Extensive C-terminal deletion in human immunodeficiency virus type 1 Env glycoprotein arising after long-term culture of chronically infected cells.

Human immunodeficiency virus type 1 (HIV-1) chronically infected (CI) cell lines were established from HIV-1HIB/LAI-infected MT-4 cells that survived acute infection. The HIV env gene expressed in the two long-term cultured cell lines differed from that of the lines cultured for shorter periods, by coding for a glycoprotein gp 160 that had the C terminus deleted. One long-term cultured cell line, CI-17, was studied in detail. An insertion of a premature stop codon in the env gene caused about 90% of gp160 molecules to be truncated (gp160x), lacking both cytoplasmic and transmembrane domains; these species were secreted into the cell medium, and could form oligomers with other truncated gp160 molecules as well as with their normal counterparts. CI-17 cells constantly yielded high levels of viral protein and relatively low quantities of infectious virus, without cytopathicity. However, acute infection of fresh MT-4 cells with CI-17-derived virus led to cytopathicity, the rate of which as well as the Env glycoprotein pattern depended on multiplicity: (i) using an infection dose of 10(-4) ID50/cell, cells died 7 to 8 days post-infection with normal gp160 synthesis predominating; (ii) with 10(-2) ID50, gp160x was produced as early as 48 h post-infection and cell death was delayed. Predominant gp160x formation occurred again when new CI cell lines were obtained with CI-17-derived virus. Thus, two human immunodeficiency virus variants, a normal and a defective one, are persistently expressed in CI-17 cells. The other long-term cultured CI cell line also expressed gp160 with a similar (albeit slightly longer) deletion of a C-terminal region in most molecules, but the cell lines that were cultured for shorter periods did not. These results suggest that the emergence of HIV variants with a C-terminal deletion in the Env glycoprotein, which coexist with normal virus, may play a role in maintaining the long-term growth capacity and viability of CI cells.

[Pathological cleavage of human immunodeficiency virus gp160 protein in infected cells as a probable factor for infection becoming chronic]. / Pathologicheskoe razrezanie belka gp160 birusa immunodefitsita cheloveka v zarazhennykh kletkakh kak veroiatnyi faktor khronizatsii infektsii.

The synthesis of HIV-1 (IIIb isolate) structural protein in chronically (CI) and acutely infected (AI) MT4 cells was studied. During long-term cultivation the CI system was characterized by high involvement of the cells into infection (up to 100%), high level of virus-specific protein synthesis, moderate virus yield, but absence of any virus-induced cytopathic effects and normal growth potential of infected cells. AI cells demonstrated a similar level of synthesis of virus specific proteins, higher virus yield, and rapid progression of cytopathicity followed by total cell death. Most of the HIV gp160 protein molecules undergo rapid cleavage in the region between the point of conventional cleavage and the transmembrane domain, being removed from the physiologically competent pool, but a small portion of gp160s undergo apparently normal intracellular development. According to our data, the two HIV variants (normal and defective) persist in CI system and pathological cleavage of defective virus gp160 protein results most probably in chronization of infection.

[Group- and type-specific antibodies to the gag-gene protein p26 of the human immunodeficiency virus immunological type 2 in infected patients]. / Gruppo- i tipospetsificheskie antitela k belku p26 gena gag virusa immunodefitsita cheloveka 2-go immunologicheskogo tipa u infitsirovannykh.

The immunoreactivity of serum samples from HIV-2 infected persons was studied by radioimmunoprecipitation assay (RIPA) in homo- and heterotypic variants. In homotypic RIPA all sera studied have precipitated the viral glycoprotein with the high molecular weight, gp170. Some samples were active for gag-gene products, p57 and p26 in homotypic RIPA. Most these samples were also active for heterotypic gag-protein of HIV-1 serotype, p55 and p24. On the other hand anti-gag reactivity of one sample was limited only by homotypic activity. Some causes of this phenomena as well as its significance for serodiagnosis of HIV infection are discussed.

[A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use]. / Metod radioimmunopretsipitatsii v serodiagnostike VICh-infektsii: razrabotka optimal'nykh uslovii postanovki i otsenka vozmozhnostei ispol'zovaniia.

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.

Consolidation of intramolecular disulfide bonds in influenza virus hemagglutinin as an element of intracellular maturation.

