amino acid, then, carrying a covalently attached amino acid, with another protein ( elongation factor EF-Tu), together with which it enters the ribosome, and then simultaneously with ribosomal RNA and mRNA in the ribosome. Although this path undoubtedly implements the functions of specific recognition of other macromolecules by tRNA molecules, for a long time it was tacitly accepted that the main role is played by the recognition of tRNA by proteins - enzymes, translation factors and ribosomal proteins, but not vice versa.

The English scientist E. Cundliffe was the first to clearly and definitely declare the ability of structured sections of ribosomal RNA to specifically recognize small ligands of a non-nucleic and non-protein nature. He presented experimental data in favor of selective interaction (binding) of sections of folded RNA, and not proteins, with a number of ribosomal antibiotics - thiostrepton, erythromycin, aminoglycosides (streptomycin, kanamycin, neomycin). Ten years later, other scientists presented direct structural evidence for the specific binding of aminoglycoside antibiotics to the small (16S) ribosomal RNA region (see also review).

The final recognition of RNA's ability to recognize a wide variety of molecules and very specifically interact with them came thanks to aptamers - small synthetic RNAs obtained by selecting from many variants of nucleotide sequences using the procedures of the so-called “cell-free evolution”, “evolution in vitro” [, ]. It turned out that it is possible to select and propagate RNAs that have the ability to selectively bind almost any type of molecule, ranging from low molecular weight organic compounds to various individual peptides and proteins (see reviews [, ]). In other words, RNA, like proteins, can indeed fully possess the function of specific molecular recognition.

Even before these studies, in the early 80s of the last century, in the laboratories of T. Check and S. Altman in the USA, a sensational discovery was made that brought about a revolution in biochemistry and molecular biology: it was shown that RNA can be a specific catalyst for biochemical reactions [ ,47]. Throughout the previous history of biochemistry, it has been argued for decades that biochemical catalysis is the “prerogative” exclusively of protein enzymes. Therefore, all theories of the origin of life were forced to proceed from the primacy of proteins as macromolecules, absolutely necessary for the emergence of biochemical metabolism (metabolism). The discovery of the catalytic function of RNA overturned all previous ideas about the exclusive role of proteins not only in the emergence of life, but also in understanding the phenomenon of life itself.

By analogy with enzyme proteins - enzymes - catalytic RNAs were named ribozymes. Apparently, almost all ribozymes that naturally exist in nature in the cells of modern organisms are in one way or another involved in processes associated with the transformations of the polynucleotide chains of RNA themselves. However, it turned out to be possible to create artificial ribozymes with a wider range of catalyzed reactions. In addition, as it turns out from the entire body of data on the structure of ribosomes and the features of the ribosome-catalyzed reaction of formation of peptide bonds in the process of protein biosynthesis, the catalytic center of this reaction (peptidyl-transferase center of the ribosome) is formed by a certain domain of large ribosomal RNA, without the fundamental participation of ribosomal proteins, then is of ribozyme nature [,50].

So, it was after the discovery of the catalytic function of RNA that the paradigm changed, and the eyes of biologists turned to RNA. In fact, RNA molecules are capable of doing everything that proteins do: folding into specific structures and determining the shape of biological particles, recognizing other macromolecules and small ligands with great accuracy and interacting with them, and finally, catalyzing covalent transformations of recognizable molecules. Of course, proteins do all this more efficiently and comprehensively than RNA. But proteins, in principle, “do not know how” to reproduce themselves - there are no protein mechanisms of their own to reproduce their structure, except through RNA. At the same time, RNA contains all the necessary structural prerequisites for accurately reproducing its own structure.

RNA is a close analogue of DNA, the modern “substance of heredity.” Structurally, nothing prevents it from forming double helices like DNA, with full compliance with the principle of complementarity due to the formation of G-C, C-G, A-U and U-A pairs between two polyribonucleotide chains. In this case, the reproduction (replication) of RNA on RNA seems to be a completely permitted process. Indeed, it is well known that RNA double helices exist in nature, and primarily as independent RNA genomes in some viruses (for example, reoviruses and rotaviruses of animals and humans). In general, genomes in the form of RNA, rather than DNA, are quite common among both animal and plant viruses, as well as bacterial viruses (bacteriophages), but in the vast majority of cases their genomic RNA is represented by a single RNA chain, on which it is built only after the virus enters the cell. complementary chain.

One way or another, virology has long proven the presence of the same genetic replicative functions in RNA as are characteristic of DNA in cellular organisms.

Apparently, modern cellular organisms could not exist without the genetic “single command” of DNA, and a strict ban was imposed on the independent reproduction of RNA by evolution, otherwise deregulation of gene activity in organisms would occur, a violation of the balance of products and processes in the cell controlled by genes and complete disorganization life. However, RNA to RNA replication can occur in normal cells in special cases. This is evidenced by the latest discoveries of new classes of small non-genetic RNAs in animal and plant cells - the so-called interfering RNAs (siRNA) and microRNA (miRNA) - with regulatory and antiviral activity: the functioning and reproduction of these RNAs requires their independent replication.

