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 antigen-presentaing 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 of HPV DNA which is a crucial step in HPV-induced carcinogenesis [
15]. Hence, cellular A3s act as antiviral restriction factors, but may also result in an increase in chromosomal instability.
Among this A3s family, A3A, A3B, A3C and A3H are expressed and localized in cytoplasm and/or nucleus of human cutaneous keratinocytes [
12,
13,
16]. The antiviral effects of A3s proteins have been reported with HPVs. Hyperedited HPV1a and HPV16 genomes were found in the specimens from HPV1a plantar warts and HPV16 precancerous cervical biopsies [
12]. Zhe Wang et al. also reported that endogenous A3A and A3G induced by IFN-β resulted in early gene 2 (E2) hypermutation of HPV16 in W12 cells [
16]. Then the group further demonstrated that hypermutation in the E2 gene of HPV16 existed in cervical intraepithelial neoplasia, which might be caused by A3s proteins [
17]. In invasive cervical cancer, the viral DNA of HR HPV is frequently integrated into the cellular host DNA, which often resulted in loss of the viral E2 gene, high-level expression and stability of transcripts encoding the E6/E7 oncoproteins [
18]. These oncoproteins are the primary viral factors to initiate and promote tumorigenesis by manipulating cell cycle regulators and inducing DNA damage and chromosomal aberrations [
19]. In contrast to HR HPVs, the E6 proteins from LR HPVs types don’t have transforming activity and/ or immortalization capacity as that of HR HPV types does [
20,
21]. This E6 protein plays a clear role in the initiation of viral DNA replication and are responsible for the pathogenicity of LR HPV types [
20,
21]. Surprisingly, genetic variations of HPV11 E6 were also observed in HPV11 DNA isolated from the clinical specimens of recurrent respiratory papillomatosis, genital warts, anal cancer and cervical neoplasia cells [
22], but the mechanism involved in these variants has not yet been well defined. Our previous study showed that HPV11 could up-regulate A3s expression (especially A3A) in HaCaT keratinocytes containing the genome of HPV 11 (HPV11.HaCaT cells) [
23]. Thus, we speculated that HPV11 E6 gene might be vulnerable to editing by cytidine deaminases of the A3s family in cutaneous keratinocytes.
The family of type I interferons (IFNs) consists of IFN-β, IFN-ε, IFN-κ, IFN-ω and 12 subtypes of IFN-α. They are produced by virus-infected cells and have been reported to exhibit anti-proliferative, immunomodulatory and antiviral activities [
24]. In our previous work, recombinant human interferon-α (rhIFN-α) showed significant inhibition of HPV11 E6/E7 mRNA expression [
25] and induced higher A3A in HPV11.HaCaT cells [
23]. Human IFN-ω, like other IFNs is secreted from cells in response to viral infection and reported as an antiviral agent for treatment of several viruses such as HCV [
26], influenza viruses [
27], HBV [
28]. IFN-ω has a 60-65% homology to human IFN-α1 and has distinguishable biological properties in type I IFNs [
29]. For example, glycosylated IFN-ω has an unique antagonism on the cytotoxic effects of ribavirin that was not observed with IFN-α [
29]. This interferon appeared more efficient than IFN-α2 against influenza viruses [
27] and showed similar antiviral potential as IFN-α2 on HBV replication in human hepatoma cells [
28]. In our laboratory, we also explored the potential antiviral effects of recombinant human interferon-ω (rhIFN-ω), which has shown obvious effects of anti-herpes simplex virus (data not shown). Thus, IFN-ω, used by itself or in combination with other antiviral therapies, might provide a useful alternative for patients who fail to respond to IFN-α or as an additional treatment option [
26]. However, studies of type I IFNs have mainly focused on the subtypes of IFN-α and IFN-β, knowledge of IFN-ω is limited. To date, only a few studies have been made to elucidate the possible mechanisms of IFN-ω-dependent antiviral activities. It remains unknown whether IFN-ω exhibits antiviral activities through the A3s system.
In this context, we investigated whether enhanced A3A could induce hypermutation in HPV11 E6 gene by using differential DNA denaturation polymerase chain reaction (3D-PCR) that can selectively amplify A3s-edited viral DNA [
12], and then explored the effects of exogenous rhIFN-ω on the expression of A3s and E6 in HPV11.HaCaT cells.
