scholarly journals Alpha-Defensin HD5 Inhibits Furin Cleavage of Human Papillomavirus 16 L2 To Block Infection

2014 ◽  
Vol 89 (5) ◽  
pp. 2866-2874 ◽  
Author(s):  
Mayim E. Wiens ◽  
Jason G. Smith
Keyword(s):  
Human Papillomavirus ◽  
Cell Surface ◽  
Critical Role ◽  
Hpv Infection ◽  
Innate Immune ◽  
Furin Cleavage ◽  
Immune Effector ◽  
Cyclophilin B ◽  
Human Papillomavirus 16 ◽  
Furin Cleavage Site

ABSTRACTHuman papillomavirus (HPV) is a significant oncogenic virus, but the innate immune response to HPV is poorly understood. Human α-defensin 5 (HD5) is an innate immune effector peptide secreted by epithelial cells in the genitourinary tract. HD5 is broadly antimicrobial, exhibiting potent antiviral activity against HPV at physiologic concentrations; however, the specific mechanism of HD5-mediated inhibition against HPV is unknown. During infection, the HPV capsid undergoes several critical cell-mediated viral protein processing steps, including unfolding and cleavage of the minor capsid protein L2 by host cyclophilin B and furin. Using HPV16 pseudovirus, we show that HD5 interacts directly with the virus and inhibits the furin-mediated cleavage of L2 at the cell surface during infection at a step downstream of the cyclophilin B-mediated unfolding of L2. Importantly, HD5 does not affect the enzymatic activity of furin directly. Thus, our data support a model in which HD5 prevents furin from accessing L2 by occluding the furin cleavage site via direct binding to the viral capsid.IMPORTANCEOur study elucidates a new antiviral action for α-defensins against nonenveloped viruses in which HD5 directly interferes with a critical host-mediated viral processing step, furin cleavage of L2, at the cell surface. Blocking this key event has deleterious effects on the intracellular steps of virus infection. Thus, in addition to informing the antiviral mechanisms of α-defensins, our studies highlight the critical role of furin cleavage in HPV entry. Innate immune control, mediated in part by α-defensins expressed in the genital mucosa, may influence susceptibility to HPV infections that lead to cervical cancer. Moreover, understanding the mechanism of these natural antivirals may inform the design of therapeutics to limit HPV infection.

scholarly journals Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Georgia Schäfer ◽  
Lisa M. Graham ◽  
Dirk M. Lang ◽  
Melissa J. Blumenthal ◽  
Martina Bergant Marušič ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Conformational Changes ◽  
Hpv Infection ◽  
Mass Spectrometry Analysis ◽  
Restriction Factor ◽  
Furin Cleavage ◽  
Spectrometry Analysis

ABSTRACT 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.

scholarly journals ADAR1 function affects HPV replication and is associated to recurrent human papillomavirus-induced dysplasia in HIV coinfected individuals

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maria Pujantell ◽  
Roger Badia ◽  
Iván Galván-Femenía ◽  
Edurne Garcia-Vidal ◽  
Rafael de Cid ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Immune Activation ◽  
Hpv Infection ◽  
Innate Immune ◽  
Type I ◽  
Innate Immune Activation ◽  
Enhanced Expression

AbstractInfection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1–24). We identified the low frequency haplotype AACCAT significantly associated with recurrent HPV dysplasia, suggesting a role of ADAR1 in the outcome of HPV infection in HIV+ individuals. In summary, our results suggest that ADAR1-mediated innate immune activation may influence HPV disease outcome, therefore indicating that modification of innate immune effectors regulated by ADAR1 could be a therapeutic strategy against HPV infection.

scholarly journals Neutralization of Human Papillomavirus with Monoclonal Antibodies Reveals Different Mechanisms of Inhibition

2007 ◽  
Vol 81 (16) ◽  
pp. 8784-8792 ◽  
Author(s):  
Patricia M. Day ◽  
Cynthia D. Thompson ◽  
Christopher B. Buck ◽  
Yuk-Ying S. Pang ◽  
Douglas R. Lowy ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Human Papillomavirus ◽  
Cell Surface ◽  
Binding Sites ◽  
Neutralizing Antibodies ◽  
Hpv Infection ◽  
Heparan Sulfate Proteoglycans ◽  
Hacat Cells ◽  
Block Association

ABSTRACT The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells. Surprisingly, two strongly neutralizing MAbs, V5 and E70, did not prevent attachment of capsids to the cell surface. However, they did block association with the ECM and prevented internalization of cell surface-bound capsids. In contrast, MAb U4 prevented binding to the cell surface but not to the ECM. The epitope recognized by U4 was inaccessible when virions were bound to the cell surface but became accessible after endocytosis, presumably coinciding with receptor detachment. Treatment of capsids with heparin, which is known to interfere with binding to cell surface heparan sulfate proteoglycans (HSPGs), also resulted in HPV16 localization to the ECM. These results suggest that the U4 epitope on the intercapsomeric C-terminal arm is likely to encompass the critical HSPG interaction residues for HPV16, while the V5 and E70 epitopes at the apex of the capsomer overlap the ECM-binding sites. We conclude that neutralizing antibodies can inhibit HPV infection by multiple distinct mechanisms, and understanding these mechanisms can add insight to the HPV entry processes.