THE WORLD OF RNA - ANCIENT AND MODERN

Thus, RNA appears to be the most self-sufficient substance of living matter. It is fundamentally capable of performing all or almost all functions that are characteristic of proteins, including shape formation and biochemical catalysis, and at the same time can be a full-fledged genetic substance with its replicative and coding functions. Awareness of these facts led biologists, chemists and geologists to the hypothesis of an ancient “RNA world”, which evolutionarily preceded our current DNA-RNA-protein life (for more details, see). In the RNA world there were no proteins or DNA, but only ensembles of different RNA molecules performing various of the above functions. These were most likely cell-free systems. The formation of cellular structures certainly requires the participation of at least proteins and lipids that did not yet exist. Compartmentalization of RNA assemblies in the form of coacervate droplets was also unlikely due to the absence of polypeptides, polysaccharides and other polymers capable of coacervation. However, in order for each RNA ensemble to exist as a system, inherit acquired characteristics useful for the entire system, and evolve, its RNA replicases, ligand-binding RNAs, RNA synthetases and synthetic products must obviously be are then united in space. Therefore, in most theories of the origin of life, the emergence of limiting membranes or at least phase interfaces is postulated as a necessary condition for the beginning of evolution, including the evolution of RNA ensembles (for example, see).

However, an alternative is possible, and in my opinion, even more probable. About ten years ago at the Institute of Protein of the Russian Academy of Sciences, my student A.B. Chetverin and his colleagues experimentally demonstrated the ability of RNA molecules to form molecular colonies on gels or other solid media, if conditions for replication were provided on these media [,55] (Fig. 5). Mixed RNA colonies on solid or semi-solid surfaces could be the first evolving cell-free ensembles, where some molecules performed genetic functions (replication of RNA molecules of the entire ensemble), while others formed structures necessary for successful existence (for example, those that adsorbed the necessary substances from the environment ) or were ribozymes responsible for the synthesis and preparation of substrates for RNA synthesis. This cell-free situation created conditions for very rapid evolution: RNA colonies were not fenced off from the external environment and could easily exchange their molecules - their genetic material. The easy propagation of RNA molecules through environments, including the atmosphere, has also been demonstrated in direct experiments. Moreover, as recent experiments by the same group of researchers have shown, RNA molecules can spontaneously exchange pieces during collisions in an aqueous environment, that is, they have the ability to undergo non-enzymatic recombination.

Rice. 5. Colonies of replicating RNA molecules on an agarose gel [, ]
Left- RNA colonies grown on a closed Petri dish
for one hour at a temperature of 25°C.
Right- RNA colonies grown on an open Petri dish under the same conditions
(infection with RNA molecules from the air)

These are exactly the conditions that K. Vuz postulated for the emergence of the Universal Predecessor of living beings on Earth: a high level of mutations (replication errors) due to the primitiveness and imperfection of the mechanisms of replication of genetic material, the free exchange of genetic material between the predecessors of cells - “progenotes” - and the communal nature of existence these predecessors, when any products and innovations of some became the property of all (“from each according to his abilities - to each according to his needs”). However, in contrast to the hypothesis of K. Vuz, I would prefer to give the role of the Universal Precursor to the precellular - acellular - the form of existence of the RNA world, when there was no DNA or protein synthesis mechanisms yet. The Universal Predecessor could be just a communal community of colony-assembles of RNA, existing and reproducing on solid or gel-like surfaces of the primitive Earth, not physically limited by any membranes or phase divisions and therefore freely exchanging both genetic material and products of catalyzed reactions.

This communal form of existence of the RNA world - a kind of Solaris - as already indicated, had to evolve very quickly. In any case, the entire path of evolution to individual organisms with a cellular structure, DNA and a modern apparatus for protein synthesis was apparently completed in less than half a billion years (the period from 4 billion to 3.5 billion years ago). The improvement of RNA colony ensembles through natural selection should have occurred in the direction of both improving catalytic mechanisms and increasing the accuracy of replication and inheritance. RNA colonies that “learned” to make protein catalysts naturally acquired a huge advantage over others in the speed and quality of catalyzed reactions and therefore quickly replaced the “inept” ones - both through competition and through the transfer of this ability to them. On the basis of RNA, the protein synthesis apparatus appeared and improved, and due to the communal and pandemic nature of the RNA world, a universal genetic code was developed.

However, encoded protein synthesis required increased fidelity of replication of genetic material and sequencing of the production of different proteins. This led to the need to differentiate part of the RNA (genetic RNA) and modify it into DNA, which has the ability to copy more accurately, and also has significantly greater chemical stability than RNA. Finally, the efficiency and stability of such systems could be significantly increased due to their isolation from the environment, and they are surrounded by membranes of a protein-lipid nature. The communal world breaks up into individual, but highly effective cells - cells, individuals, organisms, and their own evolution and their own lineages begin. From the communal Universal Predecessor, two main branches of microorganisms emerge - bacteria (eubacteria) and archaea (archaebacteria), their cellular communities are formed based on the interaction of their metabolisms, and then their symbiotic relationships lead to the appearance of chimeras, and the first eukarya - the predecessors of higher eukaryotes - arise organisms.

What happened to the RNA world after the collapse of the commune? Although the commune fell apart, the RNA world persisted in every cell of every living organism. The basis of modern life is the inherited biosynthesis of proteins, which determines all the characteristics of currently existing living organisms. The central link in this process of protein biosynthesis is a set of RNA molecules of various types interacting with each other, primarily ribosomal RNA, which forms the protein synthesis apparatus, tRNA, which delivers activated amino acids to the ribosome for the construction of polypeptide chains of proteins, and mRNA, which carries in its nucleotide sequence program for protein synthesis (see, Fig. 1). In addition to these three main representatives of the intracellular RNA world, a number of minor RNAs have been discovered that provide the processes of DNA reduplication and inheritance, gene copying and formation.

synthesis of mRNA, regulation of protein synthesis, protein transport through membranes, regulation of embryogenesis and cell differentiation, determination of life span, and so on. Every year, more and more new types of minor RNAs are discovered in the cells of modern organisms, and their most important role in the life of organisms is revealed.

