Target cell cyclophilins facilitate human papillomavirus type 16 infection

Abstract

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Figure 1. CyP facilitate HPV infection.

293TT (A), HaCaT (B), or HeLa cells (B) were infected with HPV16 (A,B,C) or HPV18 pseudovirus (B) in presence of indicated inhibitors and infection was scored at 72 hpi. (D) Effect of drugs on cell growth was determined by the MTT assay. We did not notice significant increase in cell death. CsA: cyclosporine A; VIVIT: cell permeable 11R-VIVIT; Nfdp: nifedipin; Vpml: verapamil. Representative graphs are based on three replicates each with standard deviation indicated.

Figure 2. Knock down of CyP inhibits HPV16 infection.

293TT (A,B) or HaCaT (C,D) cells were transfected with indicated siRNA and infected with HPV16 pseudovirus 48h after transfection. Infection was scored at 72 hpi. Representative graphs based on six replicates are shown (A,C). Knockdown of CyPA and CyPB was confirmed by Western blot prior to infection (B,D). Numbers indicate percent levels of protein after correcting for input (D). *: p<0.05; **: p<0.01.

Figure 3. Inhibition of CyP leads to noninfectious HPV16 internalization.

(A) HaCaT cells were infected with HPV16 pseudovirus in the absence or presence of NIM811. At 18 hpi cells were fixed and stained using conformation-dependent NmAb H16.56E or linear epitope-specific nonneutralizing 33L1-7. Cells were also stained for actin and DNA using AF⁴⁸⁸–labeled phalloidin (green) and Dapi (blue), respectively. Images were taken with a Leica DBMI6000 at 40× magnification. (B) HaCaT cells were transfected with indicated siRNA. At 48 hpTx, cells were harvested and reseeded for infection with HPV16 pseudovirions. At 18 hpi cells were fixed and stained using H16.56E (red) and CyPA or CyPB (green). Images were taken with a confocal Zeiss LSM 510 microscope at 63× magnification.

Figure 4. CyP facilitate exposure of the RG-1 epitope after cell attachment.

HPV16 pseudovirus was bound to HaCaT cells for 2 h at 4°C and chased for 4 h at 37°C in presence or absence of NIM811. (A) Cells were subsequently stained with L2-specific RG-1 and L1-specific K75 antibody. All images were taken using the same settings. (B) RG-1– and K75–specific signal strength of randomly selected cells (n>15 for each group) was measured. RG-1 reactivity normalized to K75 signal strength is plotted after subtraction of background signal. The graph shows quantifications from one experiment. However, the experiment was repeated three times with similar outcomes.

Figure 5. Effect of RG-1 on particle internalization.

(A) HPV16 pseudovirus was added to HaCaT grown on coverslips and incubated for 18 h at 37°C in the presence or absence of RG-1 (1∶10 dilution of cell culture supernatant). Samples were stained for viral particles and ECM using H16.56E and rabbit polyclonal antiserum specific for laminin 5, respectively. Note the accumulation of viral particles on ECM in presence of RG-1. As fixation prior to addition of secondary antibody destroys reactivity with RG-1 [24, and our own observations], the signal picked up by mouse-specific secondary antibody is solely attributable to H16.56E. (B) HaCaT cells were grown and infected as above with the addition of NIM811 during the incubation where indicated. Samples were stained with H16.56E. Note that the presence of RG-1 did not affect the stabilized capsid phenotype and viral internalization irrespective of NIM811 treatment. Images were taken with a Zeiss LSM at 63× magnification focusing on ECM (A) and cells (B), respectively. Actin staining with labeled phalloidin is shown in blue. (C) Neutralization of HPV16 by RG-1 at indicated dilutions was measured at 72 hpi of 293TT cells (n = 5).

Figure 6. L2 protein is the likely target of CyPB.

(A) Sequence alignment of selected PV L2 proteins with CyPA binding site of HIV capsid protein. (B) 293TT cells were infected with similar amounts of HPV16 wt and 16L2-GP-N mutant pseudovirus and scored at 72 hpi. The difference in infectivity is statistically significant (p<0.01; n = 5) based on testing two independent pseudovirus preparations. (C) 16L2-GP-N mutant pseudovirus was bound to HaCaT cells in presence or absence of NIM811 for 4 h at 37°C and subsequently stained with RG-1 and K75. All images were taken with the same settings. (D) Quantification of RG-1 and K75 signal strength using randomly selected cells (n>15).

Figure 7. Mutant pseudovirus infection is impaired by CyP inhibitors.

(A) Sensitivity of 16L2-GP-N mutant pseudovirus to CsA was determined by infection of 293TT cells. (B) 293TT cells were transfected with indicated siRNA and infected with mutant pseudovirus at 48 hpTx and scored at 72 hpi. *: p<0.01. (C) HaCaT cells grown on coverslips were infected with HPV16 wt or 16L2-GP-N mutant pseudovirus. At 18 hpi cells were stained with H16.56E (red) and labeled phalloidin (blue).