scholarly journals Analysis of mutations in the E6/E7 oncogenes and L1 gene of human papillomavirus 16 cervical cancer isolates from China

2006 ◽  
Vol 87 (5) ◽  
pp. 1181-1188 ◽  
Author(s):  
Yuping Wu ◽  
Yulong Chen ◽  
Longyu Li ◽  
Guifang Yu ◽  
Ying He ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Cancer Patients ◽  
Hpv Infection ◽  
Typing Method ◽  
Hpv16 E6 ◽  
Human Papillomavirus 16 ◽  
Human Papillomavirus Type 16 ◽  
New Variant

Human papillomavirus type 16 (HPV16) has a number of intratypic variants; each has a different geographical distribution and some are associated with enhanced oncogenic potential. Cervical samples were collected from 223 cervical cancer patients and from 196 age-matched control subjects in China. DNA samples were amplified by using primers specific for the E6, E7 and partial L1 regions. Products were sequenced and analysed. It was found by using a PCR–sequence-based typing method that HPV infection rates in China were 92·8 % in cervical cancer patients and 15·8 % in healthy controls. HPV16 was detected in 70·4 % of cervical cancer patients and in 6·1 % of controls. In HPV16-positive cervical cancers, 23·6 % belonged to the prototype, 65·5 % were of the Asian variant, 5·5 % were of African type 1 and 3·6 % were European variants, whilst only one was a new variant that differed from any variant published so far. Prevalences of HPV16 E6 D25E and E113D variants were 67·3 and 9 %, respectively. In addition to D25E and E113D, the following E6 variations were found in this study: R129K, E89Q, S138C, H78Y, L83V and F69L. The results also showed that the prevalences of three hot spots of E7 nucleotide variation, N29S, S63F and a silent variation, nt T846C, were 70·2 % (33/47), 51·1 % (24/47) and 61·7 % (29/47), respectively. The following L1 variations were found in this study: S377A, K387E, E378D, K382E and T379P. It was also found that the average age of Asian variant-positive cervical cancer patients (42·98±10·43 years) was 7·56 years lower than that of prototype-positive patients (50·54±10·91). It is suggested that the high frequency of HPV16 Asian variants might contribute to the high incidence of cervical cancer in China.

scholarly journals TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction

2021 ◽  
Vol 17 (11) ◽  
pp. e1009820
Author(s):  
Hataf Khan ◽  
Helena Winstone ◽  
Jose M. Jimenez-Guardeño ◽  
Carl Graham ◽  
Katie J. Doores ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Critical Role ◽  
Ectopic Expression ◽  
Lung Epithelial Cells ◽  
Furin Cleavage ◽  
Nuclear Receptor Coactivator ◽  
Host Immune Responses ◽  
Lysosomal Protease ◽  
Furin Cleavage Site ◽  
Endosomal Acidification

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike’s polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

scholarly journals Innate Immune Modulation by GM-CSF and IL-3 in Health and Disease

2019 ◽  
Vol 20 (4) ◽  
pp. 834 ◽  
Author(s):  
Francesco Borriello ◽  
Maria Galdiero ◽  
Gilda Varricchi ◽  
Stefania Loffredo ◽  
Giuseppe Spadaro ◽  
...  
Keyword(s):  
Immune Modulation ◽  
Cytotoxic T Lymphocyte ◽  
Critical Role ◽  
Experimental Models ◽  
Innate Immune ◽  
Immune Effector ◽  
Gm Csf ◽  
Innate Immune Effector ◽  
Macrophage Colony ◽  
Emergency Myelopoiesis

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inteleukin-3 (IL-3) have long been known as mediators of emergency myelopoiesis, but recent evidence has highlighted their critical role in modulating innate immune effector functions in mice and humans. This new wealth of knowledge has uncovered novel aspects of the pathogenesis of a range of disorders, including infectious, neoplastic, autoimmune, allergic and cardiovascular diseases. Consequently, GM-CSF and IL-3 are now being investigated as therapeutic targets for some of these disorders, and some phase I/II clinical trials are already showing promising results. There is also pre-clinical and clinical evidence that GM-CSF can be an effective immunostimulatory agent when being combined with anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) in patients with metastatic melanoma as well as in novel cancer immunotherapy approaches. Finally, GM-CSF and to a lesser extent IL-3 play a critical role in experimental models of trained immunity by acting not only on bone marrow precursors but also directly on mature myeloid cells. Altogether, characterizing GM-CSF and IL-3 as central mediators of innate immune activation is poised to open new therapeutic avenues for several immune-mediated disorders and define their potential in the context of immunotherapies.

Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

2021 ◽  
Author(s):  
Mizuki Yamamoto ◽  
Jin Gohda ◽  
Ayako Kobayashi ◽  
Keiko Tomita ◽  
Youko Hirayama ◽  
...  
Keyword(s):  
Cell Surface ◽  
Spike Protein ◽  
Dependent Manner ◽  
Furin Cleavage ◽  
Entry Pathway ◽  
Furin Cleavage Site ◽  
Metalloproteinase Inhibitors ◽  
A Cell ◽  
A Disintegrin And Metalloproteinase ◽  
Human Coronaviruses

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future.

scholarly journals E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Masahiko Ajiro ◽  
Zhi-Ming Zheng
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Critical Role ◽  
Splice Isoforms ◽  
Hpv16 E6 ◽  
Human Papillomavirus 16 ◽  
Hpv18 E6 ◽  
Splice Isoform ◽  
E6 And E7

ABSTRACTTranscripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.IMPORTANCEHPV16 is the most prevalent HPV genotype, being responsible for 60% of invasive cervical cancer cases worldwide. What makes HPV16 so potent in the development of cervical cancer remains a mystery. We discovered in this study that, besides producing two well-known oncoproteins, E6 and E7, seen in other high-risk HPVs, HPV16 produces E6^E7, a novel splice isoform of E6 and E7. E6^E7, in addition to self-interacting, binds cellular chaperone proteins, HSP90 and GRP78, and viral E6 and E7 to increase the steady-state levels and half-lives of viral oncoproteins, leading to cell proliferation. The splicingciselements in the regulation of HPV16 E6^E7 production are highly conserved in 11 oncogenic or possibly oncogenic HPVs, and we confirmed the production of HPV18 E6^E7 in HPV18-infected cells. This study provides new insight into the mechanism of splicing, the interplay between different products of the polycistronic viral message, and the role of the host chaperones as they function.

scholarly journals Molecular detection of oncogenic subtypes of human papillomavirus (HPV) in a group of women in the Amazon region of Brazil

2020 ◽  
Vol 42 ◽  
pp. e50005
Author(s):  
Alessandra Silva e Silva ◽  
Cláudia Giuliano Bica ◽  
Aniúsca Vieira ◽  
Cleiton Fantin
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Genetic Material ◽  
Hpv Infection ◽  
Cervical Cancer Prevention ◽  
Eligibility Criteria ◽  
Hpv 16 ◽  
Hpv Dna ◽  
Human Papillomavirus 16 ◽  
Hpv 18

The natural history of cervical cancer is strongly related to the presence of human papillomavirus (HPV) infection, with its relationship with cervical cancer being a matter of concern. It is estimated that 70% of all cervical cancers worldwide are caused by HPV 16 and 18. Accordingly, the present study aimed to contribute to the identification of HPV subtypes circulating in a group of women of Manaus-Brazil.  Cervical samples were collected from 49 women, following the eligibility criteria of the study, and DNA was then extracted from the samples, which were analyzed for the presence of the virus in the genetic material through the polymerase chain reaction (PCR) using generic primers (GP05/06). Finally, identification of the viral subtypes was performed using specific primers for the detection of the main subtypes already examined (16 and 18). Positive HPV DNA was detected in 100% of the samples included in the study. Human papillomavirus 16 was the most prevalent subtype in the majority of lesions, accounting for 29 (59.2%) of the positive cases, and HPV 18 was detected in four (8.2%) women. In these 4 cases there was co-infection, with the presence of both HPV 18 and HPV 16. Therefore, 40.8% (20 cases) in which HPV DNA was detected presented infection with other subtypes of HPV not included in the study. This data has clinical implications related to cervical cancer prevention, as the current prophylactic HPV vaccines are only effective against high-risk HPV 16 and 18 subtypes.

scholarly journals Incidence and Clearance of Anal Human Papillomavirus (HPV)-16 and HPV-18 Infection, and Their Determinants, Among Human Immunodeficiency Virus-Infected Men Who Have Sex With Men in France

2019 ◽  
Vol 221 (9) ◽  
pp. 1488-1493 ◽  
Author(s):  
Catharina J Alberts ◽  
Isabelle Heard ◽  
Ana Canestri ◽  
Lucie Marchand ◽  
Jean-François Fléjou ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Papillomavirus ◽  
Hpv Infection ◽  
High Grade ◽  
Hpv 16 ◽  
Human Papillomavirus 16 ◽  
Immunodeficiency Virus ◽  
Adjusted Incidence Rate ◽  
Sex With Men ◽  
Hpv 18

Abstract Background Prospective data on the natural history of anal human papillomavirus (HPV) infection are scarce in human immunodeficiency virus (HIV)-infected men who have sex with men (MSM). Methods We analyzed incidence and clearance of HPV-16 and HPV-18 in a French cohort of HIV-infected MSM, aged ≥35 years, followed-up annually (n = 438, 2014–2018). Results Human papillomavirus-16 and HPV-18 incidence were similar (~10% incident infections at 24 months). Human papillomavirus-16 incidence was higher among high-grade versus no lesion at baseline (adjusted incidence rate ratio = 3.0; 95% confidence interval, 1.07–8.18). Human papillomavirus-16 cleared significantly slower than HPV-18 (32% versus 54% by 24 months). Conclusions In conclusion, anal HPV-16 is more persistent than HPV-18, and its incidence correlates with a prior detection of high-grade lesions.

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