In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function

Area of Science:

• Molecular Biology
• Virology

Background:

• The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is crucial for viral gene expression.
• Rev protein regulates protein expression from HIV-1 RNAs containing the Rev-responsive element (RRE).
• Rev acts on both nuclear RNA splicing/transport and cytoplasmic RNA translation.

Purpose of the Study:

• To investigate the in vivo binding of the HIV-1 Rev protein to RRE-containing RNAs.
• To determine if Rev RNA binding is sufficient for its nuclear and cytoplasmic functions.

Main Methods:

• Immunoprecipitation using a monoclonal antibody specific for the HIV-1 Rev protein.
• Analysis of cellular extracts from HIV-1-infected and -transfected cells.
• Generation and analysis of a Rev mutant with retained RNA binding but lost function.

Main Results:

• Wild-type Rev showed specific binding to RRE-containing RNAs predominantly in cytoplasmic extracts.
• A Rev mutant retained in vivo RNA binding in both nuclear and cytoplasmic extracts.
• Rev RNA binding was confirmed in vivo, but it was not sufficient for Rev's nuclear or cytoplasmic functions.

Conclusions:

• Direct binding of Rev to HIV-1 RNAs in vivo is verified.
• Rev RNA binding is necessary but not sufficient for its functions in nuclear export and translation.
• These findings support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.