Electrophoretic behavior of influenza virus hemagglutinin during SDS electrophoresis in polyacrylamide gel is critically dependent on the life time in the infected cells and also on the conditions of sample preparation and analysis. During electrophoresis of total cell lysate proteins under nonreducing conditions the short-labeled hemagglutinin is detected as multiple bands, electrophoretic mobility of most of them being lower than that of hemagglutinin of viral particles. This heterogeneity failed to be detected during electrophoresis under reducing conditions which is indicative of the differences in the number or direction of intramolecular disulfide bonds between short-labeled and mature hemagglutinin molecules. After chasing at 37 or 20 degrees hemagglutinin gradually assumes an electrophoretic character identical to that of virion protein. Chasing at 0 degrees or the substitution of parafluorophenyl alanine for phenylalanine in the maintenance medium during labeling prevents maturation. At the same time, both iodacetamide perfusion of infected cells and the preparation of nuclei-free extract prior to SDS lysis result in a marked increase in the yield of disulfide mature short-labeled hemagglutinin. These results suggest that disulfide maturation in hemagglutinin proceeds in two stages: a relatively rapid (with respect to synthesis completion) formation of intramolecular disulfide bonds as such followed by a much slower consolidation of bridges against the action of endogenous cell reductants which activate during lysis. Consolidation may be caused by two factors: trimerization of hemagglutinin monomers or their covalent post-translational modifications.

[Masking of HIV antigen by specific antibodies in a model experiment]. / Maskirovanie antigena virusa immunodefitsita cheloveka spetsificheskimi antitelami v model'nom éksperimente.

An immuno-diagnostic test system of competitive EIA detecting HIV antigen in a concentration up to 1 ng/ml has been developed. Using this system, a phenomenon of binding of HIV antigen by antibody in sera from infected persons consisting in masking of antigenic determinants was demonstrated. The "undetectability" of HIV antigen in the system of competitive EIA caused by this phenomenon is considered to be a model of clearance of antigen at the excess of antibody in vitro. The experimental results are in agreement with the suggestion that repeated HIV antigenemia occurs as a result of exhaustion of specific immune responses.

[A comparison of the spectra of antibodies to individual proteins of the human immunodeficiency virus in seropositive sera collected in the territories of the USSR and England]. / Sravnenie spektrov antitel k individual'nym belkam virusa immunodefitsita cheloveka v seropozitivnykh syvorotkakh, sobrannykh na territorii SSSR i Anglii.

Antibody spectra to individual proteins of human immunodeficiency virus (HIV) in 74 seropositive serum samples collected in the USSR and 65 serum samples collected in Britain were studied by immunoblotting techniques. Most of the sera belonged to clinically healthy persons, some of the sera collected in Britain contained specific IgM antibodies. The results were evaluated qualitatively and quantitatively. In the former case the study of samples collected both in the USSR and in Britain yielded similar results which also coincided with the data of literature regarding asymptomatic virus carriers: very high content of antibodies to protein gp41 and sufficiently high content of antibodies to protein p24 were registered in all sera. But the quantitative evaluation of the results of this investigation revealed differences between serum samples collected in these two countries. The main feature of sera collected in the USSR was their noticeably greater reactivity with respect to the products of HIV gene gag: proteins p24, p53 and p22. The explanations of this phenomenon are discussed.

Breaking and rejoining of disulfide bonds in external glycoproteins of influenza virus.

The treatment of influenza virus with increasing concentrations of mercaptoethanol led to a progressive inhibition of hemagglutinating activity and infectivity and to gradual changes of electrophoretic behavior of virus glycoproteins under nonreducing conditions. These phenomena are most likely the result of consecutive destruction of disulfide bonds in the proteins. So, different disulfide bonds in glycoproteins of native viral particles differ in their sensitivity to the reductant. It has been found that broken disulfide bonds may re-form and the restoration seems to be most rapid in those disulfide bonds that are the least sensitive to the reductant under the native condition.

Controlled organization of multimolecular complexes of enveloped virus glycoproteins: study of immunogenicity.

Some technological and immunological problems facing the preparation of subunit viral vaccines are discussed. Solubilization of enveloped virus glycoproteins with various detergents has been studied. It has been demonstrated that a novel non-ionic detergent, MESK, can be used to prepare the glycoproteins of enveloped viruses in defined supramolecular forms: monomers, micelles, liposomes and multimeric complexes. These preparations have been tested for immunogenicity. It has been shown that the immunogenicity of glycoproteins in micellar form or in liposomes is comparable with that of the whole virus. The immunogenicity of the glycoprotein complex with the glycoside Quil A appeared to be significantly higher in comparison with the whole virus and was similar to the immunogenicity of glycoproteins mixed with Freund's complete adjuvant.

