Localization of constituent proteins of HIV Type 1 (HIV-1)

Abstract

The localization of the constituent proteins of HIV-1 in the virions and HIV-1-infected cells was examined by indirect immuno-gold labeling with monoclonal antibodies (MoAb) to gag proteins (p18 and p24/p53), to elucidate viral formation of HIV.Persistently HIV-1 (HTLV—IIIB,LAV-1)-infected MT-4 and MOLT-4 cell lines and their cloned cell lines were used as infected cells. Mouse MoAb against HIV-1 gag p18 (V17) and p24/p53 (V107) were used. The cells were fixed with 0.5-1% glutar-aldehyde in phosphate-buffered saline (pH 7.2), dehydrated in ethanol and embedded in epoxy (at 45° Cor 60°C) or Lowicryl K4M resin (at −30°C). The sections were incubated in MoAb at room temperature for 1 h and then incubated in anti-mouse goat IgG conjugated with gold (IgG-gold, 5 or 15 nm; Janssen) for 40 min. After being washed, the sections were stained with uranyl acetate and lead citrate, and were observed in an electron microscope with a tilting apparatus.The spećific reactions with V17 and V107 were detected on HIV-1 particles and in the infected cells. No reactivity was noted between uninfected control cells and the MoAb, or between the infected cells and normal mouse serum. More than 97% of the HIV-1 particles embedded in epoxy resinat 60°C were labeled with gold after exclusion of the HIV particles that were attached to supporting film or completely embedded in the section (Fig. 1). Increased labeling was observed with Lowicryl (Fig. 2)and epoxy resin embedding at 45°C.