Merkel cell polyomavirus (MCPyV) is the most recently discovered human oncogenic virus (1
). It has been associated with Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer (1–10
). MCPyV is also a widespread virus, detected on the skin of most healthy adults (11–13
). Serological studies have established that MCPyV infects most humans during early childhood and that viral prevalence increases as populations age (14–16
). The vast majority of MCPyV infections are asymptomatic (17
), but some result in the development of MCC (1–4
Although MCC cases are rare, the incidence of MCC has tripled over the last 20 years (18–21
) and increased by more than 95% in the United States since 2000 (19
). MCC is one of the most aggressive skin cancers, with a nearly 50% mortality rate (exceeding the rate of melanoma) and a 5-year survival rate of less than 45% (1–8
). MCC metastasizes rapidly, and currently there are no effective treatments for cases of metastatic cancers (24
). Chemotherapies have thus far failed to produce durable responses in patients with metastatic disease (25
). Recently, anti-PD1 and anti-PD-L1 treatments have shown exciting results in some MCC patients, but the durability of responses is variable, and a significant proportion of patients do not respond (27–29
). Given the high prevalence of MCPyV infection and the increasing incidence of MCC diagnoses (21
), a better understanding of the mechanisms that drive MCPyV oncogenesis is needed in order to develop more effective strategies to prevent and treat this highly lethal skin cancer.
MCPyV has a circular, double-stranded DNA genome of ~5 kb that carries an efficient repertoire of genes (3
). The viral genome is divided into early and late regions by a noncoding regulatory region (NCRR) containing the viral origin of replication and bidirectional promoters responsible for early and late gene transcription (3
). The early region encodes large tumor antigen (LT), small tumor antigen (sT), 57kT, and a microRNA and contains an alternate LT open reading frame (ALTO) (3
). The late region encodes the major and minor capsid proteins, VP1 and VP2 (35
). Of the early genes, the roles of LT and sT in promoting viral replication and host cell proliferation have been best described (22
MCPyV integration into the host genome is a key event that drives virus-positive MCC development (22
). Integrated MCPyV genome is detectable in more than 80% of MCC tumors, in which the virus maintains the expression of native sT and a truncated LT (LTT) (1
). These are the major oncogenes that support MCPyV-induced tumorigenesis (1
). Despite our knowledge of the MCPyV genome and the functions of its viral oncogenes, many aspects of its virology and infectious cycle are poorly understood. Hence, it is unclear what mechanisms and prerequisite conditions trigger MCPyV genome integration and MCC development.
MCPyV maintains asymptomatic infection in most of the general population (11–13
), but in elderly and immunosuppressed individuals, infection has a higher chance to trigger MCC development (52
). Epidemiological studies have established a strong link between immunosuppression, elevated MCPyV genome loads, and increased risk for MCC (53
). Factors that can dampen antiviral immunity, such as advanced age, chronic UV exposure, and immunosuppression, have been shown to significantly increase the risk of developing MCPyV-positive MCC (18
). MCPyV DNA prevalence and viral load on sun-exposed skin increase dramatically in individuals over the age of 40 and remain high for older age groups (55
). Within the same age range group, individuals who are immunocompromised due to HIV/AIDS, chronic lymphocytic leukemia, or treatment for autoimmunity or organ transplantation are at the greatest risk for MCC development (9
). Additionally, MCPyV DNA is more likely to be detected on the skin of HIV-positive men than healthy controls, and those with poorly controlled HIV infection frequently maintain higher MCPyV DNA loads than individuals with better-controlled infections (55
). Furthermore, viral oncogenesis is more rapid and aggressive in HIV-positive and immunosuppressed patients (60
), suggesting that the elevated MCPyV DNA loads associated with immunosuppression may contribute to the increased likelihood of MCC development. Collectively, these epidemiological studies suggest that MCPyV can strike a delicate balance with human hosts to avoid eradication and maintain persistent infection, but failure of the immune system to control MCPyV infection may enable unimpeded propagation of MCPyV. Rampant MCPyV infection could then lead to more frequent replication errors, which can in turn stimulate genome integration and ultimately promote MCPyV-induced tumorigenesis. Little is known, however, about the host immune response elicited by MCPyV. The way MCPyV interfaces with the host immune response to either establish long-term asymptomatic infection or integrate and trigger oncogenesis in different settings is also poorly understood.
