The human immunodeficiency virus (HIV)

Acquired Immunodeficiency Syndrome was isolated as a special disease in 1981 in the United States, when in a number of young people severe diseases were caused by microorganisms that are non-pathogenic or slightly pathogenic for healthy people. Investigation of the immune status of patients revealed a sharp decrease in the number of lymphocytes in general and T-helpers in particular. This condition is called AIDS (English Acquired Immune Deficiency Syndrome - Acquired Immunodeficiency Syndrome, or AIDS). The method of infection (sexual contact, through the blood and its preparations) indicated the infectious nature of the disease.

The causative agent of AIDS was discovered in 1983 independently by the Frenchman L. Montagnier, who named him LAV Lymphoadenopathy Associated Virus), as he detected a patient with lymphadenopathy; and the American R. Gallo, who called the virus HTLV-III (English Human T-lymphotropic virus III): previously he was found lymphotropic viruses I and II.

Comparison of the properties of LAV and HTLV-III viruses showed their identity, therefore in 1986 the virus was called HIV (Human Immunodeficiency Virus, or HIV) in order to avoid confusion. HIV is spherical, its diameter is 110 nm. The envelope of the virus has the form of a polyhedron composed of 12 pentagons and 20 hexagons. The molecule of the glycosylated protein gpl20 is located in the center and corners of each hexagon (the number 120 means the molecular weight of the protein in kilodaltons). A total of 72 gpl20 molecules are located on the surface of the virion, each of which is associated with the intramembrane protein gp41. These proteins together with the double lipid layer form the supercapsid (membrane) of the virion.

Proteins gpl20 and gp41 are formed as a result of cell protease cutting of the precursor protein Env. Protein gp41 forms the "stud" of the spine, being linked by the cytoplasmic domain with the matrix protein p17MA immediately below the membrane. Molecules p17, interacting with the maturation of the virion, form an icosahedron underlying the shell.

In the central part of the virion, the p24 protein forms a conical capsid. The narrowed part of the capsid with the participation of the pb protein is connected with the envelope of the virion. Within the capsid, there are two identical molecules of viral genomic RNA. They are bound at their 5 'ends to the nucleocapsid protein p7NC. This protein is interesting because it has two amino acid residues (motifs) that are rich in cysteine and histidine and contain the Zn atom - they are called "zinc fingers", since they capture the molecules of genomic RNA for inclusion in the emerging virions. The capsid also contains three enzymes. Reversase (RT), or pol-complex, includes reverse transcriptase, RNA-ase H, and DNA-dependent DNA polymerase. The revertase is present as a p66 / p51 heterodimer. Protease (PR) - pI, starts and realizes the process of virion maturation. Integration (IN) - p31, or endonuclease, ensures the inclusion of proviral DNA in the genome of the host cell. The capsid also contains a seed RNA molecule (tRNAl "3).

The RNA gene in the cell is converted by reverse transcriptase into a DNA genome (DNA-provirus) consisting of 9283 nucleotide pairs. It is limited to the left and right by the so-called long end repeats, or LTR (English long terminal repeat): S'-LTR - left and Z'-LTR - on the right. LTR contain 638 nucleotide pairs.

The HIV genome consists of 9 genes, some of which are overlapped (has several reading frames) and has an exonine structure. They control the synthesis of 9 structural and 6 regulatory proteins.

The LTR value for the viral genome is that they contain the following regulatory elements that control its functioning:

• transcriptional signal (promoter region);
• the signal for addition of poly-A;
• Capture signal;
• signal integration;
• a positive regulatory signal (TAR for TAT protein);
• element of negative regulation (NRE for NEF protein);
• the attachment site of the seed RNA (tRNA ™ 3) for the synthesis of the minus chain of DNA at the 3 'end; signal at the 5'-end of the LTR, which serves as a primer for the synthesis of the plus-strand of DNA.

In addition, the LTR contains elements involved in the regulation of mRNA splicing, packing the vRNA molecules into the capsid (Psi element). Finally, when transcribing the genome in long mRNAs, two signals are generated for the REV protein, which switch protein synthesis: CAR for regulatory proteins and CRS for structural proteins. If REV protein binds to CAR, structural proteins are synthesized; if it is absent, only regulatory proteins are synthesized.

