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.

Here, we show that the basic segment in HPV16 L2 has CPP activity that mediates passage of the C terminus of the L2 protein through the endosomal membrane into the cytoplasm after virus endocytosis. This activity allows retromer to bind L2 and initiate retrograde transport of the capsid to the TGN. Thus, the physiologic role of this CPP is to mediate transfer of a protein segment from one cellular compartment to another, thereby enabling a protein-protein interaction essential for virus infection.

The C-terminal segment of all sequenced papillomavirus L2 proteins contains a stretch of basic amino acids near the retromer binding sites ( Figure 1 A; Table S1 ). This basic segment is present in the membrane-destabilizing peptide identified byand is thought to act as a nuclear import signal during capsid assembly (). This segment resembles a class of peptides known as cationic cell-penetrating peptides (CPPs), which can transport proteins across the plasma membrane into cells (). The prototype cationic CPP from the human immunodeficiency virus (HIV) Tat protein is RKKRRQRRR (where R, K, and Q represent arginine, lysine, and glutamine, respectively) and the basic sequence in HPV16 L2 is RKRRKR. Certain hydrophobic and amphipathic sequences also display cell-penetrating activity. The mechanisms of membrane penetration are not fully understood, but most cationic CPPs appear to undergo endocytosis (). Although CPPs have been widely studied as agents to deliver molecules into cells for therapeutic purposes, there are few cases where the physiological role of cell-penetrating activity is known. In the nervous system, homeobox proteins such as Engrailed are secreted and then enter neighboring cells in a paracrine fashion to specify various aspects of neuronal function (). The Tat protein can be secreted from HIV-infected cells, but it has not been conclusively shown that secreted Tat plays a role in virus replication or pathogenesis (). A short basic sequence at the C terminus of the HPV11 L1 major capsid protein displays CPP activity, but the role of this activity in HPV infection is not known ().

It is not known how the L2 protein accesses the cytoplasm during infection. Several years ago,reported that peptides derived from the C terminus of HPV16 or HPV33 L2 destabilized membranes at low pH, resulting in cell death. Furthermore, this L2 segment was required for infection of fibroblasts by HPV16 and bovine papillomavirus 1, and pseudoviruses (PsVs) carrying mutations in this segment accumulated in an endo-lysosomal compartment. At the time of that report, papillomaviruses were thought to exit directly from the endosome into the cytoplasm, and the involvement of retromer and retrograde trafficking in infection was not known. Therefore, the contribution of this membrane-destabilizing sequence to cytoplasmic exposure of L2 was not explored.

This model poses a topological challenge because the incoming virus is in the lumen of the endosome but retromer is in the cytoplasm. To resolve this, we and others have proposed that the C terminus of the L2 protein protrudes through the endosomal membrane into the cytoplasm, so its binding sites are accessible to retromer (). Indeed, the central portion of the L2 protein also binds to additional cytoplasmic proteins such as SNX17 and SNX27 required for HPV entry (), suggesting that much of the L2 protein is in the cytoplasm. In addition, the L2 protein contains an N-terminal hydrophobic segment that can act as a TM domain, implying that the L2 protein adopts a TM existence during infection (). This model is supported by antibody staining and protease sensitivity experiments showing that the bulk of the L2 protein is cytoplasmic by 18 hr after infection, other than a short N-terminal segment upstream of the putative TM domain ().

A key step in HPV infection is the entry of the capsid into the retrograde transport pathway. This pathway normally recycles cellular proteins in the endosome to the trans-Golgi network (TGN) for reuse. A genome-wide small interfering RNA (siRNA) screen revealed that numerous retrograde transport factors are required for HPV entry and identified retromer as being essential for transport of the virion from the endosome to the TGN (). Retromer, a trimeric protein complex of Vps26, Vps29, and Vps35, resides in the cytoplasm where it binds to the cytoplasmic domain of cellular transmembrane (TM) proteins in the endosome membrane and sorts them into transport vesicles that bud from the endosome (). These vesicles are then transported to the TGN where membrane fusion deposits the protein cargo in the latter organelle. Retromer directly binds to conserved sites in the C-terminal segment of the HPV16 L2 protein ( Figure 1 A), and retromer knockdown or mutation of these binding sites causes the capsid to accumulate in the endosome and prevents it from reaching the TGN (). Thus, HPV is a novel class of retromer cargo.

(E) HeLa S3 cells were transfected with RNA-induced silencing complex (RISC)-free control siRNA (black bars) or siRNA targeting VPS29 (gray bars), followed by infection at MOI of one with wild-type or L2-Tat HPV16 PsV. Infectivity was measured (n = 3) and displayed like in (C).

(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). 48 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 (C). (D) Cells were treated and the results are displayed as in (C), except different mutants were analyzed. ∗ p < 0.05; ∗∗ p < 0.01.

(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 the position in the HPV16 L2 protein. The membrane-destabilizing sequence in HPV33 L2 is underlined.