Until recently, we knew very little about the intracellular world of RNA, and there is now a major re-evaluation of the relative contribution of non-genetic RNA to the functioning of living systems. It can be said that the collection of RNA molecules - the RNA world - still constitutes the core of life. Modern life is RNA. transferring part of its genetic functions to the related polymer it generated - DNA, and synthesizing proteins for the comprehensive effective functioning of the compartments containing it - cells and multicellular organisms.

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Cis-acting signals and replication specificity. Viral RNA replication and packaging are remarkably specific processes. Both of these processes accurately select the correct viral molecules from among the thousands of ribonucleic acids contained in the cell. This is mainly due to the presence of cis-acting signals that selectively determine viral RNA replication and virion assembly, but in most RNA virus genomes these signals have not been clearly identified.

The signals that have been characterized do not include linear nucleotide sequences, but secondary structures in the form of loops, tRNA-like structures and pseudoknots that create specific three-dimensional molecular shapes that can interact only with viral enzymes and viral structural proteins. However, understanding of the molecular basis of the specificity of RNA replication and virion assembly is limited by a lack of knowledge of the three-dimensional structures of viral RNA and its cis-acting signals.

Structural and non-structural proteins of viruses. By definition, virus-specific structural proteins are included in viral particles, and non-structural proteins are found only in infected cells. However, viruses with negative-negative, ambipolar, and double-stranded RNA genomes include RdRp and associated enzymes in their progeny virions and therefore encode predominantly or exclusively structural proteins. In addition to the polymerase, virus-encoded enzymes often include one or more proteases, RNA helicase, guanylyl and methyltransferases, poly-A polymerase, sometimes a nuclease, and in the case of retroviruses, DNA integrase. At the same time, the participation of host cell enzymes in the replicative cycle has been established for several RNA viruses.

Proteases cleave the primary translation product of which they are part at highly defined sequences (sites). In some cells infected by picornaviruses, they also selectively inhibit host cell protein synthesis by proteolyzing the cellular cap-binding protein. Helicases are required by large RNA viruses to disrupt intramolecular base pairing during RNA synthesis, although some RdRps are able to unwind RNA duplexes without its assistance. Guanylyl and methyltransferases build a 5'-cap on mRNA in almost all eukaryotic RNA viruses, except for picornaviruses, whose RNA is not capped, and orthomyxo- and bunyaviruses, which steal the cap from cellular mRNAs through a cap-specific endonuclease. At the 3' end of the mRNA of most animal viruses there is a poly-A track, while plant RNA viruses usually have a tRNA-like structure. Polyadenylation usually occurs as a result of a side reaction (slipping) of the viral RdRp, and not as a result of the work of poly-A polymerase, as in poxviruses.

Host cell proteins. Host cell proteins can play a significant role in the replication of RNA viruses. It should be noted that in different viral systems different cellular proteins are involved in this process. The most striking example is the RNA replicase of bacteriophages Qb and MS2, in which, in addition to a single phage-specific polypeptide, four cellular subunits are required to ensure polymerase activity: ribosomal protein S1 E.coli, two translation elongation factors and an RNA-binding protein. In some eukaryotic viruses, host translation factors may also be involved in RNA replication. For example, in bromoviruses (plant viruses), the initiation factor subunit eIF-3 binds to RdRp and increases its activity. In infected cells, several other host proteins interact with the terminal nucleotide sequences of viral RNAs. Among them are poly-A- and polypyrimidine-binding proteins, carleticulin and proteins Ro and L, which interact with small nuclear RNAs. Although it should be noted that distinguishing random interactions from those that play functional roles is often difficult.

Host cell membranes. Unlike phage replicases, RdRp of eukaryotic viruses is invariably associated with supramolecular structures: host cell membranes in (+)RNA viruses, the nucleocapsid in (-)RNA viruses, and subviral particles in lncRNA viruses. The intracellular membranes of cells infected with viruses with a (+)RNA genome undergo rapid redistribution, forming anchoring sites for viral replication complexes. When these complexes become detached from membranes, they lose the ability to catalyze true RNA replication, although they often retain a limited ability to copy the RNA template. In a study of nodavirus infection, the true RNA replicase activity of partially purified RdRp was restored by adding glycerol phospholipids to a cell-free extract. These results supported the idea that membrane organization plays a central role in (+)RNA replication. The same conclusion was obtained when poliovirus RNA replication was inhibited by brefeldin A, which blocks intracellular membrane interactions. Although the specific role of membranes is unclear, it is likely that they can speed up the assembly of replication complexes by reducing process time and separating daughter molecules from templates.

Mechanisms of replication of RNA genomes. As already noted, replication of RNA genomes is carried out by a virus-specific RdRp, which can be part of the virion or determined by the genome. Unlike enzymes that copy DNA using a primer, most RdRps can begin RNA synthesis de novo. An exception is the RdRp of picornaviruses, which uses a small viral protein (VPg) covalently linked to uracil to initiate synthesis. VPg is removed during genome translation but retained during genome encapsidation.

Interestingly, in togaviruses (Sindbis virus), replication of (+)RNA at the stage of minus-strand synthesis (RF formation) is carried out only by a transitional version of RdRp, which is subsequently proteolytically processed, which switches the template specificity of RdRp to the synthesis of positive strands.

Replication of (+)RNA polioviruses .

Polioviruses are small (27 nm), non-enveloped, icosahedral viruses that infect vertebrates. The genome is a linear single-stranded RNA of positive polarity. At the 5" end, the RNA is covalently linked to the terminal genomic protein through a tyrosine residue; the 3" end is polyadenylated (Figure 8).

Replication/transcription of the genome is carried out by RNA polymerase, determined by the 3" end of the genome, which is translated immediately after the virus enters the cell. At the first stage of replication, a double-stranded RF is formed due to the synthesis of the minus strand, initiated by the attachment of a uracil molecule to the 3" poly- And the end.

