Production of Furin‐Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1‐ or L2‐Specific Antibodies

Wang, Joshua W.; Matsui, Ken; Pan, Yuanji; Kwak, Kihyuck; Peng, Shiwen; Kemp, Troy J.; Pinto, Lígia A.; Roden, Richard B.S.

doi:10.1002/9780471729259.mc14b05s38

Cited by 9 publications

(9 citation statements)

References 31 publications

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“…1C ). Moreover, infection of CHO-K1 wild-type cells also resulted in a more robust (approximately 30-fold) increase of infection in the presence of FPC particles, while neutralization with the HPV16-neutralizing antibody H16.V5 (but not with the HPV18-neutralizing antibody H18.J4) abolished infectious uptake independently of furin pretreatment as expected ( 53 ) in both cell types ( Fig. 1C ).…”

Section: Resultssupporting

confidence: 75%

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions

Schäfer

Graham

Lang

et al. 2017

J Virol

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Human papillomavirus (HPV) infection is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells are controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase, and subsequent transfer of the virus to an as-yet-unidentified high-affinity receptor. In line with this model, we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were nonpermissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pulldown assays followed by mass spectrometry analysis showed that furin-precleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and uncleaved HPV16-PsVs colocalized with surface-expressed vimentin on pgsD677, HeLa, HaCaT, and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by small interfering RNA (siRNA) knockdown in HeLa and NIKS cells significantly increased HPV16-PsV infectious internalization, while overexpression of vimentin had the opposite effect. The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for the design of novel antiviral drugs preventing HPV internalization into epithelial cells.IMPORTANCE Despite HPV being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalization are poorly understood. We herein describe the identification of surface-expressed vimentin as a novel molecule not previously implicated in the infectious internalization of HPV16. Contrary to our expectations, vimentin was found to act not as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalization of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.

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Section: Resultssupporting

confidence: 75%

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions

Schäfer

Graham

Lang

et al. 2017

J Virol

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“…The cells were washed in PBS with 10 mM MgCl 2 and lysed in PBS containing 10 mM MgCl 2 , 0.5% Triton X-100, 25 mM ammonium sulfate (pH 9), and 5 mM CaCl 2 . Lysates were incubated at 37°C 48 h for maturation and spun at 7,000 rpm for 5 min (48).…”

Section: Methodsmentioning

confidence: 99%

Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

Jose

DeSmet

Androphy

2020

J Virol

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The human papillomavirus (HPV) E2 protein is a key regulator of viral transcription and replication. In this study, we demonstrate that the non-receptor tyrosine kinase Pyk2 phosphorylates tyrosine 131 in the E2 transactivation domain. Both depletion of Pyk2 and treatment with a Pyk2 kinase inhibitor increased viral DNA content in keratinocytes that maintain viral episomes. The tyrosine to glutamic acid (E) mutant Y131E which may mimic phosphotyrosine failed to stimulate transient DNA replication, and genomes with this mutation were unable to establish stable episomes in keratinocytes. Using co-immunoprecipitation assays, we demonstrate that the Y131E is defective for binding to the C-terminal motif (CTM) of Bromodomain-containing protein 4 (Brd4). These data imply HPV replication depends on E2 Y131 interaction with the pTEFb binding domain of Brd4. Importance Human papillomaviruses are the major causative agents of cervical, oral and anal cancers. The present study demonstrates that the Pyk2 tyrosine kinase phosphorylates E2 at tyrosine 131, interfering with genome replication. We provide evidence that phosphorylation of E2 prevents binding to the Brd4-CTM. Our findings add to the understanding of molecular pathways utilized by the virus during its vegetative lifecycle and offers insights into the host-virus interactome.

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“…To remove heparin sulfate proteoglycans (HSPG) that co-purify attached to the virion surface, a PsV prep was subjected to a brief trypsin and heparinase treatment prior to the addition of soybean trypsin inhibitor and purification on the iodixanol gradient and is referenced as “HSPG-” within the manuscript. Furin pre-cleaved HPV16 (fpc) PsV were generated using 293TTF cells using the protocol outlined by Wang et al ( 25 ). Assessment of L2 cleavage by furin was carried out by western blot.…”

Section: Methodsmentioning

confidence: 99%

Theta-Defensins Inhibit High-Risk Human Papillomavirus Infection Through Charge-Driven Capsid Clustering

et al. 2020

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Persistent infection with high-risk human papillomavirus (hrHPV) genotypes results in a large number of anogenital and head and neck cancers worldwide. Although prophylactic vaccination coverage has improved, there remains a need to develop methods that inhibit viral transmission toward preventing the spread of HPV-driven disease. Defensins are a class of innate immune effector peptides that function to protect hosts from infection by pathogens such as viruses and bacteria. Previous work utilizing α and β defensins from humans has demonstrated that the α-defensin HD5 is effective at inhibiting the most common high-risk genotype, HPV16. A third class of defensin that has yet to be explored are θ-defensins: small, 18-amino acid cyclic peptides found in old-world monkeys whose unique structure makes them both highly cationic and resistant to degradation. Here we show that the prototype θ-defensin, rhesus theta defensin 1, inhibits hrHPV infection through a mechanism involving capsid clustering that inhibits virions from binding to cell surface receptor complexes.

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Production of Furin‐Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1‐ or L2‐Specific Antibodies

Cited by 9 publications

References 31 publications

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions

Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein

Theta-Defensins Inhibit High-Risk Human Papillomavirus Infection Through Charge-Driven Capsid Clustering

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