The initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding

Abstract

Using a murine challenge model, we previously determined that human papillomavirus (HPV) pseudovirions initially bind preferentially to the cervicovaginal basement membrane (BM) at sites of trauma. We now report that the capsids undergo a conformational change while bound to the BM that results in L2 cleavage by a proprotein convertase (PC), furin, and/or PC5/6, followed by the exposure of an N-terminal cross-neutralization L2 epitope and transfer of the capsids to the epithelial cell surface. Prevention of this exposure by PC inhibition results in detachment of the pseudovirions from the BM and their eventual loss from the tissue, thereby preventing infection. Pseudovirions whose L2 had been precleaved by furin can bypass the PC inhibition of binding and infectivity. Cleavage of heparan sulfate proteoglycans (HSPG) with heparinase III prevented infection and BM binding by the precleaved pseudovirions, but did not prevent them from binding robustly to cell surfaces. These results indicate that the infectious process has evolved so that the initial steps take place on the BM, in contrast to the typical viral infection that is initiated by binding to the cell surface. The data are consistent with a dynamic model of in vivo HPV infection in which a conformational change and PC cleavage on the BM allows transfer of virions from HSPG attachment factors to an L1-specific receptor on basal keratinocytes migrating into the site of trauma.furin ͉ HSPG ͉ mouse model ͉ proprotein convertase P apillomaviruses (PVs) infect mucosal and cutaneous stratified squamous epithelia, resulting in benign lesions, some of which can progress to invasive cancer. Human papillomavirus (HPV) 16 and several other PVs that preferentially infect the anogenital mucosa are responsible for virtually all cases of cervical cancer, as well as a number of other mucosal epithelial malignancies. It has been recognized for decades that efficient induction of experimental PV infection of animals requires local wounding in conjunction with virus exposure (1-4). However, there has been little molecular insight into the in vivo steps by which PVs initiate the infectious process.We recently described a murine cervicovaginal challenge model of PV transmission that is amenable to examination of the early in vivo events of PV infection (5, 6). It employs high titer PV pseudoviruses, which are authentic PV capsids composed of the L1 major and L2 minor structural proteins that have encapsidated a non-PV plasmid encoding a quantifiable reporter gene to monitor successful infection (7). PV pseudoviruses abrogate the exquisite species specificity of the viral gene expression program of authentic PV virions, while retaining the early events of PV infection, including the preferential infection of keratinocytes in vivo. In the initial characterization of the mouse genital tract model, we confirmed that wounding by the transient disruption of cervicovaginal epithelial integrity, by physical or chemical means, is essential for efficient infection (5). It was shown that ...

Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2

“…18 A cross-neutralizing monoclonal anti-L2 antibody, K4L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] , highly potent at neutralizing HPV 16 and other high-risk HPV pseudovirions (PSV), fails to neutralize HPV 31. Vice versa, an anti-HPV 31 L2 neutralizing antibody (K3L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] ) is not able to neutralize HPV 16 PSV. Also, anti-HPV 16 L2 polyclonal sera neutralize HPV 31 pseudovirions with a low efficiency (Seitz et al, unpublished data).…”

“…This fact could raise some doubts about the true existence of the HPV 31 D31N variant, which might require independent confirmation, despite the fact that PSV carrying this exchange proved to be highly infectious in vitro. HPV 31 type-specific K3L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] neutralized HPV 31 reference and P34T pseudovirions as well as D31N pseudovirions albeit the latter to a lower degree. Likewise, the polyclonal sera from HPV 31 antigenimmunized mice neutralized HPV 31 reference and P34T pseudovirions equally well, while D31N was neutralized to a lesser extent.…”

“…Upon binding to heparansulfate proteoglycan, however, the capsid structure opens, and L2 neutralizing epitopes are exposed and can be bound by antibodies. 35,36 Thus, while the virus is shielding its conserved L1 motifs by heterogeneous loops, it also limits access to L2 epitopes that play a role in the entry process. Still, the broadly neutralizing L2 epitope might be considered an Achilles' heel to be targeted by prophylactic vaccines, and the existence of variants escaping cross-protective antibodies might hint at the occurrence of natural L2-based immunity.…”

“…Two monoclonal antibodies, K18L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] and K3L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] , as well as five polyclonal sera were tested on the HPV 31 reference and variant pseudovirions in a neutralization assay. Although K18L2 [20][21][22][23][24][25][26][27][28][29][30][31][32]…”

“…Neutralization of HPV 18 P33H, P33N and P29S 1 P33N L2 variants HPV 18 L2 reference, P33H, P33N and P29S þ P33N pseudovirions were tested in an in vitro neutralization assay with cross-neutralizing antibodies K4L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] and K18L2 20-38 and 10 polyclonal sera.…”