Unconventional p97/VCP-Mediated Endoplasmic Reticulum- to-Endosome Trafficking of a Retroviral Protein

Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the endo-plasmic reticulum (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, and yet it was not detectably secreted into cell superna-tants. Inhibition of intracellular trafficking by the drug brefeldin A (BFA), which interferes with the ER-to-Golgi secretory pathway, resulted in dramatically reduced intra-cellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT but was insen-sitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-de-pendent compartment. Treatment with endoglycosidase H indicated that Rem-CT does not traffic through the Golgi apparatus. 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 ungly-cosylated mutant, was colocalized with Rab5-positive (Rab5+) early endosomes. The expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (contain-ing a mutation of threonine to asparagine at position 31 [T31N]) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase p97/VCP rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5+ endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes. IMPORTANCE Mouse 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 uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nu-cleus 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 mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a trans-membrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking with-out passage through the Golgi apparatus. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host antiviral immunity.

Automated summary

The Rem-CT protein of MMTV, lacking a transmembrane domain or ER retention signal, is primarily localized in the ER and requires glycosylation and p97 ATPase for trafficking to early endosomes, suggesting a novel intracellular trafficking pathway.