The HPV virion consists of the 8-kb DNA genome in a capsid comprising 360 molecules of the L1 major capsid protein and up to 72 copies of the L2 minor capsid protein, which is largely buried in the L1 protein shell but plays an essential role in the trafficking of viral DNA to the nucleus (). HPV infection is initiated by the binding of L1 to heparan sulfate proteoglycans on the cell surface (), which triggers conformational changes in the capsid and proteolytic cleavage of L1 and the N terminus of L2 (). The capsid is then thought to be transferred to a specific cell-surface internalization receptor, whose identity remains controversial (). After endocytosis, acidification of the endosomal lumen exposes the capsid to low pH, which is required for further progression of infection (). The protease γ-secretase is also required for proper HPV trafficking by facilitating association of L2 with membranes ().

Cell membranes pose formidable barriers to the passage of proteins and non-enveloped viruses. Unlike enveloped viruses, which deliver virion contents into the cytoplasm by membrane fusion, non-enveloped viruses need to cross or disrupt membranes during virus entry (). For most non-enveloped viruses, a lytic peptide released by proteolytic cleavage of a capsid protein physically disrupts the membrane or forms a large pore, allowing passage of the capsid into the cytoplasm. Human papillomaviruses (HPVs) are responsible for 5% of human cancer, most notably cervical cancer. These non-enveloped viruses have evolved a unique mode of intracellular trafficking during entry, in which viral material is sequestered in membrane-bound retrograde transport vesicles until late during entry (). This mechanism is thought to protect the virion from cytoplasmic innate immune sensors until the viral DNA has reached safe haven in the nucleus, where viral genome expression and replication occur.

To determine whether L2 protrusion required cell-cycle progression, we treated cells with aphidocolin, which causes S-phase arrest and blocks HPV infection by inhibiting translocation of the incoming virus into the nucleus late during entry (). As shown in Figure S5 , aphidocolin inhibited infection by PsV containing GFP11 but did not decrease the reconstituted GFP signal, showing that cell-cycle progression and nuclear membrane breakdown are not required for exposure of the C terminus of L2 in the cytoplasm during infection.

(A) HeLa S3 cells were left untreated or treated with 6 μM aphidicolin. Twenty-four h later, cells were infected at MOI of one with wild-type HPV16 PsV or PsV containing L2-GFP11-CPP or L2-CPP-GFP11. Forty-eight hpi, the fraction of infected cells was determined by flow cytometry for HcRed fluorescence and normalized to untreated cells infected with wild-type HPV16 PsV. (B) HaCaT/GFP1-10NES cells were infected at an MOI of 2000 with wild-type HPV16 PsV or PsV containing GFP11-tagged L2 in the presence or absence of 6 μM aphidicolin. Three hpi, cells were examined by confocal microscopy for reconstituted GFP fluorescence. Fluorescence intensity of individual cells (∼250 for each condition) is plotted in arbitrary units from two independent experiments. Mean results and SD are indicated. There were no statistically significant differences among samples infected with either GFP11-tagged PsV in the presence or absence of aphidocolin.

To test whether the L2 CPP was required for membrane protrusion, we produced PsV in which the wild-type CPP in L2-GFP11-CPP and L2-CPP-GFP11 was replaced with three arginines ( Figure S3 A). As expected, the 3R mutation did not inhibit PsV internalization but blocked infection ( Figures S3 B and S4 B). In HaCaT/GFP1-10NES cells infected with these mutant PsVs, there was significantly less reconstituted fluorescence compared to cells infected with PsV with wild-type CPP ( Figures 7 D and 7E). This result provides direct evidence that the L2 CPP is required for cytoplasmic exposure of the L2 C terminus during infection.

We also produced PsV with GFP11 fused to the C terminus of the L2 protein from HPV5, a β-type HPV that infects skin and is associated with skin cancer. As shown in Figure 7 C, reconstituted cytoplasmic GFP was also generated by HPV5-GFP11-CPP PsV infection. Thus, early during infection by two distinct HPV types, the C terminus of at least a fraction of L2 molecules protruded into the cytoplasm.

