Inhibition of HIV-1 replication by newly developed adamantane-containing polyanionic agents.

Newly developed antiviral compounds consisting of an adamantane derivative chemically linked to a water-soluble polyanionic matrix were shown to inhibit HIV-1 infection in lymphoblastoid cells, HeLa CD4+ beta-galactosidase (MAGI) cells and macrophages. The effect of the compounds was recorded by measuring viral reverse transcriptase activity and p24 by ELISA in culture supernatant and by immunoblotting of cell lysates. In this paper we describe the data obtained with one of the most promising compounds, Amant. Amant was not toxic for the host cells at concentrations as high as 1 mg/ml. The inhibition of HIV-1 replication in MT-4 and MAGI cells was observed when Amant was added either before infection or with the virus (0 h of infection), and was expressed even when the compound added at 0 h was removed 1.5 h after infection. Its inhibitory concentration (IC50) against HIV-1 and HIV-2 replication was 2-6 and 93 microg/ml, respectively. The anti-HIV-1 effect of the compound was gradually decreased when it was added 1 and 2 h post infection, and no inhibition was observed when the compound was added 4 h after infection, suggesting that the compound as a membranotropic drug blocks an early step of replication. It completely prevented the transport of Gag proteins into the nuclei. Pretreatment of the virus with Amant did not reduce its infectious activity. The classical adamantane derivatives amantadine and rimantadine hydrochloride did not inhibit HIV replication.

Localization by immunogold labelling of HIV-1 structural proteins on Lowicryl embedded HIV-1 infected cell ultrathin sections.

Using several HIV-1-specific antibodies and the immunogold labelling technique, we have detected and localized distinct viral proteins on ultrathin sections of HIV-1 infected cells embedded in the Lowicryl K4M resin. Monoclonal antibodies (MAbs) against p24, p17 and gp160/gp41 showed a preferential labelling on viral formations still attached to the cell membrane (budding process) or free in the extracellular space. The anti-p24 and the anti-p17 MAb yielded a gold labelling not only on mature but also on immature virions where the gold particles were associated with the ring-like electron-dense material. The three HIV-1-specific antibodies against p24, p17 and p55 yielded a cross-reaction with HIV-2 in agreement with the conservation of the internal antigenic determinants of both viruses. In all instances, there was no specific immunogold labelling over the cells, suggesting that once the virus structural proteins were synthesized, they were promptly utilized for virus assembly at the plasma membrane level.

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.

[The enhancement of the penetration of the human immunodeficiency virus into cells with the aid of a helper virus]. / Usilenie proniknoveniia virusa immunodefitsita cheloveka v kletki s pomoshch'iu virusa-pomoshchnika.

Penetration of human immunodeficiency virus (HIV) into the cells of lymphoblastoid T-cell line H9 was studied using 35S-methionine-labeled virus by demonstration of virus-specific proteins in the cytoplasm of the infected cells. Purification of the virus by ultracentrifugation through 30% glycerol was shown to lead to virus aggregation and its partial destruction manifested by the loss of gp120 protein, therefore unlabeled concentrated virus was used mainly with subsequent determination of virus-specific proteins by immune blotting. The addition of Sendai virus inactivated with UV rays to HIV increased the amount of HIV associated with cells as well as the amount of virus-specific proteins in the cytoplasm of the infected cells.

[The characteristics of the interaction of the proteins comprising the virions of the human immunodeficiency virus type 1]. / Osobennosti vzaimodeistviia belkov v sostave virionov virusa immunodefitsita cheloveka tipa 1.

Treatment of virions of human immunodeficiency virus type 1 (HIV-1) with ionic and nonionic detergents (NP-40, octylglucoside, sodium deoxycholate) exerted an effect on the virus uncommon for enveloped viruses: instead of solubilization, both glycoproteins (gp120 and gp41) were found in subviral particles, whereas the core protein p24 was found in the supernatant fluid after the removal of subviral particles by centrifugation. The matrix protein p17 and unprocessed molecules of the precursor protein p55 were associated with subviral particles. The above data confirm the proposed model of the HIV-I structural organization according to which glycoproteins are incorporated into the isometric matrix formed by protein p17. Our data indicate that the core protein p24 is not incorporated into the matrix and not associated with nucleocapsid proteins.

[The RNA electrophoretypes of the rotaviruses circulating in Moscow and Leningrad in the winter of 1987-1988]. / Elektroforetipy RNK rotavirusov, tsirkuliruiushchikh v Moskve i Leningrade zimoi 1987--1988 gg.

