Dependence of conformation of D3/D4 domains of human CD4 on glycosylation and membrane attachment.

Conformational dynamics of human T-helper cell receptor protein CD4 has been studied with the help of monoclonal antibody (mAb) T6. The mAb T6 discriminates between s- and m-forms of CD4 and recognizes a specific conformation of the soluble (s) form of CD4 including the first nine amino acids of CD4 transmembrane sequence. However, change of tryptophan for serine in position 2 in this sequence destabilizes the T6-type conformation. By enzymatic deglycosylation and deletions of glycosylation sites, we show that T6-type conformation depends on glycosylation in both sites (Asn271 and Asn300). We show also that the sugars are not involved in direct binding to the antibody but stabilize the D3/D4 local conformation. Deglycosylated forms of sCD4 in vivo acquire a specific conformation similar to the wild type sCD4, which however cannot be restored after denaturation/renaturation under conditions of non-reducing Western blot. This observation indicates that the correct protein folding needs chaperone assistance and cannot be achieved in vitro. Completely non-glycosylated sCD4 is synthesized and secreted into the growth medium. In the medium, this mutant appears to be unstable and aggregates during time. In a contrast to soluble CD4, mutations in glycosylation sites abrogate expression of membrane CD4, thus demonstrating a different secretion pathways for soluble and membrane proteins.

[Autoimmune response in HIV-infected patients is directed against CD4 domain 4].

Phage display epitope library technology and a novel computer algorithm have been used for the localization of CD4 epitopes specific for monoclonal antibody (mAb) T6 and autoimmune antibodies found in an HIV infected patient. Both predicted epitope clusters have been shown to overlap and to be localized within the domain 4 of CD4. They included Cys303, Glu304, Glu330, and Lys331 amino acids. Few amino acids predicted by the algorithm as the epitope residues and two residues that did not relate to the epitope were sequentially substituted for Ala. Further analysis of the mutated forms of sCD4 expressed in 293T cells transfected with the corresponding DNAs, supported the predicted localization of the mAb T6 epitope. The results demonstrate that the autoimmune response in HIV-infected patients is directed against domain D4 of sCD4.

[Enzyme immunoassay for detection of porcine circovirus type 2, by using the recombinant capsid protein ORF-2].

Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age.

[Anti-CD4 autoimmunity in HIV-infected persons in Russia].

The frequency of anti-CD4 antibodies was determined in the sera or plasma derived from the patients infected with HIV-1 belonging to different genetic subgroups. The anti-CD4-antibodies in a dilution of > or = 1:1000 were found in 14% of the patients infected with the gagA/envA virus characteristic for injectable drug users in East Europe. The frequency of autoimmune antibodies among the HIV-infected patients with envB virus was substantially less (4.4%). Competitive ELISA using monoclonal antibodies to different CD4 domains demonstrated that irrespective of the viral genotype, the autoimmune epitope is located within the D4 or D3/D4 domains of CD4 receptor.

[DNA fragmentation of chicken adenovirus CELO by specific endonucleases R. HpaI, R. EcoRI, R. HindIII]. / Izuchenie fragmentatsii DNK ptich'ego adenovirusa CELO s pomoshch'iu spetsificheskikh endonukleaz R. HpaI, R. EcoRI, R. HindIII.

The effect of specific endonucleases on DNA chiken adenovirus CELO was studed. It was shown that endonucleases R. HpaI, R. EcoRI and R. HindIII cleaved viral DNA into 5,7 and 9 specific fragments, respectively. The sequence of fragments (physical map) was determined and found to be: D-A-E-C-B for enzyme R. HpaI; B-(EG)-C-A-D-F for enzyme R. EcoRI; H-F-A-C-G-B-D-E-I for enzyme R. HindIII.

[Simian adenovirus genome. II. Identification of specific fragments of viral DNA possessing transforming activity]. / Izuchenie genoma adenovirusa obez'ian. II. Identifikatsiia spetsificheckogo fragmenta virusnoi DNK, obladaiushchego transformiruiushchei aktivnost'iu.

The antigenic variant of simian adenovirus 7 (SA7) DNA was cleaved by restriction endonucleases EcoRI, XbaI, BamHI, SalI. The resulted digests of viral DNA were tested for transforming activity using the "calcium" technique. It was shown that BamHI. XbaI and SalI digests transformed primary baby rat kidney cells as well as native viral DNA. The transforming activity of separated BamHI and SalI fragments was tested also. The viral DNA fragments with transforming activity (BamHI-B and SalI-B) were localised on the left of the physical map of the viral genome. It was also shown that fragment-transformed cell lines were able to form colonies in 0.33% agarose medium.

[Simian adenovirus 20 genome. I. DNA mapping using restriction enconucleases BamHI, EcoRi, XbaI, HindIII]. / Izuchenie genoma adenovirusa obez'ian SV20. I. Postroenie tizicheskikh kart s pomoshch'iu restriktaz BamNI, EcoRI, KbaI, SalI HindIII.

