Characterization of the Impact of Merkel Cell Polyomavirus-Induced Interferon Signaling on Viral Infection

In our previous study, we discovered that MCPyV infection induces a robust innate immune response in HDFs driven by the cGAS-STING and NF-κB signaling pathways (64). Additionally, we found that MCPyV infection causes the upregulated expression of key ISGs and inflammatory cytokines (64). Based on this finding, we sought to further investigate the possible role of ISGs in regulating the innate immune response to MCPyV infection in this study.

ISGs are normally induced by the activation of IFN downstream signaling. IFNs secreted from the virus-infected cell are known to function via autocrine and paracrine modalities through binding of a heterodimeric cell surface receptor complex, which activates the JAK-STAT signaling pathway to stimulate the transcription of ISGs in the nucleus (65–67). Our previous studies detected global, population-level changes in innate immune response signaling in response to MCPyV infection (64). To more accurately probe the questions of how the innate immune response in infected HDFs is regulated and whether presumed cytokine signaling spreads the innate immune response to neighboring cells, the activation of innate immune effectors was examined at the single-cell level in infected HDFs by immunofluorescence (IF) staining.

Expression of several key ISGs downstream of type I and type III IFN (Mx1, ISG15, OAS1, IFI16, and viperin) was induced at the protein level by MCPyV infection (Fig. 1). We also observed that the ISGs examined in our studies were broadly expressed among cells with both detectable and undetectable expression of viral proteins (Fig. 1). This result implies that a potential antiviral response is spread to all cells through cytokine signaling, which may then control viral infection.

Because MCPyV infection induced a robust induction of ISGs in both our current and previous studies (Fig. 1) (64), we decided to further examine the level of IFNs released by MCPyV-infected cells. In our prior study (64), we found that MCPyV infection causes a modest induction of type I IFNs. In that study, HDFs treated with heat-inactivated MCPyV stocks were used as the negative control for HDFs infected with live virus in order to specifically examine the effects of viral activity and not of other components of the viral preparation on the innate immune response (64). We subsequently discovered that our heat-inactivation protocol does not completely kill MCPyV and the resulting MCPyV stocks could still induce type I IFN expression in HDFs (data not shown), contributing to the apparently low IFN induction by live virus in our prior measurements (64). In this study, we decided to reexamine the complete innate immune response to MCPyV treatment in HDFs using untreated cells—rather than cells treated with heat-inactivated MCPyV stocks—as the negative control in all experiments. In this modified infection protocol, primary HDFs were untreated or treated with live MCPyV virions in the absence of serum and presence of collagenase for 2 days to promote viral entry and then treated with fetal bovine serum (FBS) to initiate viral gene expression. By examining the relative expression of IFNs and inflammatory cytokines in MCPyV-infected HDFs, we discovered that the expression of both IFN-β and IFN-λ1 was stimulated significantly in MCPyV-treated cells compared to untreated cells (Fig. 2A). The cycle threshold (C_T) values obtained from the reverse transcription-quantitative PCR (RT-qPCR) analysis for other IFNs (IFN-α, IFN-λ2-4, IFN-ω, and IFN-γ) were all above 35, suggesting that the expression levels of these IFNs were not stimulated by MCPyV infection. MCPyV infection also stimulated the transcription of inflammatory cytokines such as interleukin 1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α), recapitulating our previous findings (Fig. 2A) (64). Furthermore, expression of IFN-β, IFN-λ, and the key downstream ISGs such as OAS1 increased dramatically during the course of viral infection, with the peak of expression coinciding with the peak of viral early gene expression (Fig. 2B). This result suggests that the IFN and ISG responses are primarily induced by MCPyV DNA replication and/or transcription but not by the inoculating virions or components of the virus preparation (Fig. 2B).