The experiments described above showed that fusion proteins containing the HPV L2 CPP could enter the cytoplasm. Next, we determined whether the C terminus of L2 carried in the intact virion protruded into the cytoplasm during infection. We inserted seven tandem copies of GFP11 at two different positions in the C terminus of the HPV16 L2 protein, between the FYL retromer binding site and the CPP (L2-GFP11-CPP) or at the extreme C terminus of L2 (L2-CPP-GFP11) ( Figures 7 A and S3 A) and tested the activity of PsV stocks containing L2 with the inserted GFP11 segments. Inserted GFP11 did not impair HPV16 PsV infectivity in HeLa cells ( Figure S3 B). In HaCaT/GFP1-10NES cells, GFP11 had a modest (<50%) inhibitory effect on infectivity, and infection remained sensitive to γ-secretase inhibition ( Figure S3 C). We then infected HaCaT/GFP1-10NES cells at high MOI with HPV16 PsV with or without GFP11-tagged L2 protein and examined the cells by confocal microscopy. As shown in Figure 7 B, fluorescence was not detectable in HaCaT/GFP1-10NES cells infected with wild-type HPV16 PsV lacking the GFP11 insert, confirming that the GFP1-10NES protein did not fluoresce on its own. Similarly, as expected, infection of unmodified HaCaT cells with HPV16 PsV containing GFP11-tagged L2 did not generate a fluorescent signal (data not shown). In contrast, infection of HaCaT/GFP1-10NES cells with PsV containing GFP11 inserted at either C-terminal position in L2 resulted in reconstituted cytoplasmic fluorescence in ∼60%–90% of cells, demonstrating that the C terminus of L2 protrudes into the cytoplasm during infection ( Figure 7 B). Reconstituted fluorescence showed either a punctate distribution or a more uniform distribution throughout the cytoplasm (or both) and was evident at a low level by 1.5 hpi and increased by 3 hpi ( Figures 7 B and S4 A). Notably, reconstituted fluorescence did not display peripheral cell-surface localization at any time point examined.

(C) Mock-infected HaCaT/GFP1-10NES cells (M) or cells infected with wild-type HPV16 PsV (WT) or PsV containing L2-GFP11-CPP (GC) or L2-CPP-GFP11 (CG) were subjected to high pH wash to remove surface-bound virions, harvested by trypsinization, and lysed at the indicated hpi. Input PsV and lysates were subjected to electrophoresis and immunoblotting with an antibody to HPV16 L2. The wild-type and GFP11-tagged L2 proteins are indicated by thin and thick arrowheads, respectively. Non-specific bands reactive with the anti-L2 antibody and present in all lysates, including those from mock-infected cells, are indicated with asterisks. Membrane was stripped and reprobed for GAPDH as a loading control.

(B) HeLa S3 cells were infected at MOI of one with the indicated HPV16 PsV containing an HcRed reporter gene. After 48 h, the fraction of fluorescent cells was determined by flow cytometry and normalized to cells infected with wild-type PsV. Mean results and standard deviation are shown (n = 3).

(D) Fluorescence of cells in images like in (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. The mean results and SDs are indicated. ∗∗∗ p < 0.0001.

(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 S3 A for sequences.

Next, we constructed fusion proteins consisting of seven tandem copies of GFP11 fused to a C-terminal segment of HPV16 L2 extending from 22 residues upstream of the CPP to the end of the basic sequence ( Figure 6 A). As a control protein, the L2 CPP was replaced with the HIV Tat CPP. Incubation of HaCaT/GFP1-10NES cells with purified GFP11 fusion proteins containing the wild-type L2 or Tat CPP generated a fairly uniform, cytoplasmic, reconstituted GFP signal ( Figure 6 B). Fluorescence was resistant to trypan blue treatment, showing that it was intracellular. Similarly, when the CPP from HPV16 L2 was replaced by the basic segment from HPV5, HPV18, or HPV31, reconstituted intracellular fluorescence was observed ( Figure 6 C), whereas GFP11 fusion proteins containing the 3R or 6A mutant HPV16 L2 CPP were markedly impaired for cytoplasmic delivery. These results demonstrate that the L2 CPP from several HPV types could deliver the C terminus of a fusion protein into the cytoplasm of HaCaT cells.

(C) Cells were treated like in (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.

We first constructed a clonal HaCaT cell line expressing GFP1-10 linked at its C terminus to a nuclear export signal (NES) ( Figure S2 A). Immunofluorescence experiments with an anti-GFP antibody confirmed cytoplasmic expression of GFP1-10NES ( Figure S2 B). As expected, cells expressing this construct without GFP11 displayed minimal fluorescence (data not shown; see also Figures S2 E, 6 B, and 6C). We then conducted control experiments to establish that GFP fluorescence in HaCaT/GFP1-10NES cells indicated the presence of GFP11 in the cytoplasm. We used CD8-cation-independent mannose phosphate receptor (CD8-CIMPR) fusion proteins consisting of the extracellular domain of CD8 fused to the TM and cytoplasmic domains of CIMPR (). We inserted seven tandem copies of GFP11 at either the N terminus or the C terminus of CD8-CIMPR to generate GFP11-CD8-CIMPR and CD8-CIMPR-GFP11, respectively ( Figure S2 C) (). When expressed, these proteins adopt a type 1 TM orientation with the GFP11 segment located in the extracellular/luminal space for GFP11-CD8-CIMPR and in the cytoplasm for CD8-CIMPR-GFP11 ( Figure S2 D). Neither construct fluoresced on its own when transfected into unmodified HaCaT cells (data not shown). We then transfected HaCaT/GFP1-10NES cells with a plasmid expressing GFP11-CD8-CIMPR or CD8-CIMPR-GFP11 and assessed fluorescence. As shown in Figure S2 E, bright cytoplasmic fluorescence was observed after expression of CD8-CIMPR-GFP11 (containing cytoplasmic GFP11), but not after expression of GFP11-CD8-CIMPR. Additional control experiments showed that GFP11-CD8-CIMPR reconstituted fluorescence in cells expressing luminal GFP1-10 ( Figures S2 C, S2D, and S2F). Taken together, these results validate the split GFP reporter system by showing that fluorescence is reconstituted in HaCaT/GFP1-10NES cells only when the GFP11 segment is in the cytoplasm and establish that GFP1-10NES does not access the luminal space.