The effect of specific restriction endonuclease on the simian adenovirus SV20 DNA was studied. It was shown that endonucleases SalI, XbaI, EcoRI, BamHI, HindIII cleaved the viral DNA into 3, 4, 5, 5, 8 specific fragments respectively. The sequence of fragments (physical map) was determined and found to be B-C-A for enzyme SalI, C-D-B-A--for enzyme Xbal, E-A-C-D-B--for enzyme EcoRI, B-E-C-A-D--for enzyme BamHI and B-E-A-C-(GH)-D-F--for enzyme HindIII. The G-C content of specific fragments was studied. The "right"-"left" orientation of the physical map of the simian adenovirus 20 DNA based on the G-C content was made in respect with the nomenclature of human adenoviruses.

[Expression of the vif gene of human immunodeficiency virus type 1 in Escherichia coli and study of the immunoreactivity of the vif protein]. / Ekspressiia gena vif virusa immunodefitsita cheloveka tipa 1 v kletkakhEscherichia coli i issledovanie immunoreaktivnosti vif-belka.

Full-sized gen vif of human immunodeficiency virus has been synthesized and cloned into plasmid pGEX-2T. Vif-gene expression was found in Escherichia coli cells resulting in production of a hybrid GST-protein. The recombinant protein studied by the immunoblotting technique reacted with 8 of 22 probes of human HIV-positive sera. The recombinant protein is specifically cut by thrombin in two proteins corresponding to GST and VIF-proteins in molecular mass.

[The immunoenzyme test system for detection of HIV-1 antigens based on using immune polyclonal anti-HIV serum and monoclonal antibodies against gene GAG HIV-1 proteins]. / Immunofermentnaia test-sistema dlia vyiavleniia antigenov VICh-1, osnovannaia na primenenii immunnoi poliklonal'noi anti-VICh syvorotki i monoklonal'nykh antitel k belkam gena GAG VICh-1.

The paper describes the enzyme immunoassay system for detection of human immunodeficiency virus antigens, which is based on the use of rabbit anti-HIV antibodies and monoclonal antibodies to HIV-1 gene proteins gag. The system may be useful in the examination of laboratory and clinical samples to reveal both free and conjugated antigens in the composition of immune complexes. The sensitivity of the assay system under development is 0.5 ng/ml at 100% specificity.

[Detailed study of the structure of simian adenovirus genome DNA and identification of its sequence in transformed cells]. / Detal'noe izuchenie struktury DNK genoma obez'ian'ego adenovirusa i identifikatsiia ee posledovatel'nostei v transformirovannykh kletkakh.

The paper describes physical maps of SA-7P DNA drawn with the help of XhoI, HpaI and BglII enzymes. DNA nucleotide sequence in the left side of the viral genome was found to have the length of 150 nucleotides. It contains codon for initiation of protein synthesis and coincides in this area with the known primary structure of Ela operon for human adenoviruses Ad5, Ad7, and Ad12. Study on the cells of two lines transformed by fragments of SA-7P DNA showed DNA of these cells to contain elements of the viral genome in different numbers of copies.

[Restriction endonuclease study of interstrain differences in the DNA of herpes simplex virus type 1]. / Issledovanie mezhshtammovykh razlichii DNK virusa prostogo gerpesa tipa 1 s pomoshch'iu restriktsionnykh éndonukleaz.

Electrophoregrams of the products of cleavage of DNA of 4 HSV-1 strains by restrictases Xbal, Hind III, and Sau 18/4 were compared. The sets of fragments obtained by the effect of Xbal restrictase on DNA of all 4 strains were found to be identical. At the same time, analysis of the products of DNA hydrolysis by Hind III and Sau 18/4 revealed significant interstrain differences.

[Identification of a new antigenic variant of simian adenovirus SA7 P]. / Identifikatsiia novogo antigennogo varianta adenovirusa obez'ian SA7 P.

Serological, biological, and physico-chemical properties of a new antigenic variant of simian adenovirus SA7P were studied. Neutralization tests with hyperimmune specific antisera demonstrated the new antigenic variant SA7P to have very significant antigenic similarity with the prototype SA7 strain. Same as the latter, SA7P does not agglutinate rat red blood cells, is highly oncogenic for newborn Syrian hamsters and capable of transforming rat kidney cell cultures. At the same time, the method of heteroduplex analysis showed SA7P DNA to be homologous to DNA of the reference SA7 strain by 85% and to contain 3 non-homologous regions in the right part of the virus genome. Comparison of the physical maps of the 2 virus DNAs by 4 restrictases established considerable differences in the number of recognition sites and their location.

[Effect of the synchronization of cell culture chronically infected with tick-borne encephalitis virus on the synthesis of virus antigen]. / Vliianie sinkhronizatsii kul'tury kletok, khronicheski infitsirovannoi virusom kleshchevogo entsefalita, na sintez virusnogo antigena

A cell culture chronically infected with tick-borne encephalitis virus was synchronized by the methods of double thymidine block and mitotic selection. There was a correlation between the time at which the cells entered the S period of the cell cycle and 4-8 fold increase in the number of cells carrying the virus antigen detectable by immunofluorescence.

[Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin]. / Konstruirovanie rekombinantnogo shtamma virusa ospovaktsiny, soderzhashchego ékspressiruemyi gen gemaggliutinina virusa grippa A.

