Targeting of the Sendai virus C protein to the plasma membrane via a peptide-only membrane anchor

Abstract

Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPSFLKKILKLRGRR . . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic alpha-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.

FIG. 1.

Schematic representation of the SeV P gene. The P gene mRNAs are shown as a horizontal line, and ORFs expressed as proteins are shown as boxes. The 5 ribosomal start codons near the mRNA 5′ end are indicated. A nested set of 4 “C” proteins (C′, C, Y1, and Y2) are expressed during infection, and their mobility and abundance are shown in the Western blot on the bottom right. Cotranscriptional editing of the P gene mRNAs (bent arrow) fuses alternate ORFs in place, yielding P, V, and W proteins from ribosomes that initiate at AUG¹⁰⁴. The two domains of the C^1-204 protein (C^1-23 and C^24-204 or Y₁), are indicated. A summary of the activities of the domains is included on the left. indep., independent.

FIG. 2.

C protein constructs containing TOM between residues 23 and 24 are biologically active. 2fTGH cells were transfected with plasmids expressing TOM, TOM-C^24-204, or C^1-23-TOM-C^24-204, along with 3X-flag-Stat1 and 3X-flag-Stat2 (to monitor transfected p-Stat1 and p-Stat2 levels induced by IFN) or pISRE-luciferase (as a reporter for ISG expression). After 48 h, half of the cultures were treated with 1,000 IU of IFN-β. Cell extracts were prepared after 45 min post-IFN, and levels of 3X-flag-pY⁷⁰¹-Stat1 or 3X-flag-pY⁶⁸⁹-Stat2 were determined by Western blotting and normalized to actin levels (top panels). To monitor reporter gene expression, cells were transfected for 30 h, IFN treated, and harvested 18 h post-IFN, and the levels of firefly and Renilla luciferase activities were determined (bottom panel). The results are shown as a bar graph; error bars in the lower panel show the ranges of two independent, parallel experiments.

FIG. 3.

Intracellular location of C^1-23-GFP in yeast and animal cells. GFP, GFP with C^1-23 fused to its N terminus (C^1-23-GFP), or GFP carrying the Φ5 mutant peptide (F⁴L⁵I⁸L⁹L¹¹ all mutated to A), were expressed in S. cerevisiae (A) or 2fTGH cells (B). 2fTGH cells were also infected with either SeV in which a GFP-C^1-204 fusion protein is the only C protein expressed (its normal C gene is closed) (panel c, right) or with otherwise wt SeV expressing an unmodified GFP transgene similarly placed between the M and F genes (panel c, left). The yeast cells were photographed in a simple fluorescent microscope. The 2fTGH cells were examined in a confocal microscope (see Materials and Methods). The photos of the different GFP fusion proteins were exposed for different times to compensate for the relative stabilities of the various fusion proteins (11).

FIG. 4.

Biochemical evidence of C^1-23-directed membrane association. RED (which forms a stable tetramer) with C^1-23 or the Φ5A mutant peptide fused to its N terminus (C^1-23-RED and Φ5-RED) or the same constructs with the CAAX domain of K-Ras (tK) fused to their carboxy termini (C^1-23-RED-tK and Φ5-RED-tK) were expressed by transfection in 2fTGH cells. Cell extracts were prepared 48 hpt, and equal amounts of the PNS (lanes PNS of the immunoblots) were centrifuged on flotation gradients (see Materials and Methods). Three fractions were collected: the top of the gradient containing membranes (float), the intermediate fraction (interm), and the gradient pellet (pellet). The proteins present in each fraction were recovered (see Materials and Methods), and the relative amounts of RED proteins, transferrin receptor (TfR), and actin (not shown) were determined by Western blotting (A). The immunoblots of the different RED fusion proteins were exposed for different times to compensate for the relative instability of proteins containing the wt C^1-23 peptide (see Fig. 7) (11). The band intensities from two independent experiments (normalized to actin levels) were determined and are plotted as a bar graph in panel B.