(C) Schematic diagram of CD8-CIMPR and calnexin (CNX) fusion proteins used to validate the split GFP system. All proteins contain a TM domain (TM) (light blue). The GFP11 segment (green) is in the luminal domain of GFP11-CD8-CIMPR and the cytoplasmic domain of the CD8-CIMPR-GFP11. Both proteins contain a WLM-to-AAA mutation (red) that inactivates the retromer binding site in the cytoplasmic segment. The GFP1-10 segment (green) is in the luminal domain of GFP1-10-CXN and the cytoplasmic domain of CXN-GFP1-10.

To directly demonstrate membrane passage of the L2 C terminus during virus entry, we adapted a split GFP assay (). A protein consisting of GFP beta strands 1 to 10 (GFP1-10) does not fluoresce, nor does the 16-residue beta strand 11 of GFP (GFP11). However, when GFP11 is in the same cellular compartment as GFP1-10, GFP is reconstituted, generating a fluorescent signal. This approach was used to demonstrate cytoplasmic delivery of soluble fusion proteins linked to CPPs ().

The 3R mutation could inhibit retromer association directly, by impinging on the retromer binding sites, or indirectly, by preventing the exposure of the binding sites in the cytoplasm. To determine whether mutations in the L2 CPP directly inhibited binding to retromer, we performed in vitro pull-down experiments. Glutathione S-transferase (GST)-tagged retromer subunits were expressed in bacteria, purified, assembled into the trimeric retromer complex, and bound to glutathione beads. The purified GFP-L2 fusion proteins containing a wild-type L2 segment, the 3R mutation, or the DM mutations in the retromer binding sites were incubated with the retromer beads at 4°C, pelleted, and subjected to western blotting with an anti-GFP antibody to detect the L2 fusion protein in the pellet. As expected, the wild-type protein bound retromer well and the DM mutant bound poorly ( Figure 5 C). Notably, the 3R mutant L2 fusion protein also bound retromer well, demonstrating that this mutation did not inhibit the intrinsic ability of L2 to bind retromer. We also incubated biotinylated wild-type and mutant C-terminal L2 peptides with detergent lysates of uninfected HeLa cells and performed streptavidin pull-down experiments and western blotting for Vps35. Similar to the results with purified fusion proteins, those for the 3R mutation indicate that it did not affect retromer binding to the L2 peptide, whereas binding was abolished by the retromer binding site mutations ( Figure 5 D). Therefore, the 3R mutation does not directly interfere with retromer binding, and we conclude that the 3R mutant displays impaired retromer association in infected cells because it cannot protrude through the endosomal membrane and access retromer in the cytoplasm.

Next, we tested whether the 3R mutation impaired association between the capsid and retromer in infected cells. HeLa cells were infected with either wild-type or mutant HPV16 PsV, and PLA was performed with an anti-L1 antibody and an antibody recognizing Vps35, a subunit of retromer. As shown in Figure 5 A, wild-type PsV generated abundant Vps35/L1 PLA signal at 8 hpi, while the signal was diminished by 16 hr, after the virus exited from endosomes and dissociated from retromer. As expected, the PLA signal for DM mutant was very weak at both 8 and 16 hr. Interestingly, we observed ∼75% reduction in the PLA signal for the 3R mutant at 8 hr, and this signal decreased further by 16 hr ( Figures 5 A and 5B), despite the accumulation of the mutant in the endosome at this later time point.

(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.