Our abilities to probe the host immune responses that keep MCPyV infection in check have been predominantly limited by a dearth of tools and systems with which to study MCPyV infection. Using ex vivo
skin sections and in vitro
cultures of cells isolated from human foreskins, we previously discovered that human dermal fibroblasts (HDFs) have the capability to support MCPyV infection activities (61–63
). The in vitro
and ex vivo
MCPyV infection models established in our studies afford a great opportunity to investigate the host innate immune response to MCPyV infection and to determine what impact those responses have on the course of MCPyV proliferation. Using this system, our additional studies demonstrated that MCPyV infection activates the cGAS-STING as well as the NF-κB signaling pathways to induce robust expression of key interferon (IFN)-stimulated genes (ISGs) and inflammatory cytokines in HDFs (64
). We also discovered that the PYHIN protein IFI16 upregulates inflammatory cytokines in response to MCPyV infection (64
In the present study, we sought to further characterize the molecular events leading to the MCPyV-induced ISG response. We also examined the impact of the MCPyV-induced ISG response on viral infection fate. We discovered that type I IFN can restrict viral infection at the early transcription stage, but growth factors that are often enriched in skin wounds can antagonize this effect to stimulate viral infection. Together, our data provide new insights for understanding how the intricate MCPyV-host relationship provides a molecular mechanism to support viral persistence.
Like many human cancers with a viral etiology, MCC develops much more frequently in immunosuppressed individuals (52
), underscoring the critical role of host immunity in viral oncogenic control. MCPyV is one of the most lethal tumorigenic viruses in immunocompromised patients, but how the virus interfaces with the host immune system to achieve persistent infection and how inadequate control of MCPyV infection encourages MCC tumorigenesis remain poorly understood.
In previous studies, we applied the MCPyV infection model established in our lab to investigate the largely unknown innate immune response to MCPyV infection. We showed that MCPyV infection generates pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) that activate STING- and NF-κB-mediated induction of cytokines and ISGs (64
). In the current study, we further discovered that MCPyV infection of primary normal HDFs stimulates expression of type I and III IFNs, which in turn stimulate robust induction of its downstream ISGs (Fig. 1
). Due to the role of IFNs and ISGs in stimulating the antiviral state of host cells (73
), we assessed the signaling pathways responsible for IFN-induced ISG induction and its functional impact on MCPyV infection. We found that treatment with IFN-β antibody, JAK inhibitors, and CRISPR KO of IFNAR1 could all dramatically repress MCPyV infection-induced ISG expression, but none of these approaches significantly elevated MCPyV replication activities (Fig. 3 to 5
). These findings suggest that IFN-induced ISG production in response to MCPyV infection is not essential for limiting viral infection in HDFs. Instead, we found that type I IFN exerts a more direct effect on MCPyV infection at the postentry stage by repressing early viral transcription (Fig. 6
). This finding is significant because MCPyV early gene transcription is critical for supporting both the MCPyV life cycle and MCC tumorigenesis. In addition to the effect of IFNs on MCPyV infection, we found that growth factors such as TGF-β and FGFs, which are present in FBS but also physiologically upregulated in the dermis during healing of skin wounds and UV-induced skin damage (70–72
), could significantly stimulate MCPyV proliferation (Fig. 8
Together, our data support a hypothetical model in which MCPyV maintains persistent infection in healthy individuals. In this model, healthy immune responses such as IFN production induced by viral activity may restrict viral propagation to reduce MCPyV burden within the host. At the same time, growth factors induced by UV irradiation or abrasion of the skin may stimulate infected dermal fibroblasts to promote MCPyV propagation. A delicate balance of these mutually antagonizing factors would allow the virus to persist at a low level in infected cells. In immunocompromised patients and those affected by MCC risk factors, such as excessive exposure to UV radiation and wounding (42
), disturbance of the virus-host balance could alter the course of MCPyV propagation to stimulate pathological rampant MCPyV replication and oncogene overexpression, which can in turn promote viral DNA integration and MCPyV-induced tumorigenesis.
