[The maturation steps of human immunodeficiency virus and the role of proteolysis].

HIV-1 virions are as immature noninfectious particles lacking a central core. Shortly after budding, virions temporally mature and acquire cores and infectious activity. The cause of maturation remains poorly studied. We have revealed that the virions produced early after infection following 24-36 hours, never mature and remain noninfectious, and only virions produced 48-72 hours after infection mature. The mature virions contain 3 times more genomic viral RNA than "early" virus. The "early" virions contain the same proteolytically cleaved Gag proteins as mature virions in contrast to the accepted version. The virus protease inhibitor Indinavir sulfate (IS) fully blocks infectivity when added early after infection. The early proteolysis of Gag precursor in the infected cells and inclusion into the virions of cellularly cleaved matrix protein (cMA) are shown in the IS-treated cells. cMA is associated with genomic viral RNA.

[Methods for diagnosis of prion diseases].

The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom, its related occurrence of a new type of Creutzfeld-Jacob disease and proven cases of this type of the disease transmitted by blood transfusion initiated intensive studies to develop a inexpensive, prompt, and sensitive method for the early lifetime diagnosis of prion diseases. This would permit initiation of the timely treatment of the patients and prevention of contamination of foodstuffs. However, despite significant progress made in this direction, this objective has not yet been achieved. The present review highlights the currently available methods for the diagnosis of transmissible spongiform encephalopathies, as well as the latest developments in the ultrasensitive detection of these diseases, which is based on the misfolded prion protein complex.

Isolation and characterization of full-length recombinant cattle PrPC protein.

Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.

[Prion diseases of man and animal]. / Prionnye bolezni cheloveka i zhivotnykh.

Summarized in the paper are data on the molecular bases of transmissible spongoid encephalopathy (TSE), its etiology, formation mechanism of prion proteins, STE-related strains, specific barriers to TSE, its pathogenesis and treatment.

[Physico-chemical properties of liposome forms of L-3,4-dihydroxyphenylalanine (DOPA) and dopamine]. / Fiziko-khimicheskie svoistva liposomnykh form L-3,4-digudroksifenilalanina (DOPA) i dofamina.

L-3,4-Dihydroxyphenylalanine (DOPA) and dopamine encapsulated in liposomes have been obtained. These preparations can be used as drugs for Parkinson's disease. The level of encapsulation of the compounds under study in liposomes and their partition coefficients have been determined. The kinetics of DOPA (or dopamine) exit from the liposomes have been studied.

[Expression of human immunodeficiency virus gag antigens and formation of virus-like particles in a cell culture, infected with recombinant vaccinia virus strains (an electron-microscopic study)]. / Ekspressiia gag-antigenov virusa immunodefitsita cheloveka i obrazovanie virusopodobnykh chastits v kul'ture kletok, infitsirovannykh rekombinantnymi shtammami virusa ospovaktsiny (élektronno-mikroskopicheskoe issldovanie).

The synthesis of gag antigens of human immunodeficiency virus (HIV) by recombinant vaccinia virus strains containing the expressed genes gag and gag-pol and the capacity of these proteins to formation of virus-like particles during infection of various cell cultures were studied. The recombinant strain containing the truncated gag gene (p48gag) was shown to effectively synthesize gag polypeptides and to form immature virus-like particles during infection of all the cell cultures tested (CV-1, Hep-2, HT-29). The morphogenesis of mature virus-like particles was detected by electron microscopy only in infection of Hep-2 cells with a strain containing a complete gag-pol sequence of HIV.

[Biochemical features of protein matrix M1 of the influenza C virus]. / Biokhimicheskie osobennosti belkovogo matriksa M1 virusa grippa C.

Influenza viruses A, B, and C belonging to Orthomyxoviridae comprise an internal ribonucleoprotein (RNP) and an outer lipoprotein envelope with surface spike glycoproteins and the M1 protein matrix. The lipoprotein envelope and spike glycoproteins are solubilized by nonionic detergent treatment in a pH-independent manner. In contrast, disassembly of the M1 protein matrix appears to depend on pH. Treatment of influenza C viruses with nonionic detergent in neutral or alkaline medium (pH 9.0-7.2) results in disintegration of the virion M1 matrix and leads to a significant release of RNP free of the M1 protein. In acidic medium (pH 6.0-5.0) the M1 matrix fails to be removed and the viral core-like complex of RNP along with the M1 matrix cover is released. Since influenza A and B viruses were characterised by acid-dependent disassembly of the virion M1 matrix, influenza C viruses seem to be more resemble the paramyxoviruses, which also show a neutral-alkaline pH dependence of the matrix disintegration. These observations suggest that uncoating of influenza C viruses and paramyxoviruses in target cells may have similar events.

