I G Mustafin

Spectrum of produced GAG gene viral proteins in lymphocytes infected HIV-1 NL4-3 strains in vitro

Aim. To study spectrum of viral proteins secreted into the culture medium by the healthy donor peripheral blood lymphocytes infected by HIV-1 NL4-3 strain in vitro. Methods. We used a primary lymphocytes culture obtained from healthy donors. For synchronous infection of primary lymphocytes culture in vitro NL4-3 strain of human immunodeficiency virus (HIV) (NIH AIDS Research & Reference Reagents Program, USA) was used. The HIV-1 titer in the supernatants was determined by enzyme immunoassay (EIA p24gag Coulter). In addition, electrophoresis of clarified culture medium proteins in 12.5% polyacrylamide gel under denaturing conditions followed by electron transport onto a nitrocellulose membrane; immunoblotting for detection of virus-specific proteins using positive control serum to HIV-1 («Bio-Rad») were performed; protein markers kits LMW 94-14.4 kD, HMW 212-53 kD («Pharmacia Biotech») were used to determine the seroactive fractions molecular weight. Results. . HIV antigens spectrum in the culture medium was studied on the 4th, 9th and 13th days after lymphocyte culture infection with HIV. Within 13 days of virus reproduction both non-structural and structural proteins of HIV-1 gag gene are detected in the culture medium, spectrum and quantitative range of which varies with time. After 4 hours of HIV reproduction in primary lymphocytes culture in vitro large number of gag gene nonstructural proteins (p40, p55) are exported from cells into culture medium. Further, gag gene non-structural proteins may be cleaved into the structural proteins (p24/25, R18) under the cellular proteases action, which is caused by the proteolytic enzymes accumulation in the culture medium. Conclusion. In HIV reproduction in primary lymphocytes cultures soluble forms of the HIV-1 gag gene proteins are secreted into culture medium; in the initial stages of the infection HIV proteins spectrum in the culture medium of lymphocytes activated with phytohemagglutinin in vitro consists of only the HIV-1 precursor proteins (p55, p40) and at a later stages - also of the gag gene viral capsid proteins (p24/25, R18).