The broad activation of ISGs in MCPyV-infected HDFs indicates that the JAK-STAT signaling pathway downstream of IFN may contribute to the induction and spread of the antiviral response between cells during infection. To determine whether JAK-STAT signaling downstream of IFNs and the IFN receptors mediates an antiviral response to MCPyV, HDFs were treated with either an IFN-β antibody or the JAK inhibitor ruxolitinib on days 2 and 3.5 of infection and collected on day 5 of infection for IF or RT-qPCR analysis (Fig. 3). Consistent with previous observations (Fig. 1) (64), MCPyV infection stimulated robust ISG expression as demonstrated by significantly increased Mx1 expression at the protein level (Fig. 3A and B). Both IFN-β antibody and ruxolitinib treatments efficiently repressed Mx1 expression in MCPyV-infected cells, suggesting that they are effective at abrogating MCPyV-induced IFN signaling (Fig. 3A and B). Because the IFN-β antibody (inhibiting type I IFN only) and ruxolitinib (inhibiting signaling downstream of both type I and III IFN) were both highly effective at repressing Mx1 expression, our data suggest that type I IFN is the primary cytokine driving ISG expression in infected HDFs (Fig. 3A and B). However, successful ablation of ISG induction by IFN-β antibody and ruxolitinib does not appear to have a significant impact on restoring MCPyV infection activity (Fig. 3C and D). From these data, we conclude that IFN-β drives the expression of infection-induced ISGs, but the ISG response does not appear to play an important role in repressing MCPyV infection in our current system.

To further examine the function of IFN downstream signaling in controlling MCPyV infection, we applied lentiCRISPR/Cas9 technology to generate stable knockouts (KOs) of IFN-α/β receptor subunit 1 (IFNAR1) in HDFs using three different single guide RNAs (sgRNAs) (Fig. S1). Of the three stable cell lines generated, the IFNAR1 KO sg2 cell line was selected for further experiments based on its ability to effectively repress IFNAR1 expression and ISG response to exogenous type I IFN compared to control sgLuc HDFs (encoding sgRNA targeting luciferase) (see Fig. S1A and B in the supplemental material). These IFNAR1 KO cells, which are incapable of “receiving” IFN signaling, were used to assess the role of type I IFN signaling in preventing MCPyV infection. IFNAR1 KO HDFs and control sgLuc HDFs were treated with MCPyV and analyzed on day 5 or 6 postinfection (Fig. 4). In the control sgLuc HDFs, MCPyV infection induced robust expression of ISGs such as OAS1, Mx1, viperin, and ISG15, at both the mRNA level and the protein level (Fig. 4A, C, and D). In response to MCPyV infection, IFNAR1 KO effectively abrogated the induction of these ISGs while displaying little effect on the IFN-β expression induced by infection (Fig. 4A, C, and D). However, inhibition of paracrine IFN-β signaling by IFNAR1 KO did not significantly affect MCPyV transcription or replication as indicated by the levels of MCPyV LT mRNA and of viral genome copy number (Fig. 4A, LT panel, and Fig. 4B). These results further suggest that the IFN downstream IFNAR1 signaling pathway is unlikely to play an important role in influencing MCPyV infection.

Unlike the other ISGs examined, ISG54 induction by MCPyV infection was not significantly inhibited in IFNAR1 KO HDFs (Fig. 4A and D), confirming an IFN-independent mechanism for inducing ISG54 expression (68). Because ISG54 has the ability to promote apoptosis (69), we next assessed its possible role as the main factor mediating the antiviral response to MCPyV infection. To accomplish this, lentiCRISPR/Cas9 technology was applied to generate stable ISG54 KO HDFs using three sgRNAs. Among these cells, the ISG54 KO sg1 cells were selected for further study based on their significantly repressed level of ISG54 expression (Fig. S2A). Still, after treatment with MCPyV, ISG54 KO HDFs did not show increased MCPyV infection and replication. Instead, these cells had a moderately reduced capacity for supporting MCPyV replication (Fig. S2B). These data suggest that ISG54 does not play a significant role in antagonizing MCPyV infection.