Figure 8 – Scheme of poliovirus RNA replication

In addition to RNA polymerase, the cell synthesizes a virus-specific terminal low-molecular protein (VPg), which binds to the uracil molecule through tyrosine. This structure is used by RNA polymerase as a primer - that is, terminal initiation occurs using a nucleotide-protein primer. Synthesis occurs with chain displacement. The resulting (+)RNA molecules are used as mRNA before the accumulation of a sufficient amount of virus-specific proteins, after which they begin to be encapsidated into the viral particle.

It should be noted that the presented scheme of replication of the genomic (+)RNA of polioviruses is not universal. Viral (+)RNA genomes differ in the organization of 5" and 3" terminal structures, which determines the features of their replication associated with the initiation of synthesis.

Replication of (-)RNA genomes .

Viral (-)RNA genomes can be continuous or segmented. In all cases, RNA is part of a ribonucleoprotein, which determines the characteristics of its replication, since deproteinized RNA cannot serve as a template for polymerase. All viruses with a (-)RNA genome have their own RNA-dependent RNA polymerase, which is part of the RNP. To obtain a full-length genome, a replicative full-length plus strand must be synthesized. However, at the first stage of the reproductive cycle, genomic (-)RNA serves as a template for transcription, which proceeds with subsequent processing of mRNA, and cannot serve as a template for the synthesis of a full-length copy. The synthesis of replicative full-length (+)RNA begins only after the accumulation of the corresponding viral proteins that suppress premature termination of RNA in the internal regions of the matrix. How this happens remains unknown. The synthesis of antigenomic and genomic RNA occurs as part of RNP.

Replication of reovirus lncRNAs .

Reoviruses are bicapsid (60-75 nm) particles with icosahedral symmetry; they infect vertebrates, invertebrates, and plants. The genome consists of 10-12 lncRNA fragments.

LncRNA replication is inextricably linked with transcription, which is its first stage.

1 Synthesis of (+)RNA on a double-stranded template proceeds in a conservative manner without strand displacement and occurs as part of a single-capsid viral particle with the participation of core proteins - viral RNA-dependent RNA polymerase (VP2) and guanidyl transferase (VP3). The mRNA leaves the particle through the pores of the internal capsid.

2 Plus-strand RNA combines with newly synthesized core and nonstructural (NS) proteins. When the virion matures, RNA polymerase synthesizes minus strands on the (+)RNA template using a repair mechanism, drawing it inside the forming capsid. The formed single-capsid particle can again begin the synthesis of plus-strand RNA.

As in the case of polioviruses, the presented method of replication of the reovirus genome is not universal for viruses with a lncRNA genome. For example, in phage φb, the synthesis of plus strands on the parental duplex occurs according to a semi-conservative model and is always associated with the displacement of the preceding strand of (+)RNA.

During the replication process, DNA viruses carry out several steps that are absent in RNA genomic viruses. For most DNA viruses, genetic strategies include: transport of virion DNA into the cell nucleus, initiation of transcription from this DNA, induction of transcription of additional viral genes, preparation of the cell for viral DNA replication, duplication of the DNA genome, packaging of DNA into virions, and release of viral particles from kernels. In addition, many DNA viruses have developed unique mechanisms for evading the body's immune defense and the ability to cause tumors in animals. In the process of intimate relationships with their hosts, viruses exploit key cellular regulatory systems and usurp important cellular processes. In this regard, studying various aspects of DNA virus replication provides new fundamental knowledge about the molecular processes occurring in the cell, including gene expression, DNA replication and control of the cell division cycle.

Preparing cells for viral DNA replication. During a productive viral infection, many DNA viruses can produce 100,000 or more genome copies within a few days from a single genome molecule. This requires the work of many proteins, including DNA-binding proteins and polymerases, as well as an abundant supply of nucleotides. Replication of some DNA viruses occurs only in cells that naturally replicate their own DNA, thereby providing the necessary cellular environment for viral DNA replication. Other DNA viruses also rely heavily on cellular DNA replication systems, but these viruses encode proteins that stimulate the cell division cycle. Finally, some of the largest DNA viruses make limited use of the cellular replication apparatus because themselves encode viral versions of many of the essential proteins.

The first group of viruses includes the simplest DNA-containing viruses of the family Parvoviridae, which have a linear single-stranded genome. Parvoviruses can replicate their DNA and carry out a complete infectious cycle only in cells that are in the DNA replication stage - that is, in the S phase of the cell cycle.

In fact, viral gene expression is not activated until the cell enters S phase and the viral DNA genome is converted into double-stranded RF, which is the template for transcription. However, unlike other viruses, which require cells to actively copy their DNA, parvoviruses are unable to stimulate cells to enter S phase. In this regard, they can carry out successful reproduction only if they enter a cell that is already synthesizing DNA. Some parvoviruses, especially adena-associated virus (AAV), have even more stringent requirements and can replicate only in the presence of a helper, an adenovirus or herpes virus, whose gene products activate parvovirus gene expression and DNA replication.

Other DNA viruses, in order to create conditions for the replication of their DNA, stimulate cells to divide. For these viruses, viral DNA replication is the result of interactions between cellular replication proteins and viral proteins that are directly involved in replication, as well as initiator proteins that are localized at the origin of replication of the viral replication apparatus. These DNA viruses rewire the cell's replication apparatus for viral replication by engaging in protein-protein interactions with key cellular regulatory molecules, some of which act as chaperones, allowing them to stabilize protein complexes. Often, these interactions lead to the neutralization of cellular tumor repressor proteins such as the transcription factor p53 and members of the retinoblastoma protein (Rb) family and, as a result, activation of cell growth.