To examine the post-internalization defect of the 3R mutant, we used the proximity ligation assay (PLA) to determine the localization of incoming wild-type and 3R mutant HPV16 PsV. PLA is an immune assay used to test whether proteins of interest are within 40 nm. PLA was performed with an anti-L1 antibody and an antibody that recognizes either EEA1, a marker of the early endosome, or the TGN marker, TGN46. The L2 double mutant (DM) mutant, which lacks retromer binding sites, was used as a control. As shown in Figure 4 A, these antibodies did not generate a PLA signal in mock-infected cells. At 8 hr post-infection (hpi), punctate intracellular EEA1/L1 PLA signal was observed in cells infected with wild-type PsV or either mutant and showed similar fluorescence intensity, confirming that the 3R mutant, like DM, efficiently entered cells and reached the endosome. At 16 hpi, the EEA1/L1 PLA signal of cells infected with wild-type PsV was significantly diminished, due to the departure of the virion from the endosome, whereas the signal for the DM in the endosome was markedly increased, reflecting endosomal accumulation because of the absence of retromer binding sites () ( Figure 4 A). Notably, the EEA1/L1 PLA signal of the 3R mutant at 16 hpi was increased ∼3-fold compared to the 8-hr signal, similar to the increase seen with DM mutant. Thus, the 3R mutant accumulated in the endosome. As expected, little TGN46/L1 PLA signal was generated at 8 hr after wild-type or mutant PsV infection ( Figure 4 B). At 16 hpi, cells infected with wild-type displayed abundant TGN46 PLA signal, reflecting delivery of L1 to this distal site. In contrast, 3R and DM mutants showed a greatly reduced TGN46/L1 PLA signal, indicating that these mutants did not arrive at the TGN. These results indicate that 3R mutation causes accumulation of the incoming virus particle in the endosome and prevents its delivery to the TGN, a phenotype similar to the mutant that cannot bind retromer. Together with the reduced infectivity of the 3R mutant and the impaired cell entry of 3R mutant fusion proteins, these data suggest that the trafficking defect displayed by 3R mutant is due to impaired endosomal membrane penetration and retromer association.

(A and B) HeLa-sen2 cells were mock-infected or infected at 37°C at MOI of 100 with wild-type, 3R, or retromer binding site (DM) mutant HPV16 PsV. At 8 and 16 hpi, PLA was performed with anti-L1 antibody and either an EEA1 (A) or a TGN46 (B) antibody. PLA signal is green. Multiple images obtained like in the 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 8 and 16 hpi, respectively. Black bars, 8 hpi; gray bars, 16 hpi. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Next, we examined the role of the L2 CPP in virus internalization. After incubation of cells with HPV16 PsVs at 4°C, we shifted cells to 37°C for 8 or 16 hr to allow internalization, which was assessed by immunofluorescence and by flow cytometry. The 3R L2 mutant internalized into cells as well as wild-type, whereas the 6A mutant showed much less internalized L1, as expected because of its cell-surface binding defect ( Figures 3 D and 3E). This result showed that the short basic segment of three arginines could support cell-surface binding and virus internalization, even though it was not sufficient to restore infectivity or mediate membrane penetration. Thus, the L2 CPP is required for an intracellular event after cell binding and internalization.

Because the 6A mutation affected binding, we also tested HPV16 PsV containing L2 with the amphipathic or hydrophobic CPP in place of the wild-type sequence. As shown in Figure 3 C, both of these mutants bound cells well, indicating that the severe infection defect of these mutants was not due to impaired cell-surface binding.

To determine the role of the L2 CPP in HPV infection, we first conducted cell binding experiments. HeLa cells were incubated with either wild-type or mutant HPV16 PsV for 2 hr at 4°C, followed by washing to remove unbound viruses. Non-permeabilized cells were stained with an L1 antibody, and immunofluorescence was performed to detect viruses stably bound to cells. As shown in Figure 3 A, similar amounts of wild-type and 3R mutant PsV bound cells. Thus, a mutation that inhibits the cell penetrating activity of L2 did not affect virus binding to cells, showing that L2 CPP activity was not required for binding. Unexpectedly, the 6A mutation resulted in a dramatic reduction of cell binding. Similar results were obtained when cell binding was assessed by flow cytometry or western blotting for L1 ( Figures 3 B and 3C). Because the cell binding defect of the 6A mutant conflicted with published studies showing efficient binding of capsids lacking L2 (e.g.,), we also tested binding with PsV comprising L1 only. As shown in Figure 3 C, L1-only PsV bound cells to a similar level as complete PsV containing L1 plus wild-type L2, far better than PsV containing the 6A mutant. These results imply that although the L2 CPP is not required for cell-surface binding, sequences in the C terminus of L2 can modulate binding.

(E) HeLa-sen2 cells were infected as described in (D) and harvested by trypsinization 6 hr after shift to 37°C. 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.

(D) HeLa-sen2 cells were mock infected or infected at MOI of 50 at 4°C for 2 hr with wild-type, 6A, or 3R HPV16 PsV and then washed and shifted to 37°C for 8 or 16 hr to allow internalization. Permeabilized cells were stained with anti-L1 antibody (green) and examined by confocal microscopy.

(C) HeLa-sen2 cells were treated as described in (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 2 hr at 4°C, 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).

(B) HeLa-sen2 cells were treated as described in (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.

