Opposing Effects of Bacitracin on Human Papillomavirus Type 16 Infection: Enhancement of Binding and Entry and Inhibition of Endosomal Penetration

Abstract

e Cell invasion by human papillomavirus type 16 (HPV16) is a complex process relying on multiple host cell factors. Here we describe an investigation into the role of cellular protein disulfide isomerases (PDIs) by studying the effects of the commonly used PDI inhibitor bacitracin on HPV16 infection. Bacitracin caused an unusual time-dependent opposing effect on viral infection. Enhanced cellular binding and entry were observed at early times of infection, while inhibition was observed at later times postentry. Bacitracin was rapidly taken up by host cells and colocalized with HPV16 at late times of infection. Bacitracin had no deleterious effect on HPV16 entry, capsid disassembly, exposure of L1/L2 epitopes, or lysosomal trafficking but caused a stark inhibition of L2/viral DNA (vDNA) endosomal penetration and accumulation at nuclear PML bodies. ␥-Secretase has recently been implicated in the endosomal penetration of L2/vDNA, but bacitracin had no effect on ␥-secretase activity, indicating that blockage of this step occurs through a ␥-secretase-independent mechanism. Transient treatment with the reductant ␤-mercaptoethanol (␤-ME) was able to partially rescue the virus from bacitracin, suggesting the involvement of a cellular reductase activity in HPV16 infection. Small interfering RNA (siRNA) knockdown of cellular PDI and the related PDI family members ERp57 and ERp72 reveals a potential role for PDI and ERp72 in HPV infection. Human papillomaviruses (HPVs) are one of the most common sexually transmitted infections in the world. HPVs are small 55-nm icosahedral nonenveloped double-stranded DNA (dsDNA) viruses that replicate in differentiating cutaneous and mucosal epithelium. Infection of mucosal epithelium by oncogenic HPV genotypes can lead to cervical, anogenital, and other head and neck cancers. HPV type 16 (HPV16) is the most common of the high-risk types and is alone responsible for over 50% of cervical cancers worldwide (77). Although HPVs have been known to be the etiological agent of cervical cancer for nearly 30 years, and despite intensive research in recent years, the infectious entry pathway of HPV16 is still not well defined. Our current understanding of HPV cellular invasion reveals a complex and prolonged process, complicated by differences between in vitro cell culture systems and the recently described in vivo mouse cervicovaginal challenge model (33,37,50,62).The HPV capsid is assembled from 360 molecules of the L1 protein, arranged as 72 pentamers. L1 monomers from neighboring pentamers are disulfide bonded to each other as dimers and trimers, providing stability to the capsid (45). The minor capsid protein L2 is localized within a central cavity beneath the L1 pentamers. L2 can be present at a maximum stoichiometry of one L2 molecule per L1 pentamer or 72 molecules per virion; however, most preparations of virus contain submaximal levels of L2, typically 20 to 25 copies per virion (6). Packaged within the capsid is the ϳ8-kb viral genome (viral DNA [vDNA]), condensed as chromatin with cellu...

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

“…1D) suggests that the viral genome may traffic from the Golgi to the ER during entry. It was previously reported that HPV16 infection was inhibited by Brefeldin A, which affects several trafficking steps including ER entry and by knock-down of protein disulfide isomerases that are primarily localized in the ER (11,42). The enrichment of potassium channels in our list of hits suggests that the channels themselves or ion fluxes play a role in infection.…”

Crystal structure of glycoprotein E2 from bovine viral diarrhea virus

“…In pestiviruses, scission of disulfide bonds in the envelope glycoproteins is thought to activate the virion for fusion, as the combination of reducing agent and low pH allows fusion with the plasma membrane ("fusion from without"), albeit at low efficiency (6). A potential precedent is human papillomavirus, which requires a cellular protein disulfide isomerase in addition to low endosomal pH for disassembly of the disulfide-linked capsid and delivery of viral DNA into the cytoplasm (14).…”

A Transmembrane Domain and GxxxG Motifs within L2 Are Essential for Papillomavirus Infection

“…2B). The HD mutation had no obvious effect on virion binding, entry, or time-dependent exposure of the buried L1-7 epitope, a marker for uncoating (44,48) (Fig. 2C).…”

“…293TT, HeLa, and HaCaT cells were maintained as previously described (44). Cloning of mutant viruses was achieved by site-directed mutagenesis of the L1/L2 expression plasmid pXULL using a QuikChange-XL II system (catalog number 200521; Agilent), and all mutant plasmids were verified by Sanger sequencing.…”

“…HPV virions containing 5-ethynyl-2=-deoxyuridine (EdU)-labeled pGL3-basic DNA were produced by CaPO 4 transfection of 293TT cells supplemented with 15 M EdU (44). HaCat cells were seeded on glass coverslips and infected with EdU-labeled viruses at 500 ng of L1/ml for 18 h. The cells were then washed once using fresh medium to remove unbound virions, medium was replaced with fresh medium, and cells were cultured for an additional 24 h. At 42 h postinfection, cells were chilled to 4°C, washed three times in cold phosphatebuffered saline (PBS), and fixed in acetone at Ϫ20°C for 5 min.…”