Identification of a Region within the Cytoplasmic Domain of the Subtype B Vpu Protein of Human Immunodeficiency Virus Type 1 (HIV-1) That Is Responsible for Retention in the Golgi Complex and Its Absence in the Vpu Protein from a Subtype C HIV-1

Abstract

The structure of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) is composed of a short Nterminal domain (NTD), a transmembrane domain (TM), and a cytoplasmic domain (CD). Previous studies have shown that the Vpu protein from subtype B HIV-1 is transported predominantly to the rough endoplasmic reticulum (RER)/Golgi complex compartments of the cell and is not incorporated into virions. Using a previously described VpuEGFP reporter system in which the Vpu protein was fused to the gene for enhanced green fluorescent protein (EGFP), we showed that the subtype B Vpu fusion protein was localized to the RER/Golgi region of the cell, similar to the native protein. In the present study, we show that fusion of the subtype C Vpu to EGFP results in a fusion protein that is transported to the cell surface. Using this reporter system, chimeric Vpu proteins in which the CD of the subtype B and C proteins were exchanged showed that the CD was sufficient for targeting the subtype B protein to the Golgi complex of the cell. Following identification of the cytoplasmic domain as being responsible for intracellular targeting, we then generated a series of mutants in which 13, 23, 31, 38, 51, and 56 amino acids were deleted from the cytoplasmic domain of subtype B Vpu. These deletion mutants were analyzed by SDS-PAGE for size, for membrane localization, and intracellular localization by confocal fluorescence microscopy. Our results indicate that the mutant with the carboxyl-terminal 13 amino acids deleted was still localized to the Golgi complex but mutants with 23, 31, 38, 51, and 56 amino acids from the carboxyl-terminus of the subtype B Vpu were transported to the cell surface. These results suggest that a signal for the retention of the subtype B Vpu within the Golgi complex resides in the second alpha-helical domain.

Equine Infectious Anemia Virus Gag Assembly and Export Are Directed by Matrix Protein through trans -Golgi Networks and Cellular Vesicles

“…The cellular distribution of viperin and its colocalization with EIAV Gag would positively support our finding that the TGN is involved in EIAV Gag assembly. Previous studies have shown that the HIV-1 Vpu protein is predominantly transported to RER/Golgi complex compartments (33). In our study, EIAV particles localized adjacent to the RER, and EIAV Gag multimers were surrounded by Vpu proteins (Fig.…”

“…4B). Previous studies have shown that the HIV-1 Vpu protein is transported predominantly to RER/Golgi complex compartments (33). Thus, we cotransfected Vpu-HA with EIAV Gag-GFP or HIV-1 Gag-GFP into 293T cells and evaluated their cellular distribution.…”

ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2

“…The prototypical BST-2 antagonist is the HIV-1 accessory protein Vpu (1,2). Vpu localizes primarily in endosomes and the trans-Golgi network (4,5), where it is thought to interact with BST-2. Vpu expression results in reduced levels of BST-2 on the host cell membrane (6 -8) and either the degradation (9 -12) or the sequestration of the host factor in intracellular compartments (7,13), leading to increased virus release.…”

Suppression of Tetherin-Restricting Activity upon Human Immunodeficiency Virus Type 1 Particle Release Correlates with Localization of Vpu in the trans -Golgi Network

“…Although Vpu appears to act on a host protein that exerts its restricting activity on HIV particle release at the cell surface, the most-studied subtype B Vpu was found to localize predominantly in the trans-Golgi network (TGN) and to a lesser extent in the ER and the recycling endosomes (23,42).…”

“…However, in contrast to the prototypical subtype B Vpu, the subtype C Vpu protein was found to localize both at the plasma membrane and in the Golgi complex (23). While ER localization is required for CD4 degradation, the artificial retention of Vpu in the ER appears to interfere with the enhancement of viral particle release, suggesting that Vpu needs to reach a post-ER compartment to mediate this function (32).…”