To confirm CPP activity of this L2 segment, we purified proteins from bacteria consisting of GFP fused in-frame to a 28-residue segment from the C terminus of L2 terminating with either the wild-type or mutant basic segment ( Figure 2 B). Microscopic examination of HeLa and HaCaT cells incubated with GFP-L2 fusion proteins showed that the protein with the wild-type L2 segment generated a strong punctate fluorescence signal inside cells and at the cell periphery ( Figure 2 C). The 3R mutation greatly impaired cellular uptake and the 6A mutation abolished it. To determine whether the fusion proteins were internalized, cells were treated with 0.04% trypan blue, a membrane-impermeable agent that quenches cell-surface GFP fluorescence, but not intracellular fluorescence. As shown in Figure 2 C, in cells incubated with the wild-type fusion protein, the signal at the cell periphery was eliminated by trypan blue, but the punctate signal persisted, demonstrating intracellular uptake of the protein. In contrast, the signal from the 3R mutant was largely eliminated. To quantify cellular fluorescence, flow cytometry was performed at 0.5 to 5 hr post-incubation following trypsinization to remove any GFP fusion protein adsorbed to the cell surface. The wild-type fusion protein translocated into cells rapidly, whereas there was no internalization of 6A and 3R fusion proteins ( Figure 2 D). Thus, the wild-type and mutant basic segments of L2 displayed cell penetration activity that correlated with the infectivity of PsV containing these segments. These results demonstrate that the L2 basic sequence has cell-penetration activity and strongly suggest that this activity is required for HPV entry.

We conducted several experiments to determine whether the basic segment of L2 has cell penetrating activity. First, we conjugated Alexa Fluor 488 onto the N terminus of a 28-residue L2 peptide that terminates with the wild-type basic segment or the corresponding peptide with the 6A or the 3R mutation ( Figure 2 A, top). As assessed by confocal microscopy, the wild-type peptide entered cells, but the peptide containing 6A mutation did not ( Figure 2 A, bottom). Cells treated with the 3R peptide displayed less fluorescence signal than cells treated with wild-type peptide, suggesting that the 3R peptide is partially impaired for cell penetration.

(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.

(C) HaCaT and HeLa S3 cells were incubated with GFP-L2 fusion proteins for 3 hr. Cells were examined by confocal microscopy and then treated with trypan blue (+TB) to quench extracellular fluorescence, and the same fields were reimaged.

(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 3 hr at 37°C and examined by confocal microscopy.

Next, we tested whether HPV16 PsV containing L2-Tat utilized a similar entry pathway as wild-type HPV16 PsV. To determine whether retromer was required for infection mediated by L2-Tat, we infected cells transfected with siRNA that knocked down the Vps29 retromer subunit (). Retromer knockdown dramatically inhibited infection by wild-type and L2-Tat PsV, indicating that the L2-Tat chimera requires retromer ( Figure 1 E). To determine whether γ-secretase was required for PsV containing L2-Tat, we treated cells with a chemical inhibitor of γ-secretase, compound XXI, and measured infectivity. XXI caused near-complete inhibition of both wild-type and L2-Tat PsV infection, showing that the L2-Tat chimera also requires γ-secretase ( Figure 1 F). Thus, the L2-Tat chimeric HPV16 PsV displays two of the key entry requirements as wild-type HPV16 PsV, dependence on retromer and γ-secretase.

We then replaced RKRRKR with various numbers of arginines or six consecutive lysines ( Figure 1 B). Published studies show that a stretch of five or more arginines can efficiently act as CPPs, whereas fewer arginines and polylysine are less effective (). Infectivity of the mutant PsV with three arginines (3R) was minimal (∼10%) and increased with the number of arginine residues, reaching a plateau with five arginines ( Figures 1 D and S1 C). The mutant with six lysines was <40% as infectious as PsV containing five or more arginines. These results demonstrate that the basic segment at the C terminus of HPV16 L2 can be functionally replaced by arginine-rich sequences with known CPP activity, and that infectivity correlated with predicted CPP activity. These results provide strong genetic evidence that the basic segment of L2 acts as a CPP during HPV16 infection.

To determine whether the L2 basic segment is important for HPV16 infection, we replaced wild-type RKRRKR with six alanines to construct the 6A mutant ( Figure 1 B). HeLa and HaCaT cells were infected with wild-type HPV16 PsV at MOI of one or with mutant PsV containing an equivalent number of encapsidated reporter plasmids, and infection efficiency was measured two days later by flow cytometry for HcRed fluorescence. As shown in Figures 1 C and S1 C, the 6A mutation abolished infectivity, showing that the basic sequence is essential for HPV16 infection. Next, we replaced the basic segment of L2 with the CPP domain of the HIV Tat protein to generate L2-Tat () ( Figure 1 B). Strikingly, HPV16 PsV with the Tat sequence in L2 infected cells as well as PsV containing wild-type L2 ( Figure 1 C). In contrast, an amphipathic or a hydrophobic CPP did not support infection. These results suggest that the basic segment of L2 acts as a cationic CPP to deliver the C terminus of L2 containing retromer binding sites through a membrane into the cytoplasm and show that only the cationic class of CPP can support HPV infection.

