Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins

ABSTRACTDespite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, thePropionibacterium shermaniitranscarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.IMPORTANCEFurin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.

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Human Papillomavirus Type 16 Minor Capsid Protein L2 N-Terminal Region Containing a Common Neutralization Epitope Binds to the Cell Surface and Enters the Cytoplasm

Journal of Virology ◽

10.1128/jvi.75.5.2331-2336.2001 ◽

2001 ◽

Vol 75 (5) ◽

pp. 2331-2336 ◽

Cited By ~ 57

Author(s):

Yukiko Kawana ◽

Kei Kawana ◽

Hiroyuki Yoshikawa ◽

Yuji Taketani ◽

Kunito Yoshiike ◽

...

Keyword(s):

Flow Cytometry ◽

Human Papillomavirus ◽

Cell Surface ◽

Capsid Protein ◽

Hela Cells ◽

Human Cancer ◽

Hpv Infection ◽

Terminal Region ◽

Neutralization Epitope ◽

Minor Capsid Protein

ABSTRACT The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4°C and to enter the cytoplasm after subsequent incubation at 37°C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.

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Generation of Antibodies against Foot-and-Mouth-Disease Virus Capsid Protein VP4 Using Hepatitis B Core VLPs as a Scaffold

Life ◽

10.3390/life11040338 ◽

2021 ◽

Vol 11 (4) ◽

pp. 338

Author(s):

Jessica Swanson ◽

Rennos Fragkoudis ◽

Philippa C. Hawes ◽

Joseph Newman ◽

Alison Burman ◽

...

Keyword(s):

Amino Acids ◽

Hepatitis B ◽

Capsid Protein ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Specific Antibodies ◽

Mouth Disease Virus ◽

N Terminus ◽

Foot And Mouth

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the economically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neutralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.

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Inhibition of Transfer to Secondary Receptors by Heparan Sulfate-Binding Drug or Antibody Induces Noninfectious Uptake of Human Papillomavirus

Journal of Virology ◽

10.1128/jvi.00998-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 10970-10980 ◽

Cited By ~ 111

Author(s):

Hans-Christoph Selinka ◽

Luise Florin ◽

Hetal D. Patel ◽

Kirsten Freitag ◽

Michaela Schmidtke ◽

...

Keyword(s):

Cell Surface ◽

Neutralizing Antibodies ◽

Structural Similarity ◽

Hpv Infection ◽

Human Papillomaviruses ◽

Laminin 5 ◽

Entry Pathway ◽

Successful Infection ◽

Major Mode ◽

Endocytic Vesicles

ABSTRACT Infection with various human papillomaviruses (HPVs) induces cervical cancers. Cell surface heparan sulfates (HS) have been shown to serve as primary attachment receptors, and molecules with structural similarity to cell surface HS, like heparin, function as competitive inhibitors of HPV infection. Here we demonstrate that the N,N′-bisheteryl derivative of dispirotripiperazine, DSTP27, efficiently blocks papillomavirus infection by binding to HS moieties, with 50% inhibitory doses of up to 0.4 μg/ml. In contrast to short-term inhibitory effects of heparin, pretreatment of cells with DSTP27 significantly reduced HPV infection for more than 30 h. Using DSTP27 and heparinase, we furthermore demonstrate that HS moieties, rather than laminin 5, present in the extracellular matrix (ECM) secreted by keratinocytes are essential for infectious transfer of ECM-bound virions to cells. Prior binding to ECM components, especially HS, partially alleviated the requirement for cell surface HS. DSTP27 blocks infection by cell-bound virions by feeding into a noninfectious entry pathway. Under these conditions, virus colocalized with HS moieties in endocytic vesicles. Similarly, postattachment treatment of cells with heparinase, cytochalasin D, or neutralizing antibodies resulted in uptake of virions without infection, indicating that deviation into a noninfectious entry pathway is a major mode of postattachment neutralization. In untreated cells, initial colocalization of virions with HS on the cell surface and in endocytic vesicles was lost with time. Our data suggest that initial attachment of HPV to HS proteoglycans (HSPGs) must be followed by secondary interaction with additional HS side chains and transfer to a non-HSPG receptor for successful infection.

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Traditional Chinese Medicine Combined with Routine Nursing on Patients with Cervical Human Papillomavirus Infection

Tobacco Regulatory Science ◽

10.18001/trs.7.6.115 ◽

2021 ◽

Vol 7 (6) ◽

pp. 6415-6421

Author(s):

Jing Ye ◽

Hui Cheng

Keyword(s):

Human Papillomavirus ◽

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Infection Rate ◽

Hpv Infection ◽

Common Disease ◽

Postoperative Rehabilitation ◽

Human Papillomavirus Infection ◽

Papillomavirus Infection ◽

Cervical Human Papillomavirus

Cervical papillomavirus infection is a common disease in women. The risk of permanent infection is the main reason for cervical HPV and its advanced acanthosis. The last time, the incidence rate of cervical papillomavirus is only different from that of malignant tumor women. The trend of young tumors is more and more obvious. Therefore, the treatment of parasitic lesions is very important to prevent or reduce the occurrence of cervical HPV. This paper aims to study the effect of traditional Chinese medicine combined with routine nursing on patients with cervical human papillomavirus infection. The application of traditional Chinese medicine therapy to promote the postoperative rehabilitation of patients with cervical human papillomavirus infection has significant effect, which is of great significance for clinicians in the future work. In this paper, through the introduction of traditional Chinese medicine and traditional Chinese medicine oral and external use combined with routine nursing methods, through the follow-up experimental investigation method to study the cervical human papillomavirus infection rate and the effect of cervical human papillomavirus infection patients after operation, it is proved that the effect of traditional Chinese medicine oral and external use combined with routine nursing on cervical human papillomavirus infection patients is better. The results show that 54% of the cervical HPV infection rate makes people pay more and more attention to cervical HPV. The infection investigation and genotyping can effectively judge the prognosis of patients, which has extremely important clinical significance.

