Characterizing the Impacts of Human Immunodeficiency Virus Type 1 (HIV-1) Rev Hydrophobic Interface Mutations on Rev Multimerization and Nuclear Export of Unspliced HIV RNAs

HIV replication requires nuclear export of intron-containing RNAs.  The viral Rev protein mediates nuclear export of unspliced RNAs via homo-oligomerization with a structured viral RNA sequence, termed the Rev response element (RRE), which can then interface with host cell nuclear export factors.  Previous biochemical, structural, and cellular reporter studies have identified key residues in the hydrophobic oligomerization domains of Rev that mediate cooperative binding of Rev subunits onto the RRE.  However, the mechanistic role of Rev/RRE oligomerization in nuclear export or other parts of the viral life cycle remains unclear.  This study investigated the functional relationship between Rev/RRE oligomerization and RNA nuclear export via site-directed mutagenesis of residues within the Rev hydrophobic oligomerization domains.  We hypothesized that progressively non-conservative mutations would confer increasingly deleterious effects on Rev multimerization and nuclear export.  To test our hypothesis, we determined the RRE-binding affinity of each mutant recombinant Rev protein via electrophoretic mobility shift assay and quantified Rev-activated nuclear export of green fluorescent protein (GFP) in a reporter T-cell line via flow cytometry.  These studies will effectively complement ongoing studies of HIV containing oligomerization-defective Rev and will help reveal the functional role of Rev multimerization in HIV-1 replication.