Direct binding of retromer to a C-terminal segment of the 473-residue HPV16 L2 protein is required for transport of the incoming virus from endosomes to the Golgi (). To test whether the basic amino acids in the C terminus of the L2 protein function as a CPP to transfer a segment of the L2 protein into the cytoplasm, we first tested whether mutations in the basic segment inhibited infectivity. For these experiments, we used PsVs comprising an HcRed reporter plasmid, wild-type HPV16 L1, and wild-type or mutant HPV16 L2 (). PsV assembly was confirmed by electron microscopy, which showed no obvious morphologic differences between wild-type and mutant PsVs ( Figure S1 A). Each PsV stock was normalized to the number of encapsidated reporter plasmids and analyzed by SDS-PAGE. Wild-type and mutant PsVs displayed comparable purity and contained similar levels of L1 and L2 ( Figure S1 B).

(A and B) HPV16 PsV containing wild-type (WT) L2 or the six alanine (6A) or three arginine (3R) mutants were examined by (A) transmission electron microscopy or (B) SDS-PAGE and staining with Coomassie blue. ( C ) HeLa S3 (black bars) and HaCaT (gray bars) cells were infected with wild-type, 6A, or 3R mutant HPV16 PsV at MOI of one. Two days later, infection was measured by flow cytometry for HcRed expression and normalized to infection by wild-type. Mean results and standard deviation are shown (n = 3). Size bars in (A) are 50 nm. Numbers in (B) show molecular mass (in kDa) of size markers.

Discussion

In this paper, we show that a short sequence of basic amino acids near the C terminus of the HPV L2 protein acts as a CPP to transfer a segment of the L2 protein into the cytoplasm where it binds retromer to initiate retrograde transport of the incoming virion to the TGN. First, we showed that the basic segment of HPV16 L2 is required for efficient infection of epithelial cells and can be replaced with the cationic CPP from HIV Tat. Like wild-type HPV16 PsV, PsV containing the Tat CPP required retromer for infection. Five or more consecutive arginine residues restored full infectivity, whereas fewer arginines or six lysines were less active, consistent with the known cell-penetrating activities of these sequences. We then used peptide and protein transduction assays to demonstrate that the basic segment of HPV16 L2 displayed CPP activity. Importantly, a truncated sequence of three arginines was defective for CPP activity and failed to support infection. The preferential uptake of the 3R peptide compared to the 3R fusion protein may reflect the higher concentration of molecules used in the peptide experiments. Taken together, these experiments demonstrated that L2 CPP activity was required for HPV infection.

HPV16 PsV containing a mutant CPP consisting of three arginines was internalized, showing the L2 CPP was not required for cell binding or endocytosis. However, in contrast to L1-only PsV, which bound cells well, the 6A mutant bound poorly. Thus, cell binding can be modulated by the L2 protein. The simplest explanation for these findings is that L1 is sufficient for binding and that the 6A mutation causes a subtle change in the structure of the capsid that inhibits L1 from binding to its receptor.

Kauffman et al., 2015 Kauffman W.B.

Fuselier T.

He J.

Wimley W.C. Mechanism matters: a taxonomy of cell penetrating peptides. The 3R CPP mutant was defective for retromer engagement, exit of PsV from the endosome, and trafficking to the TGN. The same phenotype is caused by mutations in the retromer binding sites or by retromer knockdown. However, the CPP mutation did not directly impair the ability of L2 to bind retromer, implying that during infection the retromer binding sites in the mutant were not in the same cellular compartment as retromer. To directly assay cytoplasmic exposure of L2, we developed a split GFP assay, in which fluorescence is reconstituted when a short segment of GFP at the C terminus of L2 on incoming PsV encounters GFP1-10 in the cytoplasm. Cytoplasmic exposure of HPV16 and HPV5 L2 was detectable early during infection and was impaired by replacing the CPP with three arginines. These findings show that the L2 CPP mediates passage of the C terminus of the L2 protein into the cytoplasm where it can engage retromer and enter the retrograde trafficking pathway. The presence of multiple L2 molecules in each virion ensures a high local concentration of CPPs upon infection even at low MOI, which may be important for membrane penetration (). The split GFP assay should be useful in investigating chemicals and mutations that inhibit HPV trafficking and in elucidating the molecular mechanism of this unusual process.

Inoue et al., 2018 Inoue T.

Zhang P.

Zhang W.

Goodner-Bingham K.

Dupzyk A.

DiMaio D.

Tsai B. γ-Secretase promotes membrane insertion of the human papillomavirus L2 capsid protein during virus infection. In addition to reconstituted fluorescence concentrated in discrete intracellular locations, many cells displayed a uniform signal throughout the cytoplasm (e.g., Figure 7 B), suggesting that the C-terminal segment of some L2 molecules are cleaved or released from the virion after protrusion through the membrane or that transport vesicles containing PsV rapidly distribute throughout the cytoplasm. Western blotting of infected HaCaT/GFP1-10NES cells failed to reveal shorter species of wild-type L2 or L2 linked to GFP11 at the time we measure L2 protrusion ( Figure S4 C). In addition, we recently reported that a small fraction of C-terminally FLAG-tagged L2 is cleaved by γ-secretase by 8 hpi, but cleavage is not required for infectivity (). Taken together, these results indicate that few L2 molecules are cleaved early during infection. The origin and significance of the diffuse, reconstituted GFP signal remains to be determined.