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Feline Calicivirus Can Tolerate Gross Changes of Its Minor Capsid Protein Expression Levels Induced by Changing Translation Reinitiation Frequency or Use of a Separate VP2-Coding mRNA

PLoS ONE ◽

10.1371/journal.pone.0102254 ◽

2014 ◽

Vol 9 (7) ◽

pp. e102254 ◽

Cited By ~ 3

Author(s):

Maria Haß ◽

Christine Luttermann ◽

Gregor Meyers

Keyword(s):

Protein Expression ◽

Capsid Protein ◽

Feline Calicivirus ◽

Minor Capsid Protein ◽

Expression Levels

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Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

10.20944/preprints201711.0086.v2 ◽

2017 ◽

Author(s):

Eric A. Iverson ◽

David A. Goodman ◽

Madeline E. Gorchels ◽

Kenneth M. Stedman

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Vp1 Gene ◽

Capsid Protein Gene ◽

Vp1 Protein ◽

Transposon Insertion ◽

N Terminus ◽

Capsid Protein Vp1

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene vp3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

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Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

10.20944/preprints201711.0086.v1 ◽

2017 ◽

Author(s):

Eric A. Iverson ◽

David A. Goodman ◽

Madeline E. Gorchels ◽

Kenneth M. STEDMAN

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Vp1 Gene ◽

Vp1 Protein ◽

Transposon Insertion ◽

N Terminus ◽

Capsid Protein Vp1 ◽

Virion Formation

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein VP3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

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molBV reveals immune landscape of bacterial vaginosis and predicts human papillomavirus infection natural history

Nature Communications ◽

10.1038/s41467-021-27628-3 ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Mykhaylo Usyk ◽

Nicolas F. Schlecht ◽

Sarah Pickering ◽

LaShanda Williams ◽

Christopher C. Sollecito ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Hpv Infection ◽

Rrna Gene ◽

Clinical Trial Registration ◽

Gold Standard Method ◽

Adverse Health Outcomes ◽

Papillomavirus Infection ◽

Registration Number ◽

Hpv Clearance ◽

16 S Rrna

AbstractBacterial vaginosis (BV) is a highly prevalent condition that is associated with adverse health outcomes. It has been proposed that BV’s role as a pathogenic condition is mediated via bacteria-induced inflammation. However, the complex interplay between vaginal microbes and host immune factors has yet to be clearly elucidated. Here, we develop molBV, a 16 S rRNA gene amplicon-based classification pipeline that generates a molecular score and diagnoses BV with the same accuracy as the current gold standard method (i.e., Nugent score). Using 3 confirmatory cohorts we show that molBV is independent of the 16 S rRNA region and generalizable across populations. We use the score in a cohort without clinical BV states, but with measures of HPV infection history and immune markers, to reveal that BV-associated increases in the IL-1β/IP-10 cytokine ratio directly predicts clearance of incident high-risk HPV infection (HR = 1.86, 95% CI: 1.19-2.9). Furthermore, we identify an alternate inflammatory BV signature characterized by elevated TNF-α/MIP-1β ratio that is prospectively associated with progression of incident infections to CIN2 + (OR = 2.81, 95% CI: 1.62-5.42). Thus, BV is a heterogeneous condition that activates different arms of the immune response, which in turn are independent risk factors for HR-HPV clearance and progression. Clinical Trial registration number: The CVT trial has been registered under: NCT00128661.

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Papillomavirus can be transmitted through the blood and produce infections in blood recipients: Evidence from two animal models

10.1101/541474 ◽

2019 ◽

Author(s):

Nancy M. Cladel ◽

Pengfei Jiang ◽

Jingwei J. Li ◽

Xuwen Peng ◽

Timothy K. Cooper ◽

...

Keyword(s):

Animal Models ◽

Hpv Infection ◽

Human Papillomaviruses ◽

Viral Dna ◽

Internal Organs ◽

Sexually Transmitted ◽

Potential Source ◽

Papillomavirus Infection ◽

Blood Banks ◽

Gain Access

AbstractHuman papillomavirus (HPV) infections are commonly thought to be strictly sexually transmitted. However, studies have demonstrated the presence of HPV in cancers of many non-sexual internal organs, raising the question as to how the viruses gain access to these sites. A possible connection between blood transfusion and HPV-associated disease has not received much attention. We show, in two animal models, that blood infected with papillomavirus yields infections at permissive sites. Furthermore, we demonstrate that blood from actively infected mice can transmit the infection to naïve animals. Finally, we report papillomavirus infections in the stomach tissues of animals infected via the blood. Stomach tissues are not known to be permissive for papillomavirus infection, although the literature suggests that HPVs may be associated with a subset of gastric cancers. These results indicate that the human blood supply, which is not screened for papillomaviruses, could be a potential source of HPV infection and subsequent cancers.SUMMARYHuman papillomaviruses cause 5% of human cancers. Currently, blood banks do not screen for these viruses. We demonstrate that blood transfused from papillomavirus-infected animals produces infections in recipients. Public health implications are significant if the same is true for humans.DefinitionsLocal papillomavirus infection:An infection initiated by the direct application of virus or viral DNA to the site of infectionIntravenous (IV) papillomavirus infection:An infection resulting from blood-borne delivery of virus or viral DNA to the site of infection.

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