In the regulation of the genome of the virus, the following regulating genes and their proteins play a particularly important role:

• a TAT protein that performs a positive control of the reproduction of the virus and acts through a regulatory TAR site;
• proteins NEV and VPU, carrying out negative control of reproduction through the NRE site;
• protein REV, carrying out a positive-negative control. Protein REV controls the work of genes gag, pol, env and performs a negative regulation of splicing.

Thus, the reproduction of HIV is under a triple control - positive, negative and positive-negative.

The VIF protein determines the infectivity of the newly synthesized virus. It is bound to the capsid protein p24 and is present in the virion in an amount of 60 molecules. The NEF protein is represented in the virion by a small number of molecules (5-10), possibly connected with the envelope.

VPR protein inhibits the cell cycle in G2 phase, participates in the transport of preintegration complexes into the nucleus of the cell, activates some viral and cellular genes, increases the efficiency of viral replication in monocytes and macrophages. The location of proteins VPR, TAT, REV, VPU in the virion is not established.

In addition to its own proteins, the composition of the virion membrane may include some proteins of the host cell. Proteins VPU and VPR are involved in the regulation of virus reproduction.

Antigenic variants of the human immunodeficiency virus (HIV)

The human immunodeficiency virus (HIV) is very variable. Even from the organism of one patient, strains of the virus that differ significantly in antigenic properties can be isolated. Such variability is promoted by intensive destruction of CD4 + cells and a powerful antibody response to HIV infection. Patients from West Africa have a new form of HIV, biologically close to HIV-1, but immunologically distinct from it, HIV-2. The homology of the primary structure of the genomes of these viruses is 42%. DNA-provirus HIV-2 contains 9671 bp, and its LTR - 854 bp. HIV-2 was subsequently isolated in other regions of the world. There is no cross immunity between HIV-1 and HIV-2. Two large forms of HIV-1 are known: O (Outlier) and M (Major), the latter are divided into 10 subtypes (AJ). In Russia, 8 subtypes circulate (AH).

[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]

The mechanism of interaction of HIV with the cell

Having penetrated the body, the virus first attacks cells containing a specific CD4 receptor. This receptor has a large number of T-helpers, in less - macrophages and monocytes, especially the virus-sensitive T-helpers.

The human immunodeficiency virus (HIV) recognizes CD4 receptors with its gpl20 protein. The process of interaction of HIV with the cell proceeds according to the following scheme: receptor-mediated adsorption -> bordered fovea -> bordered vesicle -> lysosome. In it, the virion membrane merges with the lysosome membrane, and the nucleocapsid, freed from the supercapsid, enters the cytoplasm; on the way to the nucleus it is destroyed, and genomic RNA and associated core components are released. Then the reverse transcriptase synthesizes the minus chain of DNA on the virion RNA, then RNA-ase H destroys the virion RNA, and the viral DNA polymerase synthesizes the plus-strand of the DNA. At the ends of the DNA-provirus, 5'-LTR and 3'-LTR are formed. DNA-provirus can be in the nucleus for a while in an inactive form, but sooner or later it integrates with the help of its integrase into the chromosome of the target cell. In it, the provirus is in an inactive state until this T-lymphocyte is activated by microbial antigens or other immunocompetent cells. Activation of transcription of cellular DNA is regulated by a special nuclear factor (NF-kB). It is a DNA-binding protein and is produced in large amounts during the activation and proliferation of T-lymphocytes and monocytes. This protein binds to specific sequences of cellular DNA and similar sequences of LTR DNA-provirus and induces transcription of both cellular DNA and DNA-provirus. By inducing transcription of the DNA-provirus, he transitions the virus from an inactive state into an active and, accordingly, a persistent infection, into a productive one. Stay provirus in the inactive state can last a very long time. Activation of the virus is a critical moment in its interaction with the cell.

From the moment the virus enters the cell, a period of HIV infection begins , a virus that can last 10 years or more; and since the activation of the virus begins the disease - AIDS. With the help of their regulatory genes and their products, the virus begins to multiply actively. TAT-protein can increase the rate of reproduction of the virus 1000 times. Transcription of the virus is complex. It includes the formation of both full-length and subgenomic mRNAs, splicing of mRNA, and further synthesis of structural and regulatory proteins.

