Unconventional ER to Endosomal Trafficking by a Retroviral Protein

Abstract

ABSTRACTMouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the ER membrane into a functional N-terminal signal peptide (SP) and the C-terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, yet was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug Brefeldin A (BFA), which interferes with the ER to Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels. A Rem mutant lacking glycosylation sites was cleaved into SP and Rem-CT, but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not exit the ER or reach a degradative compartment. BFA reduction of Rem-CT levels was not rescued by proteasome or lysosomal inhibitors. Rem-CT has simple glycans, which are necessary for Rem-CT stability and trafficking, but indicate that Rem-CT does not traffic through the Golgi. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, were co-localized with Rab5+ endosomes. Expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (T31N) mimicked the effects of BFA by reducing Rem-CT levels, suggesting that Arf1 prevents Rem-CT localization to a degradative compartment. A DN form of the AAA ATPase, p97/VCP, rescued Rem-CT in the presence of BFA or DN Arf1. Thus, Rem-CT uses an unconventional trafficking scheme, perhaps to thwart innate immunity to MMTV infection.IMPORTANCEMouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP eludes ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used multiple methods to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation for trafficking to endosomes. Blocking of retrograde trafficking through Arf1 reduced Rem-CT levels, but was not restored by lysosomal or proteasomal inhibitors. The unique trafficking of Rem-CT suggests a novel intracellular trafficking pathway, potentially impacting host anti-viral immunity.