Analysis of electrophoretypes of RNA of rotavirus which had circulated in Moscow and Leningrad in the winter of 1987-1988, detected by enzyme immunoassay (EIA), was carried out. RNA electrophoresis was performed in 10% polyacrylamide gel (PAG) followed by silver staining, Most of the strains isolated in Moscow and Leningrad had a long phoretype (67% and 77%, respectively. The greatest variations in PAG mobility were found in segments 2, 3, and 7-9, segments 1, 4, 10, and 11 showed most unchangeable mobility. According to the pattern of segment migration, 3 variants of the long phoretype and 5 variants of the short phoretype were distinguished. In Moscow the ist variant (confluent 2nd and 3rd segments as well as 7th and 8th segments) of the long phoretype was predominant, which was isolated in approximately 70% of all cases of infection; in Leningrad the dominating variant was intermediate between the 1st and 2nd phoretype (slower migration of the 2nd segment). Variants of the sport phoretype were characterized by greater variability and lack of the dominating strain. The potentials of rotavirus RNA electrophoresis as a method of molecular epidemiology are discussed.

Unusual features of protein interaction in human immunodeficiency virus (HIV) virions.

The treatment of HIV-1 virions with ionic and nonionic detergents (NP 40, octylglucoside, Na deoxycholate) resulted in an effect unusual for enveloped viruses: instead of solubilization of glycoproteins, the core protein p24 was solubilized while envelope glycoproteins with other structural proteins were found in subviral particles. These data are consistent with a model of HIV structural organization in which glycoproteins are included in the matrix formed by the protein p 17 and suggest that p24 is neither involved in the matrix nor closely bound to any viral proteins.

[Conservativism of the matrix protein of paramyxoviruses]. / Konservatizm matriksnogo belka paramiksovirusov.

Cross reactions among paramyxoviruses were determined by the immunoblot method. Human parainfluenza viruses, types 1-3, avian parainfluenza virus type 4, mumps, Sendai, and measles viruses were used. Antisera to human parainfluenza viruses were shown to cross-interact with proteins NP and M of other types, and all antisera to the members of Paramyxovirus genus cross-reacted with M proteins of other paramyxoviruses. No cross reactions with measles virus proteins were observed. It is concluded that M protein is the most conservative protein of paramyxoviruses.

[Mechanism of penetration of human type 3 parainfluenza virus into monkey kidney cells]. / Mekhanizm proniknoveniia paragrippoznogo virusa cheloveka tipa 3 v kletki pochek obez'iany.

The mechanism of human parainfluenza type 3 virus penetration into monkey kidney cells was studied by morphological and biochemical methods. The results of electron microscopic studies permit a conclusion that the virus penetrates into the cells by the mechanism of receptor endocytosis. Analysis of subvirus structures in cytosole revealed two types of particles: nucleocapsids and structures of a larger size and lower buoyant density containing, in addition to NP protein, matrix (M) protein. It is presumed that nucleocapsid is released from the endocytic vacuole into the cytosole in association with M protein which is gradually eliminated from the nucleocapsid surface.

[Proteins of the human parainfluenza virus]. / Belki virusa paragrippa cheloveka.

Proteins of human parainfluenza type 3 virus propagated in green monkey kidney cultures are described. The presence of 4 major and 3 minor polypeptides is described along with their molecular weights. Protein F1 was found as a double band, its both components having a similar intensity. Only the upper band of F1 protein was found in the virus grown in Vero cell culture which has a low sensitivity for this virus. Fractionation of the virus and analysis of the proteins of subviral structures were carried out. Nucleocapsid had a buoyant density of 1.32 g/cm3 in cesium chloride and contained major NP protein and minor P and L proteins.

[Structural organization and proteins of the human type-3 para-influenza virus]. / Strukturnaia organizatsiia i belki virusa paragrippa cheloveka tipa 3.

The structure of human parainfluenza type 3 virus was studied by electron microscopy and virion fractionation by treatment with a detergent and high ionic strength. The protein spectrum of the virus was studied. The presence of 6 structural proteins was revealed of which two, HN and F, are glycoproteins. Intracellular cleavage of F0 protein into F1+2 proteins was demonstrated in a pulse-chase experiment. A tighter binding of HN protein than of F protein with the virus lipoprotein membrane was observed which may be useful for obtaining purified F protein preparations.