The C-terminal domain of glyceraldehyde 3-phosphate dehydrogenase plays an important role in suppression of tRNA(Lys3) packaging into human immunodeficiency virus type-1 particles

Human immunodeficiency virus type-1 (HIV-1) requires the packaging of human tRNA(Lys3) as a primer for effective viral reverse transcription. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) suppresses the packaging efficiency of tRNA(Lys3). Although the binding of GAPDH to Pr55(gag) is important for the suppression mechanism, it remains unclear which domain of GAPDH is responsible for the interaction with Pr55(gag). In this study, we show that Asp(256), Lys(260), Lys(263) and Glu(267) of GAPDH are important for the suppression of tRNA(Lys3) packaging. Yeast two-hybrid analysis demonstrated that the C-terminal domain of GAPDH (151–335) interacts with both the matrix region (MA; 1–132) and capsid N-terminal domain (CA-NTD; 133–282). The D256R, K263E or E267R mutation of GAPDH led to the loss of the ability to bind to wild-type (WT) MA, and the D256R/K260E double mutation of GAPDH resulted in the loss of detectable binding activity to WT CA-NTD. In contrast, R58E, Q59A or Q63A of MA, and E76R or R82E of CA-NTD abrogated the interaction with the C-terminal domain of GAPDH. Multiple-substituted GAPDH mutant (D256R/K260E/K263E/E267R) retained the oligomeric formation with WT GAPDH in HIV-1 producing cells, but the incorporation level of the hetero-oligomer was decreased in viral particles. Furthermore, the viruses produced from cells expressing the D256R/K260E/K263E/E267R mutant restored tRNA(Lys3) packaging efficiency because the mutant exerted a dominant negative effect by preventing WT GAPDH from binding to MA and CA-NTD and improved the reverse transcription. These findings indicate that the amino acids Asp(256), Lys(260), Lys(263) and Glu(267) of GAPDH is essential for the mechanism of tRNA(Lys3)-packaging suppression and the D256R/K260E/K263E/E267R mutant of GAPDH acts in a dominant negative manner to suppress tRNA(Lys3) packaging.