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Merkel Cell Polyomavirus Infection Induces an Antiviral Innate Immune Response in Human Dermal Fibroblasts.

ABSTRACT: Merkel cell polyomavirus (MCPyV) infects most of the human population asymptomatically, but in rare cases it leads to a highly aggressive skin cancer called Merkel cell carcinoma (MCC). MCC incidence is much higher in aging and immunocompromised populations. The epidemiology of MCC suggests that dysbiosis between the host immune response and the MCPyV infectious cycle could contribute to the development of MCPyV-associated MCC. Insufficient restriction of MCPyV by normal cellular processes, for example, could promote the incidental oncogenic MCPyV integration events and/or entry into the original cell of MCC. Progress toward understanding MCPyV biology has been hindered by its narrow cellular tropism. Our discovery that primary human dermal fibroblasts (HDFs) support MCPyV infection has made it possible to closely model cellular responses to different stages of the infectious cycle. The present study reveals that the onset of MCPyV replication and early gene expression induces an inflammatory cytokine and interferon-stimulated gene (ISG) response. The cGAS-STING pathway, in coordination with NF-κB, mediates induction of this innate immune gene expression program. Further, silencing of cGAS or NF-κB pathway factors led to elevated MCPyV replication. We also discovered that the PYHIN protein IFI16 localizes to MCPyV replication centers but does not contribute to the induction of ISGs. Instead, IFI16 upregulates inflammatory cytokines in response to MCPyV infection by an alternative mechanism. The work described herein establishes a foundation for exploring how changes to the skin microenvironment induced by aging or immunodeficiency might alter the fate of MCPyV and its host cell to encourage carcinogenesis. IMPORTANCE MCC has a high rate of mortality and an increasing incidence. Immune-checkpoint therapies have improved the prognosis of patients with metastatic MCC. Still, a significant proportion of the patients fail to respond to immune-checkpoint therapies or have a medical need for iatrogenic immune-suppression. A greater understanding of MCPyV biology could inform targeted therapies for MCPyV-associated MCC. Moreover, cellular events preceding MCC oncogenesis remain largely unknown. The present study aims to explore how MCPyV interfaces with innate immunity during its infectious cycle. We describe how MCPyV replication and/or transcription elicit an innate immune response via cGAS-STING, NF-κB, and IFI16. We also explore the effects of this response on MCPyV replication. Our findings illustrate how healthy cellular conditions may allow low-level infection that evades immune destruction until highly active replication is restricted by host responses. Conversely, pathological conditions could result in unbridled MCPyV replication that licenses MCC tumorigenesis.

SUBMITTER: Krump NA

PROVIDER: S-EPMC8437356 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse (Mus musculus), rabbit (Oryctolagus cuniculus), rat (Rattus norvegicus), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), patas monkey (Erythrocebus patas), common woolly monkey (Lagothrix lagotricha), red-chested mustached tamarin (Saguinus labiatus), and tree shrew (Tupaia belangeri). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression.IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel cell carcinoma (MCC). With the increasing number of MCC diagnoses, there is a need to better understand the virus and its oncogenic potential. However, studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. To pinpoint the best candidate for developing an MCPyV infection animal model, we examined MCPyV's ability to infect dermal fibroblasts isolated from various established model animals. Of the animal cell types we tested, chimpanzee dermal fibroblasts were the only isolates that supported the full MCPyV infectious cycle. To overcome the infection blockade in the other model animals, we constructed chimeric viruses that achieved robust MCPyV entry and oncogene expression in rat fibroblasts. Our results suggest that the rat may serve as an in vivo model to study MCV oncogenesis.

Project description:Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a large tumor antigen (LT-Ag) that, together with a small tumor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G2 We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G2 arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells.IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G2 arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP).

Project description:The innate immune system provides the first response to pathogen infection and orchestrates the activation of the adaptive immune system. Though a large component of the innate immune response is common to all infections, pathogen-specific innate immune responses have been documented as well. The innate immune response is thought to be especially critical for fighting infection with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB). While TB can be a deadly disease, only 5-10% of individuals infected with MTB develop active disease, and this inter-individual variation is, at least partly, heritable. Studies of inter-individual variation in the innate immune response to MTB infection may therefore shed light on the genetic basis for variation in susceptibility to TB. Yet, to date, we still do not know which properties of the innate immune response are specific to MTB infection and which represent a general response to pathogen infection. To begin addressing this gap, we infected macrophages with eight different bacterial pathogens, including different MTB strains and related mycobacteria, and studied the transcriptional response to infection. We found that although the gene expression changes were largely consistent across the bacterial infection treatments, we were able to identify a novel subset of genes whose regulation was affected specifically by infection with mycobacteria. Genetic variants that are associated with regulatory differences in these genes should be considered candidate loci for explaining inter-individual susceptibility TB. RNA-seq of monocyte-derived macrophages isolated from 6 healthy European males at 4, 18, and 48 hours post-infection with the following 8 bacteria: Mycobacterium tuberculosis (MTB) H37Rv, Mycobacterium tuberculosis GC1237, MTB GC1237, bacillus Calmette-Guérin (BCG), Mycobacterium smegmatis, Yersinia pseudotuberculosis, Salmonella typhimurium, and Staphylococcus epidermidis. table-s1.txt is a tab-delimited text file that contains the batch-corrected log2 counts per million for each of the 156 samples, as well as the Ensembl gene ID and gene name. BCG = bacillus Calmette-Guérin GC = Mycobacterium tuberculosis GC1237 Rv = Mycobacterium tuberculosis (MTB) H37Rv Rv+ = heat-inactivated MTB H37Rv Salm = Salmonella typhimurium Smeg = Mycobacterium smegmatis Staph = Staphylococcus epidermidis Yers = Yersinia pseudotuberculosis

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Merkel Cell Polyomavirus Infection Induces an Antiviral Innate Immune Response in Human Dermal Fibroblasts.

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