Localization of reverse-transcriptase in interferon-treated mouse cells chronically infected with Moloney leukemia virus

Abstract

Interferon treatment of mouse cells chronically infected with Moloney leukemia virus (3T3/MLV) resulted in 97 per cent inhibition of infective virus release. The intracellular localization and distribution of virus reverse-transcriptase and group specific (gs) antigen were determined in interferon treated and control cells. Cytoplasm of infected cells was fractionated by isopycnic centrifugation on discontinuous sucrose gradients. Fractions were analysed for their chemical composition and characterized by the activity of membranal marker enzymes. The association and levels of viral antigens were determined in each fraction. Fractions enriched with 5' nucleotidase, specific enzyme marker for plasma membrane, were also enriched with viral proteins. In interferon treated cells, intracellular accumulation of viral proteins was specifically localized in the plasma membrane. Threefold increase in reverse-transcriptase level was the maximal accumulation found in purified plasma membranes. Intracellular enzyme levels in interferon treated cells were in accordance with the amount of cell associated infective virus particles. The small accumulation of viral proteins and infective virus particles was not sufficient to account for the great reduction in virus yield observed in the supernatants of the interferon treated cells. A possible role for interferon in modification of plasma membrane associated with virus assembly is postulated.

Glycoprotein Enrichment in Moloney Leukemia Virus Structural Proteins Released from Interferon-Treated Cells

“…18]. In a previous study we showed that in the presence of interferon, a small amount of mature MLV particles was still synthesized and released from the cell after a delay of a few hours [22]. In this study our purpose was to determine how inhibition of virus produc tion reflects itself in the composition of these viral particles.…”

“…We have shown that only a small part of synthesized proteins accumulates in the cell. Within virus particles [22] most of them are probably de graded. The incorporation of envelope glyco proteins into the assembled virus was more efficient than that of other proteins and their release as soluble glycoproteins was reduced to a lesser extent (40%).…”

Inhibition of the synthesis of Moloney leukemia virus structural proteins by N-methylisatin-beta-4',4'-diethylthiosemicarbazone

“…RNA extractions from cytoplasmic preparations (14) were performed with 2 x 108 3T3/MLV cells by the method described by Fan and Baltimore (7). Viral RNA in cytoplasmic preparations was measured by a hybridization technique (12) CaCl2, 120 mM NaCl, 5 mM KCI), and cytoplasm preparations were extracted in 2 ml of lysis buffer (40 mM Trishydrochloride, pH 7.5, 100 mM KCl, 50 mM NaCl, 3.6 mM CaCl2, 5 mM MgCl2, 6 mM 2-mercaptoethanol, 0.5% Nonidet P-40).…”