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.

[Methods of isolating and selecting recombinant vaccinia viruses expressing heterogenetic viral antigens]. / Metody polucheniia i selektsii rekombinantov virusov ospovaktsiny, ékspressiruiushchikh chuzherodnye virusnye antigeny.

The paper describes a method using plasmid construction pSC11 for generation of recombinant vaccinia viruses supporting coexpression of heterologous genes and beta-galactosidase. The Ca2+-phosphate method of cell transfection by recombinant DNAs generated on the basis of pSC11, and selection of recombinant viruses from blue plaques of virus-infected cells in the presence of X-gala are reported at length.

[Interaction of gag polyprotein-precursors p55 and p48 with p160gag-pol during formation of virus-like particles of HIV-1 with recombinant strains of vaccinia virus]. / Vzaimodeistvie gag poliproteinov-predshestvennikov p55 i p48 s p160gag-pol pri formirovanii virusopodobnykh chastits VICh-1 rekombinantnymi shtammami virusa ospovaktsiny.

The polypeptide composition of HIV-I virus-like particles produced by CV-I cells during mono- and coinfection with recombinant vaccinia virus (rVV) strains containing the whole (p55) and carboxyterminal truncated (p48) gag genes and gag-pol sequence is studied. In monoinfection both the gag-strains actively produced virus-like particles consisting of non-processed p55Gag and p48Gag polyprotein without p6 domain. In case of a coinfection of the cells with one of these strains and the rVV producing p160Gag-Pol polyprotein the virus-like particles consisted of p24 protein and a negligible amount of non-processed Gag precursors. The share of p24 protein increased in proportion to the duration of coinfection and decreased with a reduction of multiplicity of infection with rVV carrying p160Gag-Pol. Hence, the absence of p6 domain does not influence the processing of Gag proteins during virus-like particles assembly and budding. In contrast to the natural systems of HIV-I development, in the rVV expression system the p6Gag domain virtually does not contribute to reactions between Gag and Gag-Pol precursors and to the particles' morphogenesis.

[The antigenic and immunogenic properties of a recombinant protein of the HIV-1 gag-pol gene secreted by Saccharomyces cerevisiae yeast cells]. / Antigennye i immunogennye svoistva rekombinantnogo belka gena gag-pol VICh-1, sekretiruemogo drozhzhevymi kletkami Saccharomyces cerevisiae.

Analysis of the immunological properties of recombinant proteins of HIV-1 gene gag-pol secreted by yeast cells S. cerevisiae was carried out. The proteins under study interacted with antibodies from HIV-1-seropositive human subjects and with antibodies of rabbit immune serum to the native virus as effectively and specifically as natural HIV-1 proteins. The yeast gag-pol-protein complex was markedly immunogenic and induced in animals synthesis of antibodies of a certain specificity spectrum. A comparative immunochemical analysis of the properties of the recombinant proteins carried out by EIA and immune blot showed a certain degree of similarity between the yeast proteins and those of analogous construction produced in E. coli system.

[Typing of herpes simplex virus strains by analyzing the viral DNA using restriction endonucleases]. / Tipirovanie shtammov virusa prostogo gerpesa metodom analiza virusnoi DNK s pomoshch'iu restriktsionnykh éndonukleaz.

Analysis of the products of viral DNAs cleavage by restrictive endonucleases showed the strains of herpes simplex virus Us and L2 used for herpes vaccine manufacture to belong to type 1 and the VN strain to type 2 of herpes simplex virus. This method is found to be optimal for typing of herpes simplex virus strains.

[Isolation and characteristics of a mutant herpes simplex virus type 1 resistant to phosphonoacetic acid]. / Vydelenie i kharakteristika mutanta virusa gerpesa prostogo tipa 1, rezistentnogo k fosfonouksusnoi kislote.

By successive passages and triple cloning of herpes simplex virus type 1 (HSV-1) in Vero cell culture in the presence of increasing concentrations of phosphonoacetic acid (PAA) a mutant of HSV-1 resistant to PAA (PAAr) was derived and characterized. The resistance to the inhibitor was transmitted from PAAr-mutant to a sensitive strain (L2) by recombination performed by the marker rescue method using DNA fragmented by Hpa-1 restrictase. The resulting recombinant (R-551) was resistant to the inhibitor and had an altered primary structure of DNA.

[Resistance of various herpes simplex virus strains to the action of chemical inhibitors]. / Izuchenie tsoichivosti razlichnykh shtammov virusov gerpesa prostogo k deistviiu khimicheskikh ingibitorov.

The effect of chemical inhibitors on reproduction of 2 laboratory and 3 vaccine strains of herpes simplex virus (HSV), types 1 and 2, was studied. By the time of the study the vaccine strains had undergone from 27 to 69 passages in chick embryo fibroblast cultures. All the vaccine strains (L2, Us, and VN) exhibited 100-1000 fold higher resistance to phosphonoacetic acid than did the laboratory F+ and G strains, and the vaccine L2 strain (HSV-1) was also 1000-fold resistant to 1-beta-D-arabinofuranosine thymine.