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

Abstract

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.

Graphical abstract

Highlights

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  Yeast two-hybrid analysis revealed that GAPDH interacts with MA and CA region of HIV-1 precursor proteins via its C-terminal domain.

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  Docking simulation predicted that GAPDH helix 10, which is exposed on surface of its tetrameric form surface, interacts with MA and CA.

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  Mutagenesis assay on yeast two-hybrid analysis showed that D256R/K260E/K263E/E267R mutant of GAPDH lacks the binding affinity to both MA and CA.

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  D256R/K260E/K263E/E267R mutant of GAPDH acts as dominant negative effector on the packaging of tRNA ^Lys3.