Messenger ribonucleoprotein complexes containing in vitro-synthesized 26S and 42S Semliki Forest virus RNA

Abstract

An extract derived from Semliki Forest virus (SFV) infected cells is described which catalyzes the synthesis of virus-specific RNAs. The newly-synthesized 26S and 42S RNAs are found complexed with protein as messenger ribonucleoproteins (mRNPs). These mRNPs either are non-membrane bound or are associated with large cytoplasmic lipoprotein membranes, and they are found as free mRNPs as well as mRNPs bound to ribosomal subunits, ribosomes, and polysomes. Following treatment with Tween 40 and deoxycholate, membrane-bound mRNPs containing in vitro-synthesized 26S RNA are dissociated and sediment at 33S. These membrane-dissociated mRNPs contain relatively little protein. In contrast, the free or ribosome-bound mRNPs, which are isolated as 30S to 160S particles, remain heterogeneous after detergent treatment and have a much higher protein content. Addition of purified, native 40S ribosomal subunits to the extract leads to the formation of complexes between the added ribosomal subunits and the newly-synthesized viral mRNA. The in vitro-synthesized 26S and 42S RNAs participate in the assembly of translational initiation and elongation complexes.