Roles of Sialic Acid, AXL, and MER Receptor Tyrosine Kinases in Mumps Virus Infection of Mouse Sertoli and Leydig Cells

The mumps virus (MuV) causes epidemic parotitis. MuV also frequently infects the testis and induces orchitis, an important etiological factor contributing to male infertility. However, mechanisms underlying MuV infection of the testis remain unknown. Here, we describe that sialic acid, AXL, and MER receptor tyrosine kinases regulate MuV entry and replication in mouse major testicular cells, including Sertoli and Leydig cells. Sialic acid, AXL, and MER were present in Sertoli and Leydig cells. Sialic acid specifically mediated MuV entry into Sertoli and Leydig cells, whereas both AXL and MER facilitated MuV replication within cells through