Zinc-binding site of human immunodeficiency virus 2 Vpx prevents instability and dysfunction of the protein

Abstract

Human immunodeficiency virus 2 Vpx coordinates zinc through residues H39, H82, C87 and C89. We reported previously that H39, H82 and C87 mutants maintain Vpx activity to facilitate the degradation of SAMHD1. Herein, the expression of Vpx mutants in cells was examined in detail. We demonstrated that the zinc-binding site stabilizes the protein to keep its function in virus growth when low levels of Vpx are expressed. At higher levels of expression, Vpx aggregation could occur, and zinc binding would suppress such aggregation. Among the amino acids involved in zinc coordination, H39 plays the most critical role. In summary, zinc binding appears to mitigate flexibility of the three-helix fold of Vpx, thereby preventing dysfunction.

Introduction of H2C2‐type zinc‐binding residues into HIV‐2 Vpr increases its expression level

“…Previous work using sole expression from a plasmid vector also found a lower amount of HIV‐2 Vpr protein than of Vpx protein . Because Gag increases the protein amounts of Vpr and Vpx , we initially performed a similar experiment with or without the expression of HIV‐2 Gag. An expression vector for HIV‐2 Vpx (pEF‐Fvpx) or that for HIV‐2 Vpr (pEF‐Fvpr2) was transfected into 293T cells with or without the cotransfection of an expression vector for HIV‐2 Gag (pEF‐Fgag2) .…”

“…Because Gag increases the protein amounts of Vpr and Vpx , we initially performed a similar experiment with or without the expression of HIV‐2 Gag. An expression vector for HIV‐2 Vpx (pEF‐Fvpx) or that for HIV‐2 Vpr (pEF‐Fvpr2) was transfected into 293T cells with or without the cotransfection of an expression vector for HIV‐2 Gag (pEF‐Fgag2) . The expression level of Vpx protein expressed by 293T cells transfected with 25 ng of pEF‐Fvpx by this method is thought to be similar to the expression level of Vpx by a full‐genomic infectious clone , and thus 25 ng of pEF‐Fvpx or pEF‐Fvpr2 and 475 ng of pEF‐Fgag2 or pEF1/ Myc ‐HisA were used for the transfections.…”

“…Expression of HIV‐2 Vpx/Vpr in WT and mutants. (A) Immunoblot analysis of proteins in 293T cells transfected with an expression vector for HIV‐2 Vpx with a FLAG tag at its N terminus (pEF‐Fvpx) or that for HIV‐2 Vpr with a FLAG tag at its N terminus (pEF‐Fvpr2) (25 ng) along with the empty vector pEF1/ Myc ‐HisA or an expression vector for HIV‐2 Gag with a FLAG tag at its N terminus (pEF‐Fgag2) (475 ng). (B) Immunoblot analysis of proteins obtained after in vitro transcription/translation using pEF‐Fvpx or pEF‐Fvpr2 (500 or 25 ng).…”

“…with a FLAG tag at its N terminus (pEF‐Fvpx) and an expression vector for HIV‐2 Vpr (GH‐123/GL‐AN strain) with a FLAG tag at the same position (pEF‐Fvpr2) were previously constructed . An expression vector for HIV‐2 Gag (GH‐123/GL‐AN strain, the gag sequence originated from pGH‐123) with a FLAG tag at its N terminus (pEF‐Fgag2) was also previously constructed . Each mutation, 2KR, NWW, R83C, H38L, H76A or C81A, in the vpr sequence was introduced in the Eco RV– Xba I fragment of pEF‐Fvpr2 inserted into pBluescript SK(+) (Agilent, Santa Clara, CA, USA) by QuikChange site‐directed mutagenesis (Agilent), and the fragment was cloned into pEF‐Fvpr2 to yield each mutant.…”

A Dithiol Compound Binds to the Zinc Finger Protein TRAF6 and Suppresses Its Ubiquitination

“…Zinc also contributes to the protein folding, which is required for function and the stability as is the case of zinc finger proteins. These proteins do not have substrate‐binding pockets neighboring the coordinated zinc ion and thus can hardly be regarded as drug targets.…”