Bergant et al., 2017 Bergant M.

Peternel Š.

Pim D.

Broniarczyk J.

Banks L. Characterizing the spatio-temporal role of sorting nexin 17 in human papillomavirus trafficking. Campos et al., 2012 Campos S.K.

Chapman J.A.

Deymier M.J.

Bronnimann M.P.

Ozbun M.A. Opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration. Calton et al., 2017 Calton C.M.

Bronnimann M.P.

Manson A.R.

Li S.

Chapman J.A.

Suarez-Berumen M.

Williamson T.R.

Molugu S.K.

Bernal R.A.

Campos S.K. Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis. The rapid generation of reconstituted GFP fluorescent signal is consistent with the report that the L2 protein binds to the SNX17 as early as 2 hpi (). In addition, by 1.5 to 3 hpi, intracellular L1 is detectable by immunofluorescence ( Figure S4 D) and by western blotting (), providing additional evidence that HPV internalization can occur rapidly. The Campos laboratory used a related approach to determine when the L2-vDNA complex translocates across the limiting membrane to access the nucleus (). They fused the ∼30 kDa BirA biotin ligase to the C terminus of L2 and used biotinylation of a cytoplasmic target protein as a biochemical indicator of L2 translocation. Biotinylation required cell-cycle progression and was detected only after the virus reached the nucleus and the cells underwent mitosis, beginning approximately 8–10 hpi. In contrast, cytoplasmic exposure of L2 assessed by reconstituted GFP occurred earlier and did not require cell-cycle progression. Thus, biotinylation indicates that L2 molecules have been released from retrograde transport vesicles in the terminal stages of trafficking, whereas GFP reconstitution indicates the protrusion of L2 molecules into the cytoplasm to engage the retrograde trafficking machinery.

The physiological role of most CPPs is not known. Naturally occurring CPPs are usually studied as small peptide fragments removed from their protein of origin, and in many cases cell-penetrating activity may be the fortuitous consequence of a basic amino acid sequence that does not normally penetrate membranes when present in its host protein. Our results show that CPP-driven membrane penetration by L2 plays an important role in HPV infection. We propose that after the L2 CPP of endocytosed virus protrudes through the endosomal membrane into the cytoplasm, most of L2 passes through the membrane. If passage is arrested by its N-terminal TM domain, L2 will adopt a type 1 TM orientation with its N terminus in the endosomal lumen and most of the protein exposed in the cytoplasm. The exposed C terminus then binds to essential entry factors including retromer, which sorts the virus into retrograde transport vesicles. It should be pointed out, however, that it is possible that not all L2 molecules insert into membranes or bind cytoplasmic factors.

The effect of the L2 CPP on the membrane is relatively subtle and localized, in contrast to more drastic membrane disruption caused by other non-enveloped viruses, which are deposited into the cytoplasm. This allows the HPV virion to be retained in transport vesicles throughout trafficking, thereby sequestering it from cytoplasmic immune sensors during cell entry. The L2 protein may protrude into the cytoplasm in a sequential fashion, with the C terminus being exposed prior to the middle of the protein. Thus, cellular proteins may bind L2 sequentially, first retromer to the C terminus of L2 and later proteins, such as SNX17, to the middle portion of L2. Such ordered binding may be important for the assembly of the protein complexes necessary for proper trafficking.

The presence of a C-terminal basic region in all papillomavirus L2 proteins implies that the essential role of the L2 CPP has been maintained since the papillomaviruses emerged more than 250 million years ago. The sequence of the basic segment is variable, consistent with the relatively relaxed sequence requirements for CPPs. The 353 sequenced L2 proteins in the papillomavirus PaVe sequence database ( https://pave.niaid.nih.gov/#home ) contain 164 different C-terminal basic sequences, including a 10-residue poly-arginine stretch in three canine viruses, and many more CPPs are likely to exist in as-yet-uncharacterized papillomaviruses because most of these different basic sequences have been identified in only a single virus type ( Table S1 ). Moreover, sequences flanking the core basic amino acids may influence membrane penetration activity. The multitude of extant papillomavirus CPPs thus represents the results of a mutational analysis carried out over evolutionary time, revealing hundreds of presumably non-toxic sequences that should be evaluated for cargo-carrying activity for practical uses. In addition, analysis of these sequences may reveal new aspects of CPP action. For example, the ten most common L2 basic segments terminate in a C-terminal RKR sequence, implying that there is a biological advantage in maintaining RKR at the C terminus of these L2 CPPs ( Figure S4 E; Table S1 ).