Viral proteins that stimulate the cell's replicative state typically inactivate the Rb family members P105Rb, p107, and p130. Inactivation of Rb prevents repression of cell division and permits E2F-mediated transcription, which stimulates the expression of numerous cellular proteins required for S phase, including DNA polymerase α, thymidine kinase, ribonucleotide reductase, and thymidylate synthase. Some viral proteins, such as E1A of adenoviruses and E7 of human papillomaviruses, directly bind Rb proteins and inhibit their function, and thus activate E2F. Other viral proteins regulate the activity of cyclin-dependent kinases (Cdks), which catalyze Rb phosphorylation, leading to E2F activation and transcription of E2F-regulated genes. A number of viral proteins can indirectly influence the regulation of the cell division cycle. For example, E1B-55KB and E4orf6 proteins of adenoviruses and E6 of papillomaviruses inhibit the action of the p53 transcription factor through interaction with CBP/p300, which is a coactivator of the p53 gene. Abolition of p53 function leads to reduced expression of the cell division inhibitor p21 (a repressor of the Cdk–cyclin complex), thereby activating Cdk and, accordingly, the transition of cells to S phase. Similarly, adenoviral E1A binds p27, which is a Cdk inhibitor, neutralizing its effects. The large T antigen of the simian virus SV40 not only binds and inactivates Rb and p53, but also performs several functions directly required for viral DNA replication. Another mechanism is used by the middle T-antigen of polyomaviruses and the E5 protein of bovine papillomaviruses. These proteins activate the growth factor receptor-mediated signaling cascade and possibly stimulate the expression of the Cdk regulatory subunit, cyclin D, thereby stimulating Cdk activity and phosphorylation of Rb family proteins. Some herpesvirus and hepadnavirus proteins also appear to stimulate signaling cascades by activating the intracellular signal transduction proteins NFKB, P21ras, and pp60c-src.

The induction of a set of cellular replication proteins has profound consequences on the host cell, which is forced into DNA replication. When the proliferative signal is persistently maintained, for example in nonpermissive cells that are unable to support viral DNA replication, the cells may undergo persistent transformation. Thus, not only do many DNA viruses stimulate static cells to undergo repeated division cycles, they also transform cells in culture and cause tumors in animals. The reported ability of many tumor-producing DNA viruses to stimulate unlimited cell growth is not a feature of normal viral replication, but rather represents an aberrant cellular response to viral infection. Accordingly, parvoviruses, which are unable to stimulate cellular DNA replication, are among the few DNA viruses that do not transform cells. However, the ability of viruses to stimulate cellular DNA synthesis does not always correlate with their ability to transform cells. For example, some herpes viruses stimulate DNA synthesis, others do not, and yet they actually inhibit rapid cell division. Such large viruses, with their large coding capacity, are able to create the proper environment for viral DNA replication without activating the cellular replication apparatus.

Requirement of nucleotides for DNA replication. As described above, parvoviruses require cells to be in S phase for replication, and papillomaviruses, polyomaviruses, and adenoviruses stimulate cells to enter S phase, which requires high concentrations of deoxynucleoside triphosphates (dNTPs) for DNA synthesis. Through their effects on members of the Rb and E2F protein families, papillomaviruses and adenoviruses stimulate the synthesis of the enzyme ribonucleotide reductase, which is required to maintain dNTP levels sufficient for viral replication. In contrast, herpes viruses and poxviruses are able to replicate in resting cells. One reason that these viruses can bypass the S-phase requirement is their ability to encode the enzymes for dNTP synthesis, ribonucleotide reductase and thymidine kinase. In the cases of herpes virus and herpes zoster/varicella zoster virus, viral thymidine kinase is a key target for antiviral chemotherapy because this viral enzyme phosphorylates nucleoside analogs such as acyclovir more efficiently than cellular enzymes do. Converted to the phosphate form, these dNTP analogues selectively harm the DNA replication of herpes viruses.

Regardless of the type of DNA genome, the unit of its replication is the so-called replicon – a unit of the genome capable of autonomous replication. A replicon is a nucleotide sequence located between the origin of replication (origin or ori) and the end of replication (terminus). The process of DNA replication is divided into three stages: chain initiation, chain elongation and termination of synthesis. Viruses with different types of DNA genomes implement original replication strategies. In this case, the main features are observed during the initiation of synthesis.

Basic principles of replication of DNA viral genomes.

Initiation of DNA synthesis. Most eukaryotic DNA viruses (except poxviruses) copy their genomes in the nucleus. Replication of viral DNA genomes is initiated at specific ori points.

Unlike cellular origins, which are activated once during the cell cycle, viral oris can fire many times during a single replication cycle. Initiation of DNA chain synthesis can only occur in the presence of a primer for DNA polymerase. The type of primer and the method of its formation differ among different viruses and determine the uniqueness of viral replication systems. There are three main ways to initiate DNA synthesis (see paragraph 3.7.1.1, p. 63).

Chain elongation during the replication of viral genomes is not fundamentally different from the process of cellular DNA synthesis. Enzymes, accessory proteins and replication proteins belonging to both the host cell and the virus are used. DNA synthesis is usually carried out by DNA-dependent DNA polymerase α. The main property of the synthesis is its polarity, in which the next nucleotide is added to the 3’ end of the growing chain. That is, the direction of synthesis goes from the 5’- to the 3’-end, reading - from the 3’- to the 5’-end. Features of the synthesis of complementary strands are associated with the method of initiation. On the dsDNA matrix, synthesis occurs through the formation of a replication fork (Figure 9) or with strand displacement; on the ssDNA matrix, through the repair mechanism.

