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. 2012 Sep;86(18):9875-87.
doi: 10.1128/JVI.00980-12. Epub 2012 Jul 3.

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry

Malgorzata Bienkowska-Haba  1 , Carlyn Williams, Seong Man Kim, Robert L Garcea, Martin Sapp
Affiliations

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry

Malgorzata Bienkowska-Haba et al. J Virol. 2012 Sep.

Abstract

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

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Figures

Fig 1
Fig 1
Characterization of the HPV16 L2-GP-N mutant pseudovirus. (A) HaCaT cells were fixed, permeabilized, and stained for L1 (green) using MAb H16.56E at 18 hpi with wt and 16L2-GP-N mutant pseudovirus. Nuclei (blue) and filamentous actin (magenta) were stained in addition using DAPI and fluorescently labeled phalloidin, respectively. (B) L2-specific Western blotting of HaCaT cell lysate at 18 hpi with the wt and 16L2-GP-N mutant in the presence or absence of CsA. L2 protein was detected using a mix of RG-1 (22), anti-hemagglutinin, and 33L2-1 MAbs. (C) Sensitivity of wt and 16L2-GP-N mutant pseudovirus infection to 10 μM CsA.
Fig 2
Fig 2
Treatment of HPV16 particles with the Click-iT reaction cocktail denatures capsid protein L1. (A) RasMol-generated model of HPV16 L1 capsomeres (Protein Data Bank code 1dzl) highlighting in red the 33L1-7 antibody's conserved linear epitope (residues 303 to 313). (B) HPV16 pseudovirions were bound to ELISA plates and left untreated or treated with the Click-iT reaction cocktail prior to detection with indicated MAbs. Note the gain of reactivity with linear epitope-specific 33L1-7 and 16L1-312F and loss of reactivity with the conformation-dependent MAbs H16.V5, H16.E70, H16.56E, and H16.U4. (C) HaCaT cells were fixed and permeabilized at 4 hpi with wt HPV16 pseudovirions. Samples were either directly stained for conformational or linear L1 epitopes using H16.56E or 33L1-7, respectively, or treated with the Click-iT reaction cocktail prior to incubation with L1-specific antibodies. Again, note the loss of reactivity with H16.56E and gain of reactivity with 33L1-7 after treatment with the Click-iT reaction cocktail. Nuclei are stained with DAPI.
Fig 3
Fig 3
Binding, internalization, and nuclear transport of the 16L2-GP-N mutant pseudovirus. (A) HaCaT cells were fixed, permeabilized, and treated with a Click-iT reaction cocktail at the indicated times postinfection with mutant pseudovirus in the absence (control) or presence (+) of CsA prior to immunofluorescent staining using MAb 33L1-7. (B) HaCaT cells infected with mutant pseudovirus for 18 h in the absence or presence of CsA were stained for L1 using MAb 33L1-7 without prior denaturation as a measure for uncoating. (C) Delivery of viral pseudogenomes (red) was detected at 24 hpi of HaCaT cells infected with the EdU-labeled mutant pseudovirus. PML protein was stained in blue. Nuclei and filamentous actin were stained with DAPI (blue in panels A and B; gray in panel C) and phalloidin (magenta in panel B). Note the absence of the PML-NB-associated pseudogenome when infection was performed in the presence of CsA.
Fig 4
Fig 4
In vitro dissociation of HPV11 L1 from complexes of L1 and GST-L2 by CyPs. (A) Sequence comparison of putative CyP binding sites. (B) Complexes of L1 and GST-L2 bound to glutathione Sepharose beads were incubated with CyPA, CyPB, or buffer control at the indicated pH. L1 released from the complex into the supernatant was assayed by Western blotting with anti-L1 antibody. SN supernatant; A, CyPA; B, CyPB; Con, buffer-alone control; SM, starting material bound to the beads.
Fig 5
Fig 5
