ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway

Tao Zhou,Dylan A Frabutt,Kelley W Moremen,Yong-Hui Zheng

doi:10.1074/jbc.m115.675207

Abstract

Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.

Highlights

HIV-1 envelope (Env) glycoprotein is targeted to endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway for degradation after infecting cells
Treatment of these infected cells with an ERAD inhibitor kifunensine (KIF) significantly increased the Env expression in N2-NP cells (Fig. 2, C and D); KIF increased HIV-1 replication in N2-NP cells but not in N5-P cells (Fig. 2E). These results suggest that Env is degraded via ERAD, which is responsible for HIV-1 inhibition in N2-NP cells
In this report we studied the molecular mechanism of TSPOinduced HIV-1 Env degradation via ERAD and identified ER class I ␣-mannosidase (ERManI) as a critical initiator for the degradation

Summary

Introduction

HIV-1 envelope (Env) glycoprotein is targeted to endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway for degradation after infecting cells. We investigated how the four ER-associated glycoside hydrolase family 47 (GH47) ␣-mannosidases, ERManI, and ER-degradation enhancing ␣-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four ␣-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. Functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity These studies identify ERManI as a critical GH47 ␣-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process

Objectives

Results

Conclusion