The C-terminal domain of glyceraldehyde 3-phosphate dehydrogenase plays an important role in suppression of tRNALys3 packaging into human immunodeficiency virus type-1 particles

Naoki Kishimoto; Ayano Onitsuka-Kishimoto; Nozomi Iga; Nobutoki Takamune; Shozo Shoji; Shogo Misumi

doi:10.1016/j.bbrep.2016.09.015

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

Biochem Biophys Rep. 2016 Oct 6:8:325-332. doi: 10.1016/j.bbrep.2016.09.015. eCollection 2016 Dec.

Authors

Naoki Kishimoto¹, Ayano Onitsuka-Kishimoto¹, Nozomi Iga¹, Nobutoki Takamune², Shozo Shoji¹, Shogo Misumi¹

Affiliations

¹ Department of Environmental and Molecular Health Sciences, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
² Innovative Collaboration Organization, Kumamoto University, Kumamoto 860-8555, Japan.

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 Asp²⁵⁶, Lys²⁶⁰, Lys²⁶³ and Glu²⁶⁷ 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 Asp²⁵⁶, Lys²⁶⁰, Lys²⁶³ and Glu²⁶⁷ 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.

Keywords: CA, capsid; GAPDH; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HIV-1; HIV-1, human immunodeficiency virus type 1; LysRS, lysyl-tRNA synthetase; MA, matrix; tRNALys3.