Our studies also revealed a cross talk between the innate immune response and growth factor-induced signaling pathways. We previously found that MCPyV infection could activate the STING/TBK1/IRF3 and NF-κB pathways (64
). In addition to its antiviral properties, it has been well documented that the NF-κB pathway can also induce the expression of key growth factors, such as IGF-1, TGF-β, FGFs, and epidermal growth factors (EGFs), and stimulate the activities of the growth factor receptors (75–80
). These observations suggest that even in healthy unwounded skin, growth factor signaling pathways can be stimulated inside infected cells by activated NF-κB to promote viral propagation. Conversely, these growth factors and related receptor kinases can also negatively regulate NF-κB-associated pathways, providing a mechanism by which these growth factors may antagonize IFN signaling to promote MCPyV infection (81
The findings herein also pose questions regarding the role of ISGs in MCPyV infection. Previous studies have shown that overexpression of T antigens encoded by SV40, JC polyomavirus (JCPyV), or BK polyomavirus (BKPyV) in mouse embryonic fibroblasts leads to upregulation of ISGs and an antiviral state (83
). Similarly, JCPyV infection of human renal proximal tubular epithelial cells triggers IFN secretion and subsequent ISG induction, which could at least partially control viral infection (85
). BKPyV infection of microvascular endothelial cells also stimulates an IFN-mediated ISG induction that could dampen viral infection (86
). However, it is unclear why blocking the induction of ISGs in response to MCPyV infection does not appear to have a significant impact on viral activity. One explanation is that MCPyV has evolved strategies to successfully counteract the ISG-mediated host immune response. Another possibility is that some ISGs are induced by IFNs in a manner that is independent of the IFNR-JAK-STAT canonical pathway and therefore are not repressed by IFN-β antibody, JAK inhibitors, or IFNAR1 KO. ISG54 is one such ISG, which appeared to be nonessential for blocking MCPyV infection (Fig. S2). Still, it is possible that other ISGs not explored here are involved in controlling MCPyV infection and need to be investigated. Finally, it could also be that the amount of type I IFNs and ISGs induced by MCPyV infection may be too small to block viral replication and transcription (Fig. S3B).
Our observation that type I IFN can repress MCPyV early transcription is consistent with a previous study showing that type I and II IFNs exert direct inhibitory effects on MCC cell lines in vitro
and in vivo
through modulation of MCPyV LT expression (87
). Our findings add to the existing literature supporting the ability of IFNs to control the transcriptional regulation of specific genes such as T cell coinhibitory receptors, programmed death ligand 1 (PD-L1), nitric oxide synthase 2 (NOS2), and triggering receptors expressed on myeloid cells (TREM) (88–91
). However, the manner in which IFNs inhibit the MCPyV early promoter remains to be fully investigated. Nevertheless, because MCPyV-positive MCC cells are dependent on the expression of viral oncogenes to survive, our findings as well as those presented by Willmes et al. (87
) indicate that type I IFN may be a promising therapeutic option for treating patients with MCPyV-positive MCC.
In summary, by characterizing the MCPyV immune signaling pathways and examining their impact on viral infection, our studies provide new clues for understanding how an oncogenic virus such as MCPyV strikes a fine balance with the host defense and growth control mechanisms to achieve persistent infection. Our findings also shed new light on how the antiviral and anticancer properties of type I IFNs may be explored as a new strategy for preventing and treating MCPyV-positive MCC.