To this point, our data have shown that inhibiting IFN signaling in HDFs does not significantly affect MCPyV’s replicative capacity (Fig. 3 and 4). However, the inhibitors and CRISPR KO used in these experiments were applied in cells that had already been treated and possibly infected with MCPyV; these methods of inhibiting IFN signaling after establishing MCPyV infection may therefore be insufficient to block the antiviral response. We also wanted to consider the possibility that the MCPyV-induced innate immune response signals to other cells in a paracrine manner to block viral infection in naive cells. However, the paracrine response cannot be tested using our current MCPyV infection system in HDFs, which supports only a single round of infection, because the FBS present in the system inhibits further spread of infection to neighboring cells (61). Additionally, the infected HDFs may not produce or release enough newly packaged viral particles to establish infection in neighboring cells. If IFN signaling controls MCPyV infection by stimulating an antiviral state in uninfected cells and inhibiting the second round of infection, then our prior experiments and the current infection system, which examined only the first round of infection, did not accurately address this possibility.

To better recapitulate the paracrine effect of MCPyV infection, we modified our infection protocol to mimic two rounds of infection in HDFs (Fig. 5A). Normal HDFs were infected per our usual protocol for the first round of infection, and on day 5, we harvested the medium from the cells. This medium, containing cytokines and viral particles released by cells during the first round of infection, was then applied to IFNAR1 KO or sgLuc HDFs, which were treated or mock infected with MCPyV. These treated IFNAR1 KO/sgLuc HDFs from the second round of MCPyV infection were then harvested on day 5 for subsequent analysis (Fig. 5A).

In the control sgLuc HDFs, extracellular IFN released from the first round of infection was modestly effective at stimulating downstream ISG expression in neighboring cells (Fig. 5B and D, compare Mock + MCPyV Med with Mock + Mock Med), while release of viral particles did not occur at a sufficient level to induce infection in naive cells (Fig. 5B, Mock + MCPyV Med). Also in these cells, fresh MCPyV added in the second round of infection led to robust LT expression and further enhanced ISG induction (Fig. 5B and D, compare MCPyV + Mock Med and MCPyV + MCPyV Med results with Mock + Mock Med and Mock + MCPyV Med results). IFNAR1 KO was effective at inhibiting downstream ISG production induced by IFNs from both the first and second rounds of infection (Fig. 5C and the IFNAR KO data in Fig. 5D). However, these cells did not show any significant alteration in viral transcription or replication (see the LT signal in Fig. 5B and C, mRNA in Fig. 5D, and viral genome copy number in Fig. 5E). These results indicate that ISG induction downstream of paracrine IFN signaling is not sufficient to block MCPyV infection.

Inhibition of ISG signaling downstream of IFN had thus far not affected MCPyV activity. One possible explanation for our results is that, instead of maintaining an antiviral state in cells, type I/III IFN may exert a more direct effect on viral activity at a specific point during the viral life cycle. To directly test whether type I or III IFN affects MCPyV activities, we treated HDFs with MCPyV and either IFN-β, IFN-λ, or both on day 2 of infection prior to the addition of FBS to the cells. Treatment with IFN-β, but not IFN-λ, significantly repressed viral replication (Fig. 6A), indicating that type I IFN is primarily responsible for the impact on MCPyV infection.

To further investigate which stage of the MCPyV life cycle is sensitive to IFN-β’s inhibition effect, treatment was repeated at two different time points: prior to viral infection and prior to the initiation of viral gene expression and replication, as marked by the addition of FBS. Interestingly, treatment of HDFs with IFN-β prior to infection had no significant effect on viral replication, but IFN-β added to the cells 2 days posttreatment was able to significantly inhibit viral replication activities (Fig. 6B). This observation indicates that type I IFN specifically inhibits MCPyV after viral entry has already occurred.

