Development of Basal-Like HaCaT Keratinocytes Containing the Genome of Human Papillomavirus (HPV) Type 11 for Screening of Anti-HPV Effects

Wang, Yongfang; Li, Xinyu; Song, Shasha; Wu, Jianbo

doi:10.1177/1087057114536987

Cited by 5 publications

(11 citation statements)

References 27 publications

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“…HPV11.HaCaT cells were established and cultured as described previously [ 30 ]. HaCaT cells were routinely cultured and passaged by trypsinization using conventional methods.…”

Section: Methodsmentioning

confidence: 99%

HPV11 E6 mutation by overexpression of APOBEC3A and effects of interferon-ω on APOBEC3s and HPV11 E6 expression in HPV11.HaCaT cells

Wang

Song

et al. 2017

Virol J

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BackgroundCondyloma acuminatum, infected by low-risk human papillomaviruses (e.g., HPV6 and HPV11), is one of the most widespread sexually transmitted diseases. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 proteins (APOBEC3s, A3s) are cellular cytidine deaminases acting as antiviral factors through hypermutation of viral genome. However, it remains unknown whether A3s results in HPV11 gene mutations and interferon-ω (IFN-ω) exhibits antiviral activities through the A3s system. Here we investigated whether enhanced APOBEC3A (A3A) resulted in the E6 gene mutations and explore the effects of recombinant human interferon-ω (rhIFN-ω) on A3s/E6 expression in HaCaT keratinocytes containing the genome of HPV 11 (HPV11.HaCaT cells).MethodsA3A-overexpressed HPV11.HaCaT (A3A-HPV11.HaCaT) cells were established by lentiviral infection and verified by immunofluorescence and western-blotting. Cell cycle, E6 gene mutations, APOBEC3s/E6 gene expression and subcellular localization were detected by FACS, 3D-PCR and sequencing, qRT-PCR and immunofluorescence respectively.ResultsThe results suggested that A3A-HPV11.HaCaT cells were successfully established. Enhanced A3A induced S-phase arrest, G > A/C > T mutations and obvious reduction of E6 mRNA expression. A3A/A3B mRNA expression was up-regulated at 6 h and 12 h and obvious A3A staining existed throughout HPV11.HaCaT cells after rhIFN-ω treatment. RhIFN-ω could also inhibit mRNA expression of HPV11 E6 significantly.ConclusionsEnhanced A3A repressed HPV11 E6 expression through gene hypermutation, and rhIFN-ω might be an effective agent against HPV11 infection by up-regulation of A3A.

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“…HPV11.HaCaT cells were established and cultured as described previously [ 30 ]. HaCaT cells were routinely cultured and passaged by trypsinization using conventional methods.…”

Section: Methodsmentioning

confidence: 99%

HPV11 E6 mutation by overexpression of APOBEC3A and effects of interferon-ω on APOBEC3s and HPV11 E6 expression in HPV11.HaCaT cells

Wang

Song

et al. 2017

Virol J

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“…The HaCaT cells were cultured as previously described [8]. The pBR322.HPV11 plasmid [12,294 base pairs (bp)] from Escherichia coli (ATCC No.…”

Section: Methodsmentioning

confidence: 99%

“…After the above steps, the circularized HPV 11 DNA and pTK-neo DNA (Novagen, USA) were transfected into HaCaT cells. After selection with G418 (Sigma, USA), the remaining cell colonies were pooled as a cell population, which was named HPV11.HaCaT [8]. …”

Section: Methodsmentioning

confidence: 99%

“…The HaCaT and HPV11.HaCaT cells were cultured as described previously [8]. The cells were collected, resuspended with new fresh medium and subsequently counted.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we have developed a basal-like HaCaT keratinocytes containing genome of HPV 11 for screening of anti-HPV 11 effect, and proved stable replication of HPV11 DNA in definite passages of cells by detecting HPV11 DNA copy number and E1^E4 transcripts [8]. Because the complete life cycles of HPVs depends on the differentiation program of infected epithelial cells, we also observed that L1 capsid protein expression of HPV11 in the raft cultures of the recombinant cells [9].…”

Section: Introductionmentioning

confidence: 93%

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Integrity of a HPV11 infection cell model and identification of (-)-Epigallocatechin-3-gallate as a potential HPV11 inhibitor

Sun

Song

et al. 2016

Oncotarget

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BackgroundCondyloma acuminatum (CA) is one of the most common sexually transmitted diseases and induced by low-risk human papillomaviruses (HPVs), mainly HPV type 6 and 11. Here, we report the identification of (-)-Epigallocatechin-3-gallate (EGCG) by an HPV11 infection cell model.ResultsThe recombined HPV11.HaCaT cells had stable HPV 11 early genes expression. The introducing of HPV11 genome significantly increased the proliferation of HPV11.HaCaT cells, as well as the proportion of cells in S and G2/M phases. After treated with rhIFN-α 2a, IFN signaling pathway was activated in both HaCaT and HPV11.HaCaT cells, while HPV11 decreased the activation level. In addition, rhIFN-α 2a, could inhibit expression of HPV 11 E6 and E7 mRNA significantly (P<0.05). However, cell growth and cell cycle did not show statistical difference (P>0.05). Nevertheless, EGCG, a major active constituent in tea polyphenol, showed strong anti-HPV11 effect, which inhibited HPV11 E6 and E7 mRNA.MethodsGene transfection technique was used to introduce HPV11 genome into HaCaT cells, named HPV11.HaCaT cells. With the established cell model, we explore the anti-HPV11 effect of (-)-Epigallocatechin-3-gallate (EGCG) on cell growth, viability and affection on expression HPV11 E6 and E7 mRNA.ConclusionOur data collectively demonstrated that the recombinant HPV11.HaCaT cells were integral and practical to be a cell model to test anti-HPV11 agents and explore the interaction between HPV11 genes and host cells. And EGCG inhibits expression of HPV11 E6 and E7 mRNA in the recombinant HPV11.HaCaT cells.

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Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

Gilson

Culleton

Xie

et al. 2020

J Virol

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Several serine and threonine residues of the papilloma virus early E2 protein have been found to be phosphorylated. By contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this paper we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the non-phosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither bind the C-terminus of Brd4, but in all other aspects, the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in-vitro assay, while HPV-31 Y102E binds E1 and replicated albeit at lower levels than wild type. To examine effect of E2 mutations under more native-like infection conditions, a neomycin selectable marker was inserted into L1/L2 of HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days post-transfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines. IMPORTANCE Post-translational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.

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Development of Basal-Like HaCaT Keratinocytes Containing the Genome of Human Papillomavirus (HPV) Type 11 for Screening of Anti-HPV Effects

Cited by 5 publications

References 27 publications

HPV11 E6 mutation by overexpression of APOBEC3A and effects of interferon-ω on APOBEC3s and HPV11 E6 expression in HPV11.HaCaT cells

HPV11 E6 mutation by overexpression of APOBEC3A and effects of interferon-ω on APOBEC3s and HPV11 E6 expression in HPV11.HaCaT cells

Integrity of a HPV11 infection cell model and identification of (-)-Epigallocatechin-3-gallate as a potential HPV11 inhibitor

Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

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