In replication forks, one strand (the leading strand) is copied continuously in the direction from the 5'- to the 3'-end. Since the other strand (the lagging strand) must also be synthesized from the 5'- to the 3'-end, it is copied intermittently, repeatedly initiating synthesis and connecting short Okazaki fragments. DNA synthesis at the replication fork is ensured by a whole set of enzyme proteins, which can have different origins. Small DNA viruses use cellular replication proteins. The replication of the polyomavirus SV40 is the best studied, where the replication proteins involved have been identified in a cell-free system in vitro.

Figure 9 – Scheme of DNA replication using a replication fork

It has been established that 10 proteins are involved in SV40 DNA replication. Nine of them are of cellular origin: DNA polymerase α (responsible for initiation of DNA synthesis at the ori point and synthesis of the lagging strand); primase (associated with DNA polymerase and primes the synthesis of Okazaki fragments); DNA polymerase d (responsible for the synthesis of the leading strand and completion of the synthesis of Okazaki fragments); proliferative cell nuclear antigen (PCNA), which binds to DNA polymerase d and forms a ring around the DNA, increasing the processivity of the polymerase; heteropentameric replication factor C – RF-C (attaches the PCNA ring to DNA and stimulates polymerase d); RPA – ssDNA-binding protein; RNase H (removes all but one ribonucleotide of the RNA primer); exonuclease FEN-1, also known as MF-1 (removes remaining ribonucleotide); DNA ligase I (ligates Okazaki fragments); topoisomerase I and/or topoisomerase II (removes supercoiling during synthesis). The only viral protein required for SV40 DNA replication is large T antigen, which has helicase properties and ensures unwinding of the double-stranded structure at the replication fork.

Other viruses themselves provide almost all of the replication fork proteins. For example, the elongation phase of adenovirus DNA replication in vitro is mediated by one adenoviral subunit of DNA polymerase, an adenoviral single-stranded DNA-binding protein that can increase the processivity of the polymerase, and cellular topoisomerase I or II. This simplicity is due in part to the unusual nature of adenovirus DNA replication, in which there is no lagging strand synthesis.

Large DNA viruses provide themselves with replication enzymes to an even greater extent. For example, herpes viruses encode DNA polymerase, an elongation factor, a primase-helicase complex, a single-stranded DNA-binding protein, and probably a number of other viral proteins that have not been identified.

Termination of synthesis. In the case of circular genomes, the end of synthesis and divergence of genomes are simplified, since the synthesis of the daughter strand proceeds in a circle and at the end of a full turn at the ori point or during bidirectional replication in the middle of the ring, the 3' and 5' ends of the newly synthesized strand are aligned and ligated. Pairwise linked rings are separated by topoisomerase. In linear DNA synthesized using RNA primers, things are more complicated. Removal of the RNA primer produces a DNA molecule with an overhang at the 3' end and a gap at the 5' end. Two methods have been proposed for completing replication with the formation of a complete copy of the template strand: using concatamers or through the formation of a hairpin.

Basic replication schemes of DNA genomic viruses.

1 Terminal initiation using a self-priming mechanism.

2 Terminal initiation using a protein-nucleotide (B-N) primer.

3 Rolling ring mechanism.

4 Kearns scheme.

5 Replication through integration.

1 Replication using terminal initiation using a self-seeding mechanism (Figure 10). Parvoviruses have this type of genomic DNA replication, in which the genome is represented by linear ssDNA, which has self-complementary sequences at both ends that form hairpin structures. The 3' end of DNA has a unique sequence of 125 nucleotides in size, forming a double-stranded T-shaped hairpin structure, which acts as a primer for DNA polymerase.

DNA polymerase, as a result of the repair synthesis of the complementary chain, recreates a duplex, both chains of which are covalently connected at one end. In this case, the 3'-terminal segment of the parental genome is not used as a template. Consequently, complete reproduction of the viral genome has not yet occurred. In the next step, a virus-specific enzyme introduces a break in the parent strand at the boundary between the replicated and non-replicated sections of the sequence (between 125 and 126 nucleotides).

Figure 10 – Scheme of the first stages of single-stranded DNA replication of parvoviruses

The terminal 125 nucleotides of the parental genome become a conditional part of the newly synthesized chain, and the 3’ end of the parental chain thus formed is used for its regeneration. As a result of these reactions, a dispersed double-stranded replicative form of viral DNA appears (Figure 10). This is followed by a chain of reactions, including the formation of a DNA primer in the form of “rabbit ears” at one end, the synthesis of a new chain with the displacement of the parent, and the formation of another replicative form. The second replicative form of DNA is used as a template for further synthesis of viral DNA, and the single-stranded molecule displaced from the duplex either enters the replicative cycle or becomes part of the daughter viral particle.

2 Replication using terminal initiation using a protein nucleotide primer (Figure 11). This type of genomic DNA replication is observed in adenoviruses, the genome of which is represented by linear dsDNA, which has inverted repeats at the 5’ ends and covalently attached genomic proteins with mm. 55 kDa.

In a cell infected with an adenovirus, a virus-specific protein weighing 80 kDa is synthesized, which binds through serine to deoxycytidine. The resulting structure B-Ser – dCTP is a primer that, through cytosine, complementarily binds to the 3'-terminal guanosine of the genome and initiates the synthesis of the DNA chain.

Initiation can occur at either end of the parent DNA and can occur either simultaneously or sequentially. With sequential initiation, the synthesis of a daughter chain is accompanied by the displacement of one of the parent chains, and the synthesis of a complementary chain occurs on a single-stranded matrix using a repair mechanism. At the same time, another mechanism for the synthesis of the second strand is being discussed. The substituted parental single-stranded DNA has self-complementary inverted repeats at the ends, which anneal, restoring the double-stranded point ori, recognized by the initiation proteins that ensure the synthesis of the parent-daughter duplex. Thus, each parent duplex is copied semi-conservatively.