Synthesis of structural proteins occurs as follows. First, the polyprotein precursor Pr55Gag is synthesized (protein with a mass of 55 kD). It contains 4 main domains: matrix (MA), capsid (CA), nucleocapsid (NC) and domain pb, from which, as a result of Pr55Gag slicing with a viral protease (it self-cut from another precursor protein - Gag-Pol), respectively, structural proteins p17 , p24, p7 and pb. The formation of the polyprotein Pr55Gag is the main condition for the formation of viral particles. It is this protein that determines the program of morphogenesis of the virion. It involves successively the steps of transporting the Gag polyprotein to the plasma membrane, interacting with it and protein-protein interactions during the formation of the viral particle and its budding. Pr55Gag is synthesized on free polyribosomes; the protein molecules are transported to the membrane on which they anchor themselves with their hydrophobic patches. The main role in creating a native conformation of the Gag protein is played by the CA domain. The NC domain ensures the inclusion (with the help of its "zinc fingers") of 2 molecules of genomic RNA in the composition of the emerging viral particle. The polyprotein molecule is first dimerized due to the interaction of matrix domains. The dimers are then combined into hexameric (from 6 units) complexes as a result of the interaction of the CA and NC domains. Finally, hexamers, connecting with the lateral surfaces, form immature spherically shaped virions, inside which there is a genomic viral RNA captured by the NC domain.

Another protein precursor, Prl60Gag-Pol (a protein with a mass of 160 kD) is synthesized by shifting the reading frame of the ribosome during translation of the 3 'end of the gag gene in the region immediately before the site encoding the pb protein. This Gag-Pol polyprotein contains an incomplete sequence of Gag protein (1 - 423 amino acids) and Pol sequences, which include the PR, RT and IN domains. Molecules of the polyprotein Gag-Pol are also synthesized on free polyribosomes and transported to the plasma membrane. The polyprotein Prl60Gagpol contains all the sites of intermolecular interactions inherent in the polyprotein Gag and membrane binding sites. Therefore, the Gag-Pol polyprotein molecules merge with the membrane and, along with the Gag molecules, are incorporated into the developing virions, resulting in an active protease and the virion ripening process begins. The HIV-1 protease is highly active only in the form of a dimer, therefore, for its self-excision from Prl60Gag-Pol, dimerization of these molecules is required. Maturation of the virion is that the released active protease cuts prl60Gag-Pol and Gag55 into recognizable sites; proteins p17, p24, p7, p6, revertase, integrase are formed and their association in the viral structure takes place.

The Env protein is synthesized on ribosomes bound to the membranes of the endoplasmic reticulum, then it is glycosylated, cut by the cellular protease onto gp120 and gp41 and transported to the cell surface. In this case, gp41 permeates the membrane and binds to the matrix domains of the Gag protein molecule associated with the inner surface of the membrane. This relationship persists in the mature virion.

Thus, the assembly of viral particles is the aggregation of progenitor proteins and associated RNA molecules on the plasma membrane of the host cell, the formation of immature virions and their release by budding from the cell surface. When budding, the virion surrounds itself with a cell membrane, in which gp41 and gp120 molecules are embedded. During budding, or possibly after the liberation of the virions, their maturation takes place, which is carried out with the help of a viral protease and consists in proteolytic cutting of the precursor proteins Pr55Gag and Prl60Gag-Pol into mature virus proteins and their association into specific structural complexes. The leading role in the processes of virus morphogenesis is played by the polyprotein precursor Pr55Gag, which organizes and assembles an immature virion; The process of its maturation is completed by a specific viral protease.