FIG. 4.

Biochemical evidence of C^1-23-directed membrane association. RED (which forms a stable tetramer) with C^1-23 or the Φ5A mutant peptide fused to its N terminus (C^1-23-RED and Φ5-RED) or the same constructs with the CAAX domain of K-Ras (tK) fused to their carboxy termini (C^1-23-RED-tK and Φ5-RED-tK) were expressed by transfection in 2fTGH cells. Cell extracts were prepared 48 hpt, and equal amounts of the PNS (lanes PNS of the immunoblots) were centrifuged on flotation gradients (see Materials and Methods). Three fractions were collected: the top of the gradient containing membranes (float), the intermediate fraction (interm), and the gradient pellet (pellet). The proteins present in each fraction were recovered (see Materials and Methods), and the relative amounts of RED proteins, transferrin receptor (TfR), and actin (not shown) were determined by Western blotting (A). The immunoblots of the different RED fusion proteins were exposed for different times to compensate for the relative instability of proteins containing the wt C^1-23 peptide (see Fig. 7) (11). The band intensities from two independent experiments (normalized to actin levels) were determined and are plotted as a bar graph in panel B.

FIG. 5.

Colocalization of C^1-23-TOM-C^24-204 and Stat1-GFP at the plasma membrane. Plasmids expressing TOM-C^24-204 (a and b) or C^1-23-TOM-C^24-204 (c and d) were transfected into 2fTGH cells along with either GFP (a and c) or Stat1-GFP (b and d), as schematized on the left. The transfected cells were examined by confocal microscopy at 24 hpt.

FIG. 6.

C^1-23 of C^1-23-TOM-C^24-204 can be replaced with carboxy-terminal Hor K-Ras CAAX domains for colocalization with Stat1-GFP at the PM. Plasmids expressing P⁸P⁹-TOM- C^24-204 (a and b) or P⁸P⁹-TOM-C^24-204 in which the H- or K-Ras CAAX domains were fused to the carboxy termini of C^24-204 (P⁸P⁹-TOM-C^24-204-tH [c and d] and P⁸P⁹-TOM-C^24-204-tK [e and f]) were transfected into 2fTGH cells along with either GFP or Stat1-GFP as indicated on the left. The transfected cells were examined by confocal microscopy 24 hpt.

FIG. 7.

C^1-23 of C^1-23-TOM-Y₁ can be replaced with the H- or K-Ras CAAX domains for IFN-independent pY⁷⁰¹-Stat1 formation. (A) Plasmids expressing TOM, TOM-C^24-204, C^1-23-TOM-C^24-204, or P⁸P⁹-TOM-C^24-204 or the same constructs in which the H- or K-Ras CAAX domains (tH/tK) were fused to the carboxy termini of TOM or C^24-204 (as indicated) were transfected into 2fTGH cells along with 3X-flag-Stat1. Cell extracts were prepared at 48 hpt, and equal amounts of total cell proteins were examined by Western blotting for their levels of bulk 3X-flag-Stat1 (Stat1), p-3X-flag-Stat1 (p-Stat1), actin, and TOM proteins. The p-Stat1 levels (normalized to actin) are reported as a bar graph below. As a positive control, TOM-transfected cells were treated with 1,000 IU of IFN-β at 48 hpt and harvested 45 min later (IFN Ctrl); the endogenous p-Stat1, which is visible here, is indicated. This level of p-3X-FLAG-Stat1 was set to 100. The results shown are representative of three separate experiments. All the immunoblots were exposed for the same time. (B) Plasmids expressing TOM, C^1-23-TOM-C^24-204, C^1-23-TOM, TOM-C^24-204, or a mixture of the latter two constructs, as indicated, were transfected into 2fTGH cells along with 3X-flag-Stat1. For further details, see legend to panel A. The symbols beside the various TOM bands (lower panel) refer to the constructs listed below the lane numbers.