Figure 11 – Scheme of adenovirus genome replication

However, the process proceeds without the synthesis of the lagging chain, i.e. without the formation of multiple initiation sites and the synthesis of Okazaki fragments.

3 Replication of circular genomes using the rolling ring mechanism (Figure 12). Rolling ring is a mode of replication in which the replication fork makes many turns on a circular template. The strand synthesized in each cycle displaces the previous (homologous) chain of the double-stranded molecule synthesized in the previous cycle, forming a tail consisting of a set of sequences complementary to the single-stranded template ring. In general terms, rolling circle replication has the following stages:

Figure 12 – Scheme of DNA genome replication using the rolling ring mechanism

1 A virus-specific enzyme introduces a single-strand break at a unique site in the parental strand of the replicative form.

2 The enzyme remains bound to the 5'-end, the released 3'-terminal nucleotide serves as a primer for DNA polymerase.

3 DNA polymerase adds nucleotides complementary to the closed strand, that is, only the leading strand is synthesized. The 5' end of the parent strand is displaced. The formation of sigma molecules (δ) is observed.

4 After the replication fork has completed a little more than a full turn, the displaced strand closes into a ring, and the enzyme moves to the newly synthesized strand and the cycle repeats. Thus, the newly synthesized strand, which has a genomic sequence, becomes a component of the RF, and the previous (parental) one appears in a free form.

Viruses contain only one type of nucleic acid – DNA or RNA. Viral DNA can be single or double stranded and linear or circular in shape. Viral nucleic acids encode virus-specific proteins and enzymes necessary for virus replication in the host cell.

Replication of DNA viruses proceeds according to a semi-conservative mechanism common to all DNA. On the viral DNA matrix, mRNA is first synthesized, and then the formation of viral proteins occurs. This process is completely ensured by the metabolic apparatus of the host cell.

Replication of RNA viruses happens in two ways.

First occurs with the participation of RNA-dependent RNA polymerase (RNA synthase or RNA replicase). It is inherent in influenza and measles viruses. Viruses are distinguished:

• containing (+) - RNA strand (plus strand), which serves as both mRNA and genome, and viruses,
• containing a (-) RNA strand (minus strand), which serves only as a genome.

There are also viruses that contain double-stranded RNA.

1. The (+)-RNA strand of the virus can be directly used in translation as mRNA. Therefore, when a (+)-RNA virus (polymyolitis virus, hepatitis A virus) enters a cell, its RNA binds to the cell’s ribosomes and is translated into a protein chain. This chain is broken by the protease of the viral particle into 7 proteins, one of which is RNA synthase. After the appearance of RNA synthase, viral RNA replication begins. At the first stage, a (-) RNA chain is formed on the (+) chain as a template, and at the second stage, the (-) chain serves as a template for the synthesis of (+) RNA chains identical to the viral one.
2. Rhabdoviruses (rabies, Ebola, Marburg viruses) and paramyxoviruses (parainfluenza, measles, mumps viruses) have a (-)-strand of RNA, which cannot be directly translated into protein. Instead of translation, this (-)-RNA is used as a template for transcription of (+)-RNA. Transcription is carried out by RNA synthase, which is present in the viral particle. The synthesized viral (+)-RNA is further used as a template for the ribosomal synthesis of viral proteins and as a template for the synthesis (replication) of a (-)-RNA strand identical to the viral one.
3. (+-)-RNA (double-stranded RNA) is found in reoviruses that cause respiratory infections. The principle of reproduction of these viruses is the same as the replication of double-stranded DNA, but instead of DNA polymerase, RNA polymerase (RNA synthase) functions.

Second way occurs with the participation of reverse transcriptase (RNA-dependent DNA polymerase, reversease). It is inherent in retroviruses (immunodeficiency virus) and some oncogenic viruses. The enzyme catalyzes three processes in sequence:

• synthesis of (-) DNA chain on the viral (+) -RNA matrix;
• destruction of viral RNA as part of the resulting RNA-DNA hybrid;
• synthesis of (+) -strand DNA onto (-) -strand DNA to form double-stranded DNA.

This DNA from the cytoplasm enters the nucleus, integrates into the host genome and serves as a template for the synthesis of viral RNA with the participation of the host cell RNA polymerase system. The resulting viral RNAs enter the cytoplasm, where they initiate the translation of viral proteins. These proteins and RNA are assembled into viral particles that can infect new cells.

Virus replication 99

bacteria replicate using the theta mechanism. The rolling ring mechanism has been studied primarily on staphylococcal and streptococcal plasmids.

4.8 Virus replication

Virus replication occurs in several stages:

1. Adsorption: the virus contacts the cell with specific molecules on its surface: for example, orthomyxoviruses and paramyxoviruses are adsorbed using glycoproteins, and adenoviruses are adsorbed using penton fibers. Specific cellular receptors are involved in adsorption: glycoproteins, phospholipids or glycolipids.

Adsorption can be disrupted by antibodies binding to the viral envelope or the host cell itself.

2. Penetration immediately follows adsorption. After this, the viral particle can no longer be separated from the host cell without damaging it. Penetration mechanisms:

A. Direct penetration: The capsid remains associated with the outer surface of the cell membrane, and its contents are released into the cell.

b. Fusion with the membrane. V. Endocytosis.

3. Shell destruction occurs due to acidification of the endosome environment in which the viral particle is located to pH = 5. Proton pumps H+ -ATPase in endosome membranes are responsible for this. Low pH values ​​lead to a change in the conformation of the components of the viral shell, which, with their hydrophobic regions, begin to contact the endosome membranes, this leads to the entry of the virus into the cytosol.

