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

Virology Journal, Nov 2017

Condyloma 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). A3A-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. The 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. Enhanced 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 E6 mutation by overexpression of APOBEC3A and effects of interferon-ω on APOBEC3s and HPV11 E6 expression in HPV11.HaCaT cells

Wang et al. Virology Journal HPV11 E6 mutation by overexpression of APOBEC3A and effects of interferon-ω on APOBEC3s and HPV11 E6 expression in HPV11.HaCaT cells Yongfang Wang 0 Xinyu Li 0 Shasha Song 0 Yang Sun 0 Jiafen Zhang 0 Changming Yu 1 Wei Chen 1 0 Department of Pharmacology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College , 12 Jiang Wang Miao Street, Nanjing 210042 , China 1 Laboratory of Vaccine and Antibody Engineering, Beijing Institute of Biotechnology , Beijing 100071 , China Background: Condyloma 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). Methods: A3A-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. Results: The 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 upregulated 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. Conclusions: Enhanced A3A repressed HPV11 E6 expression through gene hypermutation, and rhIFN-ω might be an effective agent against HPV11 infection by up-regulation of A3A. Condylomata acuminata; Human papillomavirus 11; HaCaT keratinocytes; Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3; Early gene 6; Interferon-ω Background Human papillomaviruses (HPVs) are a group of small, double-stranded DNA viruses and display strict tissue specificity [ 1 ]. They only infect mucosal or cutaneous epidermal tissues of humans and cause a wide range of apparent epithelial lesions [ 2 ]. According to oncogenic potential, HPVs can be classified as the high-risk (HR) and low-risk (LR) genotypes. HR HPVs (e.g., HPV16, HPV18) are commonly associated with cervical cancers, while LR HPVs (e.g., HPV6 and HPV11) mainly induce benign lesions, such as condylomata acuminate (CA) [ 3 ]. CA has become one of the most widespread sexually transmitted diseases and present a serious threat to social public health because of the increasing incidence and the high recurrence rate throughout the world [ 4 ]. It means that HPVs had evolved different ways to avoid detection and clearance by both the innate and adaptive immune system, leading to recurrent and protracted illness [ 5, 6 ]. LR HPVs are self-limiting and will usually be cleared by the host immune system in most infections. However, among susceptible populations, such infections tend to become persistent, a likely prerequisite for malignant progression [7]. Thus, the interaction of viral and host immune status especially at the specific sites of infection may play important roles in disease susceptibility and progression of CA. When LR HPVs invade epithelium through skin wounds, basal keratinocytes are the primary targets of HPVs infection. Recent study reinforces the importance of the keratinocytes as immune sentinels in producing innate immune mediators, acting as non-professional antigenpresentaing cells and instigators of inflammation [ 8 ]. The family of Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 proteins (APOBEC3s, A3s) plays an important roles in innate immune system [ 9 ]. The family comprises seven members: A, B, C, DE, F, G and H [ 9 ]. The A3s system is widely expressed in different tissues and cell types, especially dendritic cells, macrophages, CD4+ T cells and keratinocytes [ 10–12 ]. These members can edit single-stranded DNA (ssDNA) and/or RNA substrates of different viruses by converting cytidines to uridines (C to U) or deoxycytidines to deoxyuridines (dC to dU) [ 9 ]. They act as potent innate antiviral factors against exogenous viruses such as HIV, HBV and HPV [ 12, 13 ]. The family may also induce mutation clusters in different types of cancer, for example, cervical, bladder and breast cancers [14]. Recently, APOBEC3A (A3A) has been reported to be strongly correlated with the integration (...truncated)


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Yongfang Wang, Xinyu Li, Shasha Song, Yang Sun, Jiafen Zhang, Changming Yu, Wei Chen. HPV11 E6 mutation by overexpression of APOBEC3A and effects of interferon-ω on APOBEC3s and HPV11 E6 expression in HPV11.HaCaT cells, Virology Journal, 2017, pp. 211,