HIV-1 gag proteins in virions and in infected cell fractions.

The relation of the initial products of the HIV-1 gag gene to the final products was determined in virus samples and cell fractions of infected H9 and Jurkat-tat cell cultures. The proteins were identified by immunoblotting with pooled sera from AIDS patients or monoclonal antibodies. The proportion in the virions of gag precursor proteins and the products of their proteolytic cleavage varied according to the maturity of the virus particles as determined by electron microscopy. The distribution of viral gag proteins in the cell fractions was determined 2, 4, and 24 h after infection. Treatment of cells with cycloheximide to block de novo protein synthesis did not significantly affect the results. Gag proteins containing the N terminus of the precursor p55 (including p55, the intermediate precursors p41(45) and p39, and mature protein p17) were found in the cell nuclei up to 24 h after infection. The major core protein p24 was located in the cytoplasmic fraction. These data strongly suggest that gag precursors from the p55 N terminus and the matrix protein p17 enter the infected cell separately from the major core protein p24, or become separated from it in the cytoplasm.

[Formation of virus-like particles by HIV-1 Gag proteins, expressed by a recombinant vaccinia virus]. / Obrazovanie virusopodobnykh chastits belkov Gag VICh-1, ékspressiruemym rekombinantnym virusom ospovaktsiny.

Monkey kidney cells CV-1 were infected with recombinant vaccinia virus carrying HIV-1 gag gene with a deletion of 230 nucleotide pairs from the 3'-terminus. The main gene product detected in the lysates of infected cells was the gag precursor rp50. The protein was accumulated on the cell membranes suggesting that it had a myristylated N-terminus, and was cleaved by a recombinant virus specific protease with the formation of two proteins, p17 and p24 corresponding in molecular masses to mature gag proteins. Virus-like particles similar to immature HIV virions were budding from the surface of infected cells. They look like the ring of optically dense material covered with a lipid bilayer, of the same size (100-120 nm) and of the same density in a sucrose gradient (1.16-1.18 g/ml) as HIV-1 virions. The particles contained rp50 and cellular heterogeneous RNA. Thus, the unprocessed gag precursor with deleted 77 amino acid residues from the C-terminus is able to form virus-like particles in the absence of env proteins and virus-specific RNA, and these particles are budding from the cell surface. The question about the use of extracellular Gag-particles for AIDS diagnostic work and construction of vaccines is discussed.

[Study of influenza virus hemagglutinin H3 avidity using monoclonal antibodies]. / Izuchenie avidnosti gemaggliutinina H3 virusa grippa s pomoshch'iu monoklonal'nykh antitel.

The results of a comparative study of the features of antigenic determinants of avid and non-avid variants of influenza virus hemagglutinin H3 and of the influence of the mode of antigen presentation on the degree of its avidity are presented. The avidity of influenza virus hemagglutinin was shown to be determined by the capacity of individual antigenic determinants to interaction with antibodies. The antigenic determinants of avid hemagglutinin possessing a high functional activity in interaction with antibodies may have the spatial configuration which does not change in different modes of the antigen presentation. Isolation of hemagglutinin from virions of non-avid virus variants may lead to increased functional activity of individual antigenic determinants (and the antigen molecule as a whole) probably due to an increased degree of exposure and/or complementarity of the determinants for active centres of antibody.

[The detection of viral antigens and ribosomal RNA in cells by using low-temperature media]. / Vyiavlenie virusnykh antigenov i ribosomal'noi RNK v kletkakh s ispol'zovaniem nizkotemperaturnykh sred.

A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl-embedded HIV-infected cells. A genomic probe containing ribosomal sequences and labeled with biotin was used to hybridize rRNA molecules in sections of animal cells embedded in Lowicryl. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression and viral proteins at the ultrastructural level.

[The use of colloidal gold-labelled antibodies for demonstrating viral antigens]. / Primenenie mechennykh kolloidnym zolotom antitel dlia indikatsii virusnykh antigenov.

The principal possibility of using antibodies conjugated with colloid gold for better visualization of viruses, viral antigens, and their immune complexes in electron microscope was studied on the experimental model of HBs-antigen and Venezuelan equine encephalomyelitis virus. The same method was used for the study of the thin structure of influenza A (H1N1) virus hemagglutinins using monoreceptor antisera.

[Isolation of polymeric glycoprotein complexes of enveloped viruses using the Quill A glycoside and a study of their immunogenic activity]. / Poluchenie mul'timernykh kompleksov glikoproteidov obolochechnykh virusov s glikozidom Kvil A i izuchenie ikh immunogennoi aktivnosti.