4. Viral genome replication becomes possible due to the switching of cellular synthesis systems to viral replication and transcription. To do this, the virus stops protein synthesis by the cell and dissociates polyribosomes. Some viruses not only do not block cellular synthesis, but also accelerate it.

5. Virion assembly.

6. Release of virions from the cell.

A Genome replication DNA viruses

In animal DNA viruses, the processes of transcription and translation are not coupled (except for poxviruses): transcription occurs in the nucleus, and translation occurs in the cytoplasm. Viral DNA serves as a template for the synthesis of viral mRNA, which is a template for the synthesis of viral proteins. Viral DNA contains "early" and "late" genes that are transcribed at different times.

- “Early” genes encode proteins and enzymes necessary to begin replication of the viral genome.

- “Late” genes encode proteins involved in the maturation and assembly of viral particles.

Replication of dsDNA viruses similar to normal cellular DNA replication. The genome of most of these viruses enters the nucleus, where it is transcribed and replicated by cellular polymerases. This is how, for example, herpes viruses and papillomaviruses replicate. However there are two exceptions:

1. Each part of the poxvirus virion is synthesized and assembled in the cytoplasm. The nucleus does not participate in their replication.

2. The hepatitis B virus genome replicates differently: it is synthesized RNA is an intermediary, and then, during reverse transcription, DNA is synthesized into

RNA matrix.

Replication of viruses with ssDNA also occurs in the nucleus, where viral DNA penetrates after entering the cell. There, a second strand of DNA is synthesized, complementary to the viral one. Together they form dsDNA. Then everything happens according to the mechanism described above: protein synthesis, viral DNA replication and virion assembly.

Examples of replication in various virus families:

1. Adenoviruses replicate their genome asymmetrically: replication begins at the 3’ end of one of the strands using a protein primer. The growing daughter DNA strand displaces one mother strand and forms a full duplex with the other mother strand. The displaced strand also replicates and forms a duplex.

2. Herpesviruses have a linear genome with terminal repeats. After entering the nucleus, these repeats are partially cleaved off and joined together to form a circular DNA duplex. Next, replication occurs using the “rolling ring” mechanism. During the maturation of the viral particle, the circular DNA is cut and becomes linear again.

3. Papovaviruses have circular DNA, and its replication occurs by the theta mechanism (symmetrically and bidirectionally).

4. Parvoviruses have single-stranded DNA (positive or negative), so their replication begins when two strands (“+” and “–”) from different viral particles form a duplex DNA helix.

5. Poxviruses have unusual double-stranded DNA whose ends are linked. Their replicated DNA intermediates, found in the cytoplasm, are concatemers connected head-to-head or tail-to-tail.

6. Hepadnaviruses, such as the hepatitis B virus, use reverse transcription for replication. Their genome consists of partially double-stranded circular DNA with a complete negative strand and an incomplete positive strand. After entering the cell, the positive strand is completed and transcribed. RNA transcripts become the template for DNA synthesis during reverse transcription using viral enzymes.

Virus Replication 101

B Replication of the genome of RNA viruses

RNA viruses can be divided into 4 groups (see Fig. 71 ▼):

1. Positive single-stranded RNA (ssRNA+) viruses.

2. Retroviruses (ssRNA+ variety).

3. Negative-single-stranded RNA viruses(ssRNA–).

4. Double-stranded RNA (dsRNA) viruses.

Single-stranded RNA that can be a template in protein biosynthesis (i.e. act as mRNA) is called positive RNA or RNA+. Respectively, negative RNA or RNA– is not capable of serving as a template in protein synthesis.

Replication of ssRNA+ viruses . Once viral ssRNA+ enters the host cell, it is immediately translated into protein by ribosomes. It encodes capsid and viral proteins. RNA polymerase. Direct replication of the viral ssRNA+ occurs in two stages:

1. First, a complementary strand is synthesized on the positive viral ssRNA+ templatenegative RNA(ssRNA– ). This synthesis is carried out by the viral RNA polymerase.

2. This negative RNA is then transcribed and new molecules are formedpositive ssRNA+. They participate in the assembly of virio-

new This process is unique to viruses because no cell transcribes RNA from RNA.

An example of a ssRNA+ virus is poliovirus (poliomyelitis virus). Retrovirus replication. Retroviruses also contain ssRNA+. However, in

Unlike other similar viruses, they do not use it as mRNA. Retrovirus replication proceeds as follows:

1. Reverse transcriptase The virus, contained inside its capsid, synthesizes DNA on a ssRNA+ matrix.

2. This DNA then serves as a template in the synthesis of new ssRNA+, acting as mRNA and simultaneously forming new virions.

An example of a retrovirus is HIV.

Replication of viruses with ssRNA– . The RNA of these viruses cannot be translated directly into protein because it is not recognized by ribosomes. These viruses replicate using their own RNA-dependent RNA transcriptases(located inside the capsid and enters the cytoplasm along with the viral genome after penetration into the cell):

1. RNA transcriptase synthesizes ssRNA+ on a viral ssRNA– template.

2. The synthesized ssRNA+ acts as mRNA and serves as a template in the synthesis of new ssRNA– . The latter are included in the virions.

Examples of ssRNA viruses are influenza and rabies viruses. Replication of dsRNA viruses. The double-stranded RNA of these viruses consists of

RNA+ and RNA– chains. Their replication proceeds according to the following scenario:

1. After entering the cytoplasm, the viral RNA polymerase uses dsRNA to synthesize ssRNA+ (the negative strand of RNA serves as the template). The ssRNA+ chain acts as mRNA, i.e. translated by ribosomes to