Colocalization of pseudogenome with capsid proteins. (A) EdU-labeled wt pseudoparticles were bound to ECM-coated coverslips and stained for DNA (red) and capsid proteins (green) using 33L1-7 and 33L2-1 for L1 and L2, respectively. Scale bar, 10 μm. (B to E) HaCaT cells were infected with EdU-labeled wt pseudovirus in the absence (B and D) or presence of bafilomycin A1 (C and E). Cells were fixed, permeabilized, and stained as above for the pseudogenome (B to E) and L1 (B and C) or for the pseudogenome and L2 (D and E) at 18 hpi. Nuclei were stained using DAPI (in blue). Areas highlighted by rectangles are magnified in the smaller panels shown to the right. Merged images are shown. (F) Extent of L1/DNA and L2/DNA colocalization as determined by using LAS AF software.
Fig 6
Fig 6
CsA interferes with dissociation of L1 from the L2/DNA complex during infectious entry of 16L2-GP-N mutant pseudovirus. (A to D) HaCaT cells were fixed, permeabilized, and sequentially stained for DNA (red) and L1 (green) (A and B) or DNA and L2 (green) (C and D) using a Click-iT reaction cocktail and MAbs 33L1-7 or 33L2-1 at 18 hpi with EdU-labeled 16L2-GP-N mutant pseudovirus in the absence (A and C) or presence (B and D) of CsA. Areas highlighted by rectangles are magnified in the smaller panels shown to the right. (E) L1/DNA and L2/DNA colocalizations were manually quantified as outlined in Materials and Methods. P values were determined by a Student's t test.
Fig 7
Fig 7
CsA interferes with dissociation of L1 from the L2/DNA complex during infectious entry of wt pseudovirus. (A to D) HaCaT cells were fixed, permeabilized, and sequentially stained for DNA (red) and L1 (green) (A and B) or L2 (green) (C and D) using a Click-iT reaction cocktail and MAb 33L1-7 or 33L2-1 at 18 hpi with EdU-labeled wt pseudovirus in the absence (A and C) or presence (B and D) of CsA. Areas highlighted by rectangles are magnified in the smaller panels shown to the right. (E) L1/DNA and L2/DNA colocalizations were manually quantified as outlined in Materials and Methods. P values were determined by a Student's t test.
Fig 8
Fig 8
Cyclophilin knockdown interferes with dissociation of L1 from the pseudogenome. (A) HaCaT cells were transfected with CyP (broad) siRNA. At 48 h posttransfection, cells were infected with EdU-labeled 16L2-GP-N mutant pseudovirus for 18 h. Cells were sequentially stained for EdU-labeled DNA (red), L1 (green), and CyPB (blue) as described in Materials and Methods. DAPI-stained nuclei are depicted in gray. (B) L1/DNA colocalization was quantified in CyPB-positive and -negative cells. (C) Representative graphs of signal intensity in a single punctum in CyPB-positive and -negative cells. Note the increased CyPB signal in EdU-positive puncta. P values were determined by a Student's t test.
Fig 9
Fig 9
Partial dissociation of HPV16 L1 capsomeres from pseudovirions in vitro by CyPA. (A) HPV16 pseudovirus was treated with DNase I and DTT or left untreated. After adjustment to pH 6.0, samples were incubated with GST or CyPA and analyzed by sedimentation through sucrose gradients. Gradients were fractionated from the top, and fractions were assayed for the presence of L1 and L2 by Western blotting following concentration of proteins by trichloroacetic acid precipitation. (B) GFP-specific DNA fragments were amplified by PCR from the indicated fractions and analyzed by agarose gel electrophoresis.
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References

    1. Baker TS, et al. 1991. Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophys. J. 60:1445–1456 - PMC - PubMed
    1. Bergant Marusic M, Ozbun MA, Campos SK, Myers MP, Banks L. 2012. Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17. Traffic 13:455–467 - PMC - PubMed
    1. Bienkowska-Haba M, Patel HD, Sapp M. 2009. Target cell cyclophilins facilitate human papillomavirus type 16 infection. PLoS Pathog. 5:e1000524 doi:10.1371/journal.ppat.1000524 - DOI - PMC - PubMed
    1. Buck CB, et al. 2008. Arrangement of L2 within the papillomavirus capsid. J. Virol. 82:5190–5197 - PMC - PubMed
    1. Buck CB, Pastrana DV, Lowy DR, Schiller JT. 2004. Efficient intracellular assembly of papillomaviral vectors. J. Virol. 78:751–757 - PMC - PubMed

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