Specific multimer complexes of glycoproteins of enveloped viruses were prepared by treatment of suspensions of purified concentrated virus with a non-ionic detergent MECK mixed with 0.2% glycoside Quil A. Mixed complexes of glycoproteins with glycoside Quil A were formed upon removal of the detergent from the mixture of solubilized glycoproteins and glycoside Quil A. According to the results of electron microscopy, the formed complexes differed morphologically from the conventional micelles of glycoproteins and presented spherical reticular structures 20-30 nm in diameter. The immunogenic potency of the complexes was much higher than that of virus particles and micelles of purified glycoproteins and was comparable to the immunogenic potency of glycoproteins mixed with complete Freund adjuvant. The protective activity of the complex of protein G of rabies virus with glycoside Quil A was higher than that of the subunit rabies vaccine adsorbed on aluminium hydroxide.

[Characteristics of different recombinants of the Philippine variety of influenza virus H3N2]. / Kharakteristika razlichnykh rekombinantov filippinskoi raznovidnosti virusa grippa H3N2.

The paper presents the results of generating influenza virus recombinants by hybridization of the laboratory A/PR8/34 strain with epidemic A/Philippines 2/82 virus and studies of a number of their biological properties. A highly temperature-sensitive recombinant with mutational damages in the hemagglutinin gene was detected.

[Isolation and characteristics of endocytic vacuoles containing the influenza virus]. / Vydelenie i kharakteristika éndotsitarnykh vakuolei, soderzhashchikh virus grippa.

Endocytic vacuoles (receptosomes) containing influenza virus were isolated from the cytoplasm of Ehrlich ascitic carcinoma cells and characterized. In the sucrose density gradient, the virus-containing material was detected in two peaks with a buoyant density of 1.175-1.16 and 1.155-1.135 g/cm3 with which the activity of marker enzymes of cell plasma membranes was associated. The virus was present in receptosomes in morphologically and electrophoretically intact condition. Examinations for the lipid composition of endocytic vacuoles showed the presence in their membranes of large amounts of cholesterol and glycolipids, particularly asialo-GM1 which, according to some authors may enhance the fusion of viral and cell membranes.

[Enhanced immunogenicity of influenza virus surface proteins when incorporated into liposomes]. / Povyshenie immunogennosti poverkhnostnykh belkov virusa grippa pri ikh vkliuchenii v liposomy.

A method for incorporation of influenza virus surface proteins into monolayer liposomes has been developed on the basis of removal of dialysed non-ionic detergent (beta-octyl glucoside) from a mixture of viral proteins and lipids. The resulting lipid-protein complexes (virosomes) are similar to intact virus particles by their morphological parameters. The immunogenicity of hemagglutinin incorporated into virosomes was significantly higher than that in the preparation of surface proteins isolated from virions which was inoculated into mice with or without complete Freund's adjuvant. The high immunogenicity of hemagglutinin incorporated into liposomes correlated with their high hemagglutinating activity. The possibility of using virosomes as subunit influenza vaccines is discussed.

[Effectiveness of incorporation of spermine-condensed viral DNA into liposomes. Interaction of liposomes with cells]. / Effektivnost' vkliucheniia kondensirovannykh sperminon virusnykh DNK v liposomy. Vzaimodeistvie liposom s kletkami.

DNA from bacteriophage lambda or monkey adenovirus type 7 have been condensed with spermine and included into three types of liposomes: ethereal, obtained by inverted phases technique and by Ca++ fusion. Compact DNA form presents a tor with 100 nm external diameter and 430 nm width. It can be included into 100 nm or larger liposomes. Total preparation contains 15-18% of the required liposomes. Liposomal fractions with included DNA were separated from empty liposomes by step gradient of ficoll 400. Liposomal fraction having included DNA contains 15-18% of common lipid. Liposomal interaction with monkey cell cultures has been studied.

[Reversible dissociation of influenza virus ribonucleoprotein]. / Obratimaia dissotsiatsiia ribonukleoproteida virusa grippa.

Dissociation of influenza virus RNP under the effect of salt was studied. Separation of RNA and protein components of influenza virus RNP was shown to occur in a linear 15-30% sucrose concentration gradient containing 1.1 M NaCl. Upon RNP dissociation, protein-protein interactions between individual molecules of the structural protein were retained. The sedimentation coefficient of the protein component was 52S. The possibility of reassociation of the RNA-protein complex was studied. More complete reassociation was observed to occur in the presence of 0.1 M NaCl. The resulting RNA-protein complex morphologically is similar to the native RNP of influenza virus.