Causes of immunodeficiency

One of the main causes of immunodeficiency in HIV infection is the mass death of T-helpers. It occurs because of the following events. First, T-helper viruses infected by the virus die due to apoptosis. It is believed that in AIDS patients, viral replication, apoptosis and a decrease in the number of T-helpers are related. Secondly, T-killers recognize and destroy T cells infected with the virus or carrying adsorbed gpl20 molecules, as well as virus-infected and non-infected T helper cells, which form symplasts (syncytium) consisting of several dozen cells (part of they die as a result of the multiplication of viruses in them). Due to the destruction of a large number of T-helpers, the expression of membrane receptors in B-lymphocytes is reduced to interleukin-2, the synthesis of various interleukins (growth and differentiation factors of B-lymphocytes - IL-4, IL-5, IL-6, etc.) as a result of which the function of the T-killer system is violated. Suppression of the activity of complement and macrophage systems occurs. Virus-infected macrophages and monocytes do not die for a long time, but they are not able to remove the virus from the body. Finally, due to the structural and antigenic similarity of gpl20 to the receptors of certain epithelial cells of the organism (including the receptors of trophoblasts mediating transplantation of HIV transmission), antiserum antibodies with a wide spectrum of action are synthesized. Such antibodies can block various cellular receptors and complicate the course of the disease with autoimmune disorders. The consequence of HIV infection is the defeat of all major parts of the immune system. Such patients become defenseless against a wide variety of microorganisms. This leads to the development of opportunistic infections and neoplastic diseases. For patients with HIV infection, at least three types of cancer have been at increased risk: Kaposi's sarcoma; carcinomas (including skin cancer); B-cell lymphoma arising from malignant degeneration of B-lymphocytes. However, HIV has not only lymphocyte, but also neurotropic. It penetrates into the cells of the central nervous system (astrocytes) both through receptor-mediated endocytosis and in phagocytosis by astrocytes of virus-infected lymphoblasts. When the virus interacts with astrocytes, symplasts are also formed, which facilitate the spread of the pathogen through the intercellular channels. In macrophages and monocytes, the virus can persist for a long time, so they serve as a reservoir and spread it in the body, being able to penetrate all tissues. Infected macrophages have a major role in the migration of HIV in the central nervous system and its defeat. In 10% of patients, primary clinical syndromes are associated with CNS damage and manifest as dementia (dementia). Thus, for people affected by HIV infection, there are 3 groups of diseases - opportunistic infections, tumor diseases and CNS damage.

[11], [12]

Epidemiology of HIV infection

The source of HIV infection is only a person - a sick or a virus carrier. The human immunodeficiency virus (HIV) is found in the blood, semen, cervical fluid; in nursing mothers - in breast milk. Infection occurs sexually, through the blood and its drugs, as well as from mother to child before delivery, during and after childbirth. Cases of infection by the virus through food, beverages and through insect bites are not known.

Drug addiction contributes to the spread of AIDS. HIV infection is increasing every year. According to WHO, from 1980 to 2000, 58 million people were infected with HIV. Only in 2000, 5.3 million people were infected in the world, and 3 million people died of AIDS. In Russia, as of January 1, 2004, there were 264,000 HIV-positive people registered. Half of those infected with HIV die within 11-12 years of infection. In early 2004, out of every 100,000 Russian citizens, about 180 lived with the diagnosis of "HIV infection." It is projected that at this level of incidence, the total number of HIV-infected people in Russia by 2012 will be 2.5-3 million people. The complexity of the fight against HIV infection depends on a number of reasons: first, there are no effective methods of its treatment and specific prevention; Secondly, the incubation period for HIV infection can exceed 10 years. Its duration depends on the moment of activation of the T-lymphocyte and the DNA-provirus contained in its chromosome. It is not clear yet whether each virus infected with AIDS is doomed or is likely to have a long-term virus without disease (which seems unlikely). Finally, there are several human immunodeficiency viruses (HIV-1, HIV-2), antigenic differences between which prevent the formation of cross immunity. Detection of the immunodeficiency virus of monkeys (SIV) shed light on the origin of HIV. The SIO for organizing the genome is similar to HIV, but it differs significantly in the nucleotide sequence. HIV-2 serologically occupies an intermediate position between HIV-1 and SIV, and the nucleotide sequence was closer to SIV. In this regard, VM Zhdanov suggested that the viruses HIV-1, HIV-2 and SIV originated from a common ancestor. It is possible, according to R. Gallo, that one of the SIVs somehow got into the human body, where he underwent a number of mutations, which resulted in HIV-1, HIV-2 and other forms of it.

[13], [14],

Symptoms of HIV infection

The virus of human immunodeficiency is characterized by certain features, on which the pathogenesis of the disease largely depends. The virus has a very high rate of reproduction, determined by its regulatory elements (5,000 virions are synthesized within 5 minutes in the active stage). Due to the presence of the fusion protein (gp41), the virus induces the formation of extensive syncytial structures due to the fusion of infected and uninfected T-helpers, which results in their mass death. Large-molecule gpl20 molecules freely circulate in the blood and bind to receptors of uninfected T-helpers, as a result of which they are also recognized and destroyed by T-killers. The virus can spread through the intercellular channels from the cell to the cell, in this case it becomes less readily available to the antibodies.

