FIG. 1.
HIV-1 Pol proteins RT and IN are required for RNA dimer maturation. (A) Schematic representation of HIV-1 WT and mutant proviral DNA constructs. HIV-1 WT pDRNL4.3 was used as a control. PR(−) has a D25N substitution at the active site of PR ↑↑ preventing proteolytic activity. GagUAA has a termination codon × inserted immediately after the Gag coding region. PRUAG has a termination codon ⧫ inserted at the end of PR. RTUAG has a termination codon inserted at the end of the RT ▴ coding sequence. The UAA/G insertions prevent translation of proteins located downstream. (B) Western blot analysis was performed on purified virions. Proteins were resolved by 10% Tris-glycine SDS-PAGE and then probed with pooled sera from HIV-1-infected individuals or anti-integrase monoclonal antibodies as described in Materials and Methods. (C) Virion RNA was resuspended in RNA dimerization buffer and heat denatured for 10 min at the indicated temperatures. Dimers and monomers were electrophoresed in a 1% native agarose gel and Northern analysis performed using an HIV-1-specific riboprobe.