To elucidate which aspects of the MCPyV viral life cycle are suppressed by IFN, we first performed IF analysis of MCPyV-infected and IFN-treated HDFs to examine viral protein expression. Treatment with IFN-β and not IFN-λ on day 2 postinfection significantly repressed the expression of both MCPyV LT and VP1 at the protein level (Fig. 7A). Since protein translation occurs downstream of transcription, and since MCPyV replication is dependent on LT expression, we also investigated whether type I IFN antagonizes MCPyV infection at the level of viral transcription. Stable HDFs expressing either a luciferase reporter or the MCPyV LTT and sT genes, all under the regulation of the MCPyV early promoter, were used to examine the direct effects of IFN on MCPyV promoter-driven gene expression (Fig. 7B and C). Treatment of both of these cell types with type I IFNs inhibited MCPyV early promoter-driven expression. In contrast, ISRE-driven luciferase reporter gene expression in stable HDFs is stimulated by IFN (Fig. S3A). Our studies therefore demonstrate that type I IFNs antagonize MCPyV at the level of early viral transcription, a crucial process during the viral life cycle (Fig. 7B and C).

Throughout our studies, we observed that while IFN treatment could significantly inhibit MCPyV replication and transcription, it did not completely abrogate these viral activities (Fig. 6 and 7). These results suggest that other factors in our infection system may potentially antagonize type I IFN during MCPyV infection. A key component of our MCPyV infection system is the addition of FBS to cells after they have been treated with MCPyV in serum-free medium (61). We found that FBS can inhibit viral entry but promote viral gene expression (61). FBS contains many growth factors, such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta (TGF-β), and fibroblast growth factor 2 (FGF-2), which are among the cocktail of growth factors enriched near proliferative fibroblasts at a wound site in damaged human skin (70–72). We therefore tested if FBS or its contained growth factor elements were involved in stimulating MCPyV activity during infection.

As we described previously (61), FBS added at a 20% final concentration to MCPyV-treated cells was sufficient to stimulate robust MCPyV gene expression, but reducing FBS concentration to 2 to 10% caused a significant reduction in LT and VP1 expression (Fig. 8A). This observation supports the idea that some of its components could directly upregulate MCPyV infectious activity in HDFs. To probe which growth factors contained in FBS are involved, we utilized inhibitors against the receptors of each growth factor to determine whether downregulation of a specific growth factor activity has any repressive effects on MCPyV and its proliferation. We found that blocking the function of the TGF-β and FGF receptors with the respective inhibitors LY2109761 and AZD4547 significantly repressed MCPyV protein expression and replication (Fig. 8B and C). These studies therefore identify TGF-β and FGFs as the key growth factors that stimulate MCPyV gene expression and replication. Importantly, we found that combining IFN-β with either AZD4547 or LY2109761 further inhibited MCPyV replication, providing further support for our hypothesis that the growth factors induced in wounded or UV-irradiated skin promote MCPyV propagation by antagonizing the IFN response (Fig. 8C).

HDFs were isolated from human foreskin tissues as described in our previous studies (61, 63) and were maintained in DMEM (Invitrogen) supplemented with 10% FBS (HyClone), 1× nonessential amino acids (Gibco), and 1× GlutaMAX (Gibco).

HDFs or lentiCRISPRv2 sgRNA stable HDFs were infected with MCPyV using the method described previously (61, 63), which is outlined briefly here for convenience. HDFs were seeded with 1.2 × 10⁴ cells each in a 96-well plate in DMEM–F-12 medium (11330-032; Thermo Fisher Scientific) containing 20 ng/mL EGF (236-EG-200; R&D Systems), 20 ng/mL basic FGF (bFGF) (PHG0368; Thermo Fisher Scientific), 3 μM CHIR99021 (13122; Cayman Chemical), and 1 mg/mL collagenase type IV (17104019; Thermo Fisher Scientific). The same amounts of MCPyV virions (10⁸ to 10⁹ viral genome equivalents) were added to each 96-well plate for viral infection, and 10⁹ viral genome equivalents were used for each 96-well plate for Fig. 1 and Fig. 2 as well as the first HDF infection for Fig. 5. A dose of 10⁸ viral genome equivalents per 96-well was used in the rest of the MCPyV infection experiments. After the cells were incubated at 37°C in 5% CO₂ for 2 days, FBS was added to each well to reach a 20% final concentration. Depending on the experiment, the cells were incubated for 3 to 5 days before harvest.