Clinical criteria for HIV infection

In adults, HIV infection is established if they have at least two serious symptoms combined with at least one minor symptom and in the absence of other known causes of immunodeficiency (cancer, congenital immunodeficiency, severe form of starvation, etc.). Serious symptoms include:

• weight loss by 10% or more;
• prolonged fever, intermittent or persistent;
• chronic diarrhea.

Minor symptoms: persistent cough, generalized dermatitis, recurrent herpes zoster, candidiasis of the oral cavity and pharynx, chronic herpes simplex, generalized lymphadenopathy. The diagnosis of AIDS is made with the presence of only Kaposi's sarcoma, cryptococcal meningitis, pneumocystis pneumonia. The clinical picture of the disease is influenced by an opportunistic infection.

[15], [16], [17], [18],

Methods of cultivation of the human immunodeficiency virus (HIV)

HIV-1 and HIV-2 can be cultured in the cells of only one clone of TCB4-lymphocytes - H9, obtained from leukemic TCV4-lymphocytes. For the same purposes, monolayer cultures of astrocyte cells can also be used, in which HIV-1 multiplies well. From animals to HIV-1 susceptible chimpanzees.

The resistance of the virus in the external environment is low. He dies under the influence of sunlight and UV irradiation, is destroyed at 80 ° C for 30 minutes, when treated with commonly used disinfectants - for 20-30 minutes. To disinfect the virus-containing material, it is necessary to use mycobactericidal disinfectants, since they are effective against microorganisms with the highest resistance.

Laboratory diagnosis of HIV infection

The main way to diagnose virus and HIV infection is the enzyme immunoassay. However, in view of the fact that gpl20 has a structural and antigenic similarity to the receptors of some human cells, including receptors that transport immunoglobulins through mucosal epithelial cells, antibodies related to antibodies against gpl20 may appear in the body. In this case, there may be false positive results of the IFM. Therefore, all positively reacting sera of the studied are subjected to additional analysis by the immunoblot method, or western blotting. This method is based on the identification of the antibodies to be studied after electrophoretic separation and subsequent testing with labeled anti-virus antibodies. The virological method is of little use because of the complexity of the culture of the virus. A clone of H9 lymphocytes is used to obtain viral antigens - the necessary components of diagnostic test systems. The CDR method makes it possible to detect the virus already at an early stage of viremia.

Treatment of HIV infection

It is necessary to find or synthesize drugs that effectively inhibit the activity of reverse transcriptase (revertase) or viral protease. They would prevent the formation of DNA-provirus and (or) inhibit the intracellular multiplication of the virus. The current strategy for the treatment of HIV-infected people is based on the principle of the combined use of drugs that inhibit the viral protease (one of the drugs) and the reversion (2 different drugs), combined (triple) therapy. In Russia, the use of two home drugs: phosphazide and crixivan, specifically inhibiting HIV reproduction in early and late stages of reproduction, especially with reduced azidothymidine activity, is recommended for the treatment of HIV-infected people.

The problem of specific prevention is the need to create a vaccine that would ensure the formation of effective cell-mediated immunity based on virus-specific cytotoxic lymphocytes without any significant production of antibodies. Such immunity is provided by Thl-helpers. It is possible that antibodies, including virus neutralizing, are not only ineffective in suppressing HIV infection, but at a high level they suppress cell-mediated immunity. Therefore, the anti-HIV vaccine should primarily meet two basic requirements: a) be absolutely safe and b) stimulate the activity of T-cytotoxic lymphocytes. The effectiveness of various variants of vaccines obtained from killed (inactivated) viruses and from individual antigens with high protective properties is studied. Such antigens can either be isolated from the virions themselves, or synthesized chemically. A vaccine based on genetic engineering methods is proposed. It is a recombinant vaccinia virus that carries HIV genes responsible for the synthesis of antigens with strong immunogenic properties. The decision on the effectiveness of these vaccines takes considerable time because of the long duration of the incubation period of HIV infection and the high variability of the pathogen. Creating a highly effective vaccine against HIV is an urgent fundamental problem.