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.

In this study, we performed a mutational analysis to determine whether the mechanism by which HIV-2 Vpx confers the capacity for infectivity and viral replication in macrophages is solely dependent on its ability to degrade the host antiviral factor SAMHD1. Contrary to expectations, we demonstrated that P(109) in the C-terminal poly-proline motif of HIV-2 Vpx has two unique roles: to facilitate the specific degradation of SAMHD1 in macrophages, and to facilitate multimerization of Vpx, therefore preventing SAMHD1 degradation in the presence of high levels of Vpx.

Human immunodeficiency virus type 2 has two structurally similar proteins, Vpx and Vpr. Vpx degrades the host anti‐viral protein SAMHD1 and is expressed at high levels, while Vpr is responsible for cell cycle arrest and is expressed at much lower levels. We constructed a Vpr mutant with a high level of expression by replacing the amino acids HHCR/HHCH with a putative H2C2‐type zinc‐binding site that is carried by Vpx. Our finding suggests that during the evolution of Vpr and Vpx, zinc‐binding likely became a mechanism for regulating their expression levels.

Nef is one of the accessory proteins of the human immunodeficiency virus type 1 (HIV-1). Nef is translated from multiple-spliced mRNAs transcribed from the viral genome, whose mRNAs have a relatively long 5' untranslated region (5'UTR). Here, we identified a cis element in the 5'UTR of Nef mRNA essential for efficient Nef translation, which was named the Nef-translation essential region (NER). Mutants with a deleted NER in the 5'UTR of the HIV-1 NL4-3 strain showed an almost undetectable Nef expression owing to a low Nef translation efficiency. The NER of the NL4-3 strain was predicted to form putative stem loops. Although the 5'UTR showed significant but relatively low internal ribosome entry site (IRES) activity, the mechanism of 5'cap-dependent translation mainly contributed to the Nef translation from its Nef mRNA. Altogether, it was clarified that not only the 5' cap but also the NER in the 5'UTR is an essential cis element for efficient Nef translation, which is not a typical 5'-cap-dependent mechanism, and that there must be an as yet unknown mechanism using the NER for efficient Nef translation.

Nef is one of the accessory proteins of human immunodeficiency viruses. Here, we noted that the relative expression level of Nef(NL4-3) is much lower than that of NefJR-CSF in HEK293 cells. By evaluating the expression level using a Nef mutant, it was indicated that amino acids 129-206 of Nef(NL4-3), that is, the C-terminal region named NLAA129-206, could contain the region responsible for the induction of the low protein expression level. In addition, the expression levels of the enhanced green fluorescent protein and Renilla luciferase became extremely low with the fusion of NLAA129-206. Interestingly, the NLAA129-206-corresponding sequences of other Nef variants with relatively high expression levels also induced the extremely low protein expression level by fusion. These results suggest that the C-terminal region of Nef can generally induce an extremely low protein expression level. Here, we propose that the C-terminal region of Nef could become an excellent tool for the induction of an extremely low expression level of arbitrary proteins by attachment as fusion proteins.