Rev protein suppression of complex formation between nuclear proteins and rev-responsive element-containing RNA of human immunodeficiency virus-1

Abstract

The Rev protein from human immunodeficiency virus type 1 (HIV-1) is known to bind Rev responsive element (RRE) sequence of HIV-1 mRNA. This interaction is thought to enhance expression of viral structural proteins but the mechanism for this effect is uncertain. The aim of this study was to investigate (i) whether other cellular proteins also bind to the RRE sequence and (ii) whether binding of cellular proteins to RRE RNA is influenced by Rev protein. Our results revealed that a variety of RNA-protein complexes are formed when in vitro transcribed RRE-containing RNA is incubated with proteins present in HeLa nuclear extracts. The molecular masses of the most prominent bands in RNase protection assays were ∼90, 82, 67, 50, and 35–45 kDa. Addition of recombinant Rev protein suppressed the complex formation between RRE RNA and nuclear proteins. The strongest effect was observed when the RNA was preincubated with Rev before addition of HeLa proteins. Rev protein bound to the RRE-containing RNA under formation of oligomeric complexes with a Svedberg coefficient of 4.0–4.5 S. The β-galactosidespecific nuclear carbohydrate-binding protein 35 (CBP35), which is present in nuclear ribonucleoprotein (RNP) particles, was identified in the complexes formed between RRE RNA and HeLa nuclear protein. Binding of these complexes to a galactose affinity matrix was abolished by addition of Rev protein. We propose that binding of Rev protein to RRE-containing HIV transcripts prevents RNP complex formation between these transcripts and cellular proteins; this may inhibit spliceosome assembly allowing the transport of the unspliced or incompletely spliced HIV-1 mRNA into the cytoplasm. Such an effect could be expected to promote expression of viral structural proteins.