Discussion
The A3s system is a family of cellular cytidine deaminases that act on single-stranded DNA (ssDNA) or RNA substrates, providing intrinsic immunity to the host against viral infection. The main function of A3s is to inhibit the replication of different viruses through hypermutation of viral genome, such as HIV, HBV and HPV. To date, most studies of A3-mediated hypermutation in HPV have focused on HR genotypes because of their capabilities to cause carcinogenesis. The E6/E7 genetic variations of HPV11 were detected in HPV11 DNA isolated from HPV11-related epithelial lesions [
22], but the details involved in these variants still remain unknown. Our previous results indicated that A3s immune system (especially A3A) is triggered by HPV11 [
23]. A3A was a powerful restriction factor resulting in inhibition of infection by HPV16 and efficient knockdown of this protein has significantly increased viral infectivity [
31]. A3A protein is mostly localized in the cytoplasm, but becomes cell-wide during telophase [
32]. Thus we hypothesized that the HPV11 DNA might be vulnerable to editing by some of the A3s, raising a concern about whether enhanced A3A could result in HPV11 E6 hypermutation and could inhibit the expression of E6 gene.
In this study, both the immunofluorescence and western blotting results indicated that A3A-HPV11.HaCaT system was successfully established and might be used for further research. Here we also observed that overexpressed A3A repressed the mRNA expression of HPV11 E6, which implied that enhanced A3A could inhibit the expression of HPV11 early genes.
HPV6 or HPV11 infection resulted in cell cycle disruption which is similar to HR-HPVs infection, although there existed some differences in the expression of P53 and cyclin D1 [
33]. HPV18 established its initial infection during S phase, reached its maximum level during G2 phase, and then followed the replication pattern of cellular DNA during S phase in the stable maintenance phase [
34]. We previously observed that the proportion of cells in S-phase and G2/M-phase was increased in HPV11.HaCaT cells compared with that in HaCaT cells, with the G1 phase fractions decreased [
25]. Here we also observed that enhanced A3A induced S-phase arrest in A3A-HPV11.HaCaT cells. All the above findings indicated that transiently exposed viral ssDNA during replication might have more possibilities to be edited by enhanced A3A activities caused by HPV11 when HPV11 manipulated cell cycle of the host cells to established its replication.
Next, we investigated whether enhanced A3A could hypermutate HPV11 E6 gene. As described above, a single G > A edited sequence was detected in HPV11.HaCaT (P8), while no hypermutant was observed in P3, indicating the presence of the E6 mutant in basal-like HaCaT [
30] under the condition of long-term carrying HPV-11 episomes. Thus we speculated that endogenous A3s proteins (especially A3A) triggered by HPV11 might be responsible for this mutation. Then we detected mutation of E6 gene in A3A-HPV11.HaCaT cells. Two C-T/G-A mutants were observed after amplification by 3D-PCR. The above results indicated that HPV11 E6 could be edited by enhanced A3A. The observation that enhanced A3A leaded to a substantial reduction in E6 expression of HPV11 suggested that activated A3A during HPV11 replication is likely to serve a protective function in HPV11.HaCaT cells. Moreover, because the advantage of uracil DNA glycosylase over the activities of cytidine deaminase might cover up the E6 hypermutation [
16], further investigations are required to confirm the involvement of A3A in HPV11 E6 mutation by inhibiting uracil DNA glycosylase and transfection of small interfering RNAs against A3A.
Finally we investigated whether A3s family was induced by rhIFN-ω and explored the effects of the agent on E6 gene expression. IFNs are potent mediators involved in cell-to-cell signaling and trigger various pathways to block intracellular replication of viruses and to impede the infection of surrounding cells [
35,
36]. IFN-ω, like other IFNs, is a cytokine produced by host cells in defense against viruses and has non-specific antiviral activity [
26,
35]. In vitro studies indicated a strong activation of IFN signaling by IFN-ω, but IFN-κ exhibited weaker activation [
36]. In this study, rhIFN-ω treatment of HPV11.HaCaT cells could lead to upregulation of A3A mRNA expression and obvious increasement of A3A staining throughout the cells. This agent could also significantly inhibit mRNA expression of HPV11 E6. Based on these results, we speculated that rhIFN-ω might inhibit viral gene expression by enhanced A3A.
All the above results suggested that enhanced A3A might be one of the mechanisms of immunomodulation function and antiviral activities of rhIFN-ω. However we need to further confirm whether there exists mutation of HPV11 episomal DNA in HPV11.HaCaT cells after rhIFN-ω treatment, further studies are required to elucidate the function of A3s in HPV11-related epithelial lesions as well as in anti-HPV11 effects of rhIFN-ω.