To analyze MCPyV genomic DNA levels, harvested cell pellets were lysed in QuickExtract DNA extraction solution (Biosearch Technologies) according to the manufacturer’s instructions. The extracted DNA samples were then subjected to real-time qPCR using a QuantStudio 3 real-time PCR system (Applied Biosystems) with SYBR green master mix (Applied Biosystems). The MCPyV genomic DNA levels were detected with NCRR-targeted primers and normalized to the level of genomic glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both NCRR and genomic GAPDH primer sequences can be found in reference 64.

Ruxolitinib (Cayman Chemical), AZD4547 (LKT Laboratories), and LY2109761 (Sigma) powders were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. Normal goat IgG control (AB-108-C; R&D Systems) and human IFN-β antibody (AF814-SP; R&D Systems) lyophilized powder were reconstituted with sterile phosphate-buffered saline (PBS) to a stock concentration of 0.5 μg/μL for final use at 2 μg/mL for blocking the IFN pathway response. Recombinant human IFN-β (300-02BC; PeproTech) and recombinant human IFN-λ1 (300-02L; PeproTech) lyophilized powders were reconstituted with DMEM–F-12 medium supplemented with 1% FBS to 10⁶ U/mL. Universal type I interferon (IFN-α; 11200-2; PBL Assay Science) was also diluted to 10⁶ U/mL with DMEM–F-12 medium. Stock solutions of all chemicals, antibodies, or other reagents were stored in small aliquots at −80°C.

sgRNAs were cloned into the lentiCRISPR v2 plasmid (Addgene) following protocols from the Feng Zhang lab (92). Oligonucleotides for the generation of sgRNA cassettes targeting the IFNAR1 or ISG54 genes were selected from a CRISPick search, the Brunello library, or the GeCKO v2 library (93–95) and are as follows: IFNAR1 sg1, 5′-GACCCTAGTGCTCGTCGCCG-3′; IFNAR1 sg2, 5′-TAGATGACAACTTTATCCTG-3′; IFNAR1 sg3, 5′-TCATTTACACCATTTCGCAA-3′; ISG54 sg1, 5′-GCACCTCAAAGGGCAAAACG-3′; ISG54 sg2, 5′-TTTCACCTGGAACTTGATGG-3′; and ISG54 sg3, 5′-AGCCACAATGTGCAACCTAC-3′.

For cloning of the pLenti-MCPyVEP-Luciferase-IRES-Puro construct, the firefly luciferase gene was PCR amplified from pGL3 (Promega) and subcloned into the pLenti-MCVEP-RFP-IRES-Puro vector (96) using the AgeI and ClaI sites.

For cloning of the pLenti-MCVEP-ST/LTT-IRES-Puro construct, the MCPyV early promoter (MCVEP) and the LTT/ST gene fragment were PCR amplified from MCPyV genomes religated from the pR17b plasmid (kindly provided by Christopher B. Buck [NCI]). The resulting fragment was subcloned using the XbaI/SpeI and MluI sites into the pLenti-IRES-Puro vector, which was modified from the pTRIPZ vector (Open Biosystems) by removing the fragments between the MluI and NotI sites.

For cloning of the pTRIPZ-ISRE-Luc construct, a synthesized promoter fragment including 10 copies of ISRE (interferon-sensitive response element with the sequence GATCAAAGTGAAAGG) and a minimal promoter sequence were cloned into the XbaI and AgeI sites to replace the inducible promoter region in the pTRIPZ vector to generate the pTRIPZ-ISRE-RFP construct. The firefly luciferase gene was PCR amplified from pGL4 (Promega) and subcloned into the AgeI/XmaI and MluI sites to replace the red fluorescent protein (RFP) gene in pTRIPZ-ISRE-RFP.