[The low frequency of false positive results in the serological detection of infectivity with the human immunodeficiency virus in collectives of healthy young people]. / Nizkaia chastota lozhnopolozhitel'nykh rezul'tatov pri serologicheskom vyiavlenii infitsirovannosti virusom immunodefitsita cheloveka v kollektivakh zdorovykh molodykh liudei.

Among 1002 foreign students examined in Odessa 11 subjects with antibody to HIV, i.e. infected with HIV, were detected. A complete agreement of the results of the enzyme immunoassay and immune blotting test was observed. The reasons of this are discussed. A specific regional distribution of seropositive subjects by their permanent residence places was revealed.

[Preparative isolation of purified antigens of the herpes simplex virus type I and a study of their immunogenic properties]. / Preparativnoe poluchenie ochishchennykh antigenov virusa prostogo gerpesa tipa I i izuchenie ikh immunogennykh svoistv.

The HSV-I-containing material prepared in chick embryo fibroblast cultures was concentrated on nuclear filters followed by chromatography on a column with large-pore silica. The MESK detergent was used for isolation of glycoproteins. The glycoprotein preparation was highly immunogenic in experimental animals.

[Conditions for efficient antibody binding with proteins of the human immunodeficiency virus studied by immunoblotting]. / Izuchenie uslovii éffektivnogo sviazyvaniia antitel s belkami virusa immunodefitsita cheloveka v immunoblote.

The authors have investigated the antibody binding with individual HIV protein by "western" blotting method. The optimal condition for binding was incubation of nitrocellulose strips at 37 degrees C for 2 hrs followed by incubation at 4 degrees C overnight. Differences in the serologic activities of a number of sera in "western" blotting were shown by using two different antigens. It was concluded that mixing of different antigens before electrophoresis may be useful for more effective "western" blotting analysis.

[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.

[Use of an immune blotting method (western) for studying viral antigens and for detecting antibodies to viral proteins]. / Primenenie metoda immunnogo ("vestern") blotinga dlia izucheniia virusnykh antigenov i vyiavleniia antitel k virusnym belkam.

The technique of the procedure and the results of the use of immune ("western") blotting method for studies on viral antigens and detection of antibodies to individual proteins of viral particles are described. The possibility of detection and study of individual viral antigens in the whole plasma of patients or carriers is demonstrated by the example of HBs antigen of human hepatitis B virus. The method of immune blotting was used for screening of human sera for the detection of antibodies to the AIDS virus proteins. The sera under study positive for antibodies to AIDS virus by preliminary solid-phase enzyme-immunoassay were shown to contain actually the antibodies to different proteins of AIDS virus. The antibody levels to individual AIDS virus proteins varied in different sera. Some sera positive for antibodies to AIDS virus by the solid-phase enzyme-immunoassay contained no antibody to AIDS virus proteins but reacted with cellular proteins present in the antigen. The immune blotting method was also used for determinations of the spectrum of antibodies in animal sera produced by immunization with different viral antigens.

[Disulfide maturation of the glycoproteins of influenza virus hemagglutinin and neuraminidase in infected cells]. / Disul'fidnoe sozrevanie glikoproteidov gemaggliutinina i neiraminidazy virusa grippa v zarazhennykh kletkakh.

Fractionation by polyacrylamide gel electrophoresis (PAGE) demonstrated that in the infected cells the newly synthesized influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase (NA), differ from mature proteins of virus particles. After some time of life in the cells the differences are levelled. Since this phenomenon was demonstrable only in an analysis under the conditions favourable for the retention of disulphide bonds, it was designated as "disulphide maturation" of glycoproteins. Two causes of disulphide maturation of HA are considered: posttranslational folding of molecules conducive to drawing closer of the oxidizable thiol groups, and gradual loss of sensitivity to endogenous reducing agents. As for NA, the observed maturation here is the result of disulphide dimerization of monomers. Some factors affecting disulphide maturation of glycoproteins have been studied.

[Expert diagnosis of infectivity with the AIDS virus using immune blotting: the development of a system and assessment of its parameters]. / Ekspertnaia diagnostika infitsirovannosti virusom SPID s pomoshch'iu immunnogo blotinga: razrabotka sistemy i otsenka ee paranetrov.

A system for serodiagnosis of infection with AIDS viruses by means of immune ("western") blotting has been developed. The system has been found to be highly sensitive and suitable for expert serodiagnosis of AIDS disease and infection with human AIDS viruses.