Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function

Abstract

Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the N-terminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavage-site mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function.

Fig. 1.

MMTV mRNAs expressing either Rem or Env generate functional SP. (A) Domain structure of Rem and SP. The Rem C terminus contains an in-frame fusion of deleted versions of the Env protein (SU and TM proteins). Stars indicate glycosylation sites. ARM, arginine-rich RNA-binding motif; NES, nuclear export sequence; NoLS, nucleolar localization sequence. (B) MMTV-infected and transfected mammary cells express SP. GR-B2 mammary tumor cell extracts (lane 1) were compared with extracts from HC11 cells infected with MMTV (lane 2) or stably transfected with HYB-MTV (lane 3) by Western blotting with SP-specific antibody. Uninfected HC11 cell extracts are in lane 4. PrEnv, envelope precursor. (C) Structure of the pHMRluc reporter vector relative to the MMTV env and rem mRNAs. The reporter vector is derived from the 3′ end of the C3H-MMTV genome inserted downstream of the CMV promoter. SA, splice acceptor; SD, splice donor; x, mutation of the env splice donor. (D) Dose-dependent activity of the rem and env mRNAs on the pHMRluc vector. HC11 cells were transiently transfected with the indicated amount of expression plasmid. The averages of triplicate assays ± SDs are shown after normalization. Assays using the pHMRluc vector alone (in the absence of Rem or Env) were assigned a relative value of 1. (E) Western blotting of rem- and env-transfected HC11 cells. Western blots of extracts from transfected cells were incubated with SP-specific antibody. SP was not detected in cells transfected with 10 ng of the rem and env expression vectors (lanes 1 and 4).

Fig. 2.

Glycosylation of the Rem C terminus is required for Rem function but not for cleavage. (A) Western blotting of 293T cells transfected with 250 ng of pHMRluc, 250 ng of pGL3, and 5.5 μg of wild-type rem or glycosylation-site mutants as indicated. The three forms of Rem detected with SP-specific antibody (upper arrows) include Rem glycosylated at positions 127 and 143 (lane 2; 38-kDa band), at amino acid 143 or 127 only (lanes 3 and 4), or at neither position (lane 5). (B) Rem function requires at least one functional glycosylation site. Transient transfections of 293T cells were performed using the indicated amount of wild-type rem or glycosylation-site mutant constructs. Luciferase assays are reported as in Fig. 1D.

Fig. 3.

Signal peptidase cleavage is required for Rem function. (A) Rem diagram showing signal peptidase cleavage-site mutations. (B) Western blotting with SP-specific antibody reveals that cleavage-site mutations in either the rem or env expression vectors prevent SP generation. Constructs were transiently transfected in HC11 cells. Wild-type, G98R, or V96RG98R proteins are in lanes 1 and 4, lanes 2 and 5, and lanes 3 and 6, respectively. In the upper panel, the positions of the Env precursor (PrEnv), glycosylated Rem, unglycosylated Rem, and SP are shown. Rem detected in lanes 5 and 6 results from additional splicing of env mRNA. The lower panel shows Western blotting of the same extracts using actin-specific antibody. (C) Mutation of the signal peptidase cleavage site in either the rem or env expression construct prevents SP function. Transient transfections in HC11 cells were performed with concentrations of each expression plasmid as indicated. Luciferase assays are reported as in Fig. 1D.

Fig. 4.

GFP-tagged rem constructs defective for signal peptidase cleavage lack fluorescence. (A) Single and double cleavage-site mutants lack fluorescence in 293T cells. Cells were transiently transfected with 0.25 μg of wild-type rem or 6 μg of mutant constructs together with 0.2 μg of the expression construct ER-mCherry. Cells were stained with DAPI and visualized by fluorescence microscopy. (B) Quantitation of fluorescence by wild-type EGFP-tagged Rem compared with cleavage-site mutants. The percentage of mCherry+ and GFP+ cells compared with the number of DAPI+ cells in each of three fields was determined. The averages and SDs are given. The percentage of mCherry+ cells was statistically lower for the double mutant than for the wild-type rem or G98R constructs. The percentage of GFP+ cells was statistically lower for the mutants than for wild-type transfectants. (C) Western blotting of GFP-tagged Rem and cleavage-site mutants. Cells were transfected as described in A (ca. 20-fold higher levels of mutant DNA than in the wild type); extracts were used for Western blotting with GFP- or actin-specific antibodies.

Fig. 5.

A DN ATPase p97/VCP decreases SP activity but not cleavage. (A) Western blotting of 293T cells transfected with different amounts of GFPrem construct in the absence or presence of a DN p97 (p97QQ). Extracts were subjected to Western blotting with antibodies specific for GFP (Top), actin (Middle), or p97 (Bottom). (B) DN p97QQ inhibits Rem function. Two different amounts of GFPrem were transfected with or without the p97QQ expression construct. Luciferase activity is reported as in Fig. 1D.