Stable cells were prepared as described previously (64, 96). To package lentiviruses, HEK293T cells at 90% confluence were transfected with the lentivirus construct together with psPAX2 and pMD.2G using Lipofectamine 2000 (Invitrogen). At 6 to 8 h posttransfection, the culture medium was changed to fresh medium. At 2 days posttransfection, lentiviruses in the supernatant were harvested, filtered through a 0.45-μm syringe filter, and concentrated with Lenti-X concentrator (TaKaRa) according to the manufacturer’s instructions. Early-passage HDFs were transduced with concentrated lentivirus supplemented with 8 μg/mL Polybrene (Sigma). Starting on day 2 after transduction, cells were selected using 1 μg/mL puromycin for 2 to 3 weeks until the cells proliferated.

Total RNA was isolated using the NucleoSpin RNA XS kit (Macherey-Nagel) following the manufacturer’s instructions. Reverse transcription was performed using a 20-μL reaction mixture containing 350 ng of total RNA, oligo(dT) primers (Invitrogen), deoxynucleoside triphosphates (dNTPs; Invitrogen), and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen). Real-time qPCR was performed using a QuantStudio 3 real-time PCR system (Applied Biosystems) with SYBR green master mix (Applied Biosystems). The mRNA level of each gene was normalized to the GAPDH mRNA level. The RT-qPCR primer sequences used in this study are as follows (also available in reference 64): IFN-β F, 5′-ACTGCCTCAAGGACAGGATG-3′; IFN-β R, 5′-AGCCAGGAGGTTCTCAACAA-3′; IFN-λ1 F, 5′-TTCCAAGCCCACCACAAC-3′; IFN-λ1 R, 5′-TCCCTCACCTGGAGAAGC-3′; LT F, 5′-TGACTTCTCTATGTTTGATGAGGTTGAC-3′; LT R, 5′-GACCCATACCCAGAGGAAGAG-3′; MMP-3 F, 5′-TGGGCCAGGGATTAATGGAG-3′; MMP-3 R, 5′-GGAACCGAGTCAGGTCTGTG-3′ (F indicates forward primers, and R indicates reverse primers).

Cells cultured on coverslips were fixed with 3% paraformaldehyde in PBS for 20 min. Immunofluorescent staining was performed as previously described (64). The following antibodies were used as primary antibodies: anti-MCPyV LT (1:250; sc-136172; Santa Cruz Biotechnology), anti-MCPyV VP1 (1:2,000; Christopher Buck laboratory), anti-Mx1 (1:1,000; 37849S; Cell Signaling Technology), and anti-IFI16 (1:1,000; ab55328; Abcam). Alexa Fluor 594 goat anti-mouse IgG (1:500; A11032; Thermo Fisher Scientific), Alexa Fluor 488 goat anti-rabbit IgG (1:500; A11034; Thermo Fisher Scientific), Alexa Fluor 594 goat anti-rabbit IgG (1:500; A11012; Thermo Fisher Scientific), and Alexa Fluor 488 goat anti-mouse IgG (1:500; A11029; Thermo Fisher Scientific) were used as secondary antibodies. All immunofluorescent images were captured using an inverted fluorescence microscope (IX81; Olympus) as described previously (64). The scale bars were added using ImageJ software.

Preparation of whole-cell lysates and Western blot analyses were performed as described previously (64). The primary antibodies used in this study include anti-IFNAR1 (1:2,000; ab247992; Abcam), anti-ISG54 (1:1,000; HPA003408; Sigma), anti-OAS1 (1:500; sc-374656; Santa Cruz Biotechnology), anti-MCPyV LT (1:500; sc-136172; Santa Cruz Biotechnology), anti-Mx1 (1:2,000; 37849S; Cell Signaling Technology), antiviperin (1:2,000; 13996S; Cell Signaling Technology), and anti-GAPDH (1:2,000; 2118S; Cell Signaling Technology). Horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:3,000; 7074S; Cell Signaling Technology) and HRP-linked anti-mouse IgG (1:3,000; 7076S; Cell Signaling Technology) were used as secondary antibodies. Western blots were developed using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific), and images were captured using an Amersham Imager 600 (GE Healthcare).

Statistical analysis was performed using the unpaired t test formula of Microsoft Excel software to compare the data from the control and experimental groups. A two-tailed P value of <0.05 was considered statistically significant.