Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Sep 6;174(6):1465-1476.e13.
doi: 10.1016/j.cell.2018.07.031. Epub 2018 Aug 16.

Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking

Affiliations
Free PMC article

Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking

Pengwei Zhang et al. Cell. .
Free PMC article

Abstract

Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.

Keywords: HIV Tat; HPV; cell-penetrating peptide; protein transduction domain; proximity ligation assay; retrograde; retromer; split GFP; virus.

Conflict of interest statement

Declaration of Interests

P.Z., G.M.D.S., and D.D. are inventors on a provisional patent application related to this work.

Figures

Figure 1.
Figure 1.. The basic region can be replaced by a cationic cell-penetrating motif.
(A) Sequence of the C-terminus of the L2 protein of various HPV types. Basic amino acids (red) downstream of the major FYL retromer binding site (purple) are shown. Numbers indicate position in the HPV16 L2 protein. The membrane-destabilizing sequence in HPV33 L2 is underlined. (B) Sequence of the C-terminus of wild-type and mutant HPV16 L2 proteins. (C and D) HeLa S3 cells were infected with wild-type (WT) or mutant HPV16 PsV stocks containing equal numbers of the HcRed reporter plasmid (corresponding to MOI of one for wild-type). Forty-eight hpi, flow cytometry was used to determine the fraction of fluorescent cells. The results were normalized to the fraction of cells infected by wild-type. The mean results and standard deviation of three or more independent experiments for each sample are shown. *p<0.05; **p<0.01. (E) HeLa S3 cells were transfected with RISC-free control siRNA (black bars) or siRNA targeting Vps29 (grey bars), followed by infection at MOI of one with wild-type or L2-Tat HPV16 PsV. Infectivity was measured (n=3) and displayed as in panel (C). (F) HeLa S3 cells were untreated (black bars) or treated with 250 nM γ-secretase inhibitor XXI for one h at 37°C (grey bars), and then infected as in panel E. Infectivity was measured (n=3) and displayed as in panel C. See also Figure S1 and Table S1.
Figure 2.
Figure 2.. The L2 basic sequence displays cell-penetrating activity.
(A) Amino acid sequence of HPV16 L2 C-terminal peptides containing the wild-type (WT) basic sequence or the six alanine (6A) or three arginine (3R) mutations conjugated to Alexa Fluor 488. 293T cells were mock-treated or incubated with fluorescent peptides for three h at 37°C and examined by confocal microscopy. (B) Schematic diagram and C-terminal sequences of GFP-L2 fusion proteins. (C) HaCaT and HeLa S3 cells were incubated with GFP-L2 fusion proteins for three h. Cells were examined by confocal microscopy, then treated with trypan blue (+TB) to quench extracellular fluorescence, and the same fields were re-imaged. (D) HeLa S3 and HaCaT cells were incubated with GFP-L2 fusion proteins for various times. Cells were then treated with trypsin, and fluorescence was measured by flow cytometry. Mean fluorescent intensity (MFI) was plotted at the indicated time periods.
Figure 3.
Figure 3.. The basic region of HPV16 L2 is not essential for virus binding and internalization.
(A) HeLa-sen2 cells were mock-infected or incubated at 4°C for two h at MOI of 20 with wild-type, 6A, or 3R HPV16 PsV. Non-permeabilized cells were stained with anti-L1 antibody (green) and examined by confocal microscopy. (B) HeLa-sen2 cells were treated as described in panel A, detached with EDTA, and stained with anti-L1 antibody. MFI of the cells was measured by flow cytometry and normalized to cells incubated with wild-type HPV16 PsV. Mean results and standard deviation are shown (n=3). ***p<0.001; ns, not significant. (C) HeLa-sen2 cells were treated as described in panel A with the following PsVs: wild-type HPV16 (WT), 6A mutant, 3R mutant, mutant with amphipathic CPP (Am), mutant with hydrophobic CPP (Hy), and L1-only PsV (L1). After two h at 4ºC, cells were washed, lysed, and bound virus was assessed by SDS-PAGE and blotting for L1 (top panel). GAPDH is a loading control (bottom panel). (D) HeLa cells-sen2 were mock-infected or infected at MOI of 50 at 4°C for two h with wild-type, 6A or 3R HPV16 PsV and then washed and shifted to 37°C for eight or 16 h to allow internalization. Permeabilized cells were stained with anti-L1 antibody (green) and examined by confocal microscopy. (E) HeLa-sen2 cells were infected as described in panel D and harvested by trypsinization 6 h after shift to 37ºC. Permeabilized cells were stained with anti-L1 antibody and analyzed by flow cytometry. MFI of the cell populations was normalized to cells infected with wild-type HPV16 PsV. Mean results and standard deviation are shown (n=3). *p<0.05. See also Figure S4.
Figure 4.
Figure 4.. The 3R mutant accumulates in endosomes.
HeLa-sen2 cells were mock-infected or infected at 37ºC at MOI of 100 with wild-type, 3R or retromer binding site (DM) mutant HPV16 PsV. At eight and 16 hpi, PLA was performed with anti-L1 antibody and either an EEA1 (panel A) or a TGN46 (panel B) antibody. PLA signal is green. Multiple images obtained as in left panels were processed with BlobFinder software to determine the fluorescence intensity per cell. The graphs in the right panels show mean results and standard deviation (n=3), in which the results for the EEA1/L1 samples and the TGN46/L1 samples were normalized to those of cells infected with wild-type HPV16 PsV at eight and 16 hpi, respectively. Black bars, 8 hpi; grey bars, 16 hpi **p<0.01; ***p<0.001.
Figure 5.
Figure 5.. The 3R mutant is defective in accessing retromer during HPV infection.
(A) HeLa-sen2 cells were infected as in Figure 4. At eight and 16 hpi, PLA was performed with anti-L1 antibody and an antibody recognizing Vps35 to assess L1 in proximity to retromer. (B) Multiple images obtained as in panel A were processed and displayed as in Figure 4 with the mutant samples normalized to that of cells infected with wild-type PsV at eight hpi. Black bars, wild-type; dark grey bars, 3R mutant; light grey bars, DM mutant. *p<0.05; **p<0.01; ***p<0.001. (C) GST-tagged retromer adsorbed to glutathione beads was incubated with GFP-L2 fusion proteins. After pull-down, proteins bound to retromer were separated by SDS-PAGE and detected by blotting with anti-GFP antibody (top panel). Bottom panel shows input GFP-L2 fusion proteins. (D) Top. Sequences of wild-type and mutant biotinylated peptides. Bottom. Biotinylated peptides were incubated with extracts of uninfected HeLa S3 cells, pulled-down with streptavidin, and separated by SDS-PAGE. Retromer associated with the peptide was detected by blotting for Vps35.
Figure 6.
Figure 6.. Cell-penetrating peptides for several HPV types can deliver fusion proteins into the cytoplasm.
(A) Schematic diagram of GFP11 fusion proteins containing seven tandem copies of GPF11 (green) linked to amino acids 434 to 455 of HPV16 L2 (blue) and a CPP (red). (B) HaCaT/GFP1–10NES cells were mock-incubated or incubated with GF11–16L2–16CPP or GFP11–16L2-TatCPP for three h. In bottom panels, cells were then treated with trypan blue to quench extracellular fluorescence. Cells were examined by confocal microscopy. (C) Cells were treated as in panel B except there was no trypan blue treatment. GFP11 fusion proteins containing the following CCPs were used: HIV Tat, HPV16 (wild-type, 6A, and 3R mutants), HPV5, HPV18, and HPV31. See Figure S2.
Figure 7.
Figure 7.. Reconstitution of split GFP by infection with PsV containing L2-GFP11.
(A) Schematic diagrams of L2 protein with tandem GFP11 (green) inserted upstream of RKRRKR (L2-GFP11-CPP) or at the C-terminus of L2 (L2-CPP-GFP11). The bulk of the L2 protein is shown in blue; CPP in red; and retromer binding sites in purple. See Figure S3A for sequences. (B) Clonal HaCaT/GFP1–10NES cells were infected at MOI of 2000 with untagged HPV16 PsV or with a PsV containing GFP11-tagged L2. Three hpi, cells were examined by confocal microscopy for GFP fluorescence. (C) As in panel (B), except cells were infected with HPV5 PsV containing untagged or GFP11-tagged L2. (D) Fluorescence of cells in images as in panel B was quantified for ~250 cells for each condition. Results show the fluorescence intensity of individual cells plotted in arbitrary units from three independent experiments. ***p<0.0001. (E) Cells were infected as described in panel B with GFP11-tagged PsV containing the wild-type or the 3R mutant CPP. Three hpi, cells were examined by confocal microscopy for GFP fluorescence. See Figures S2 to S5.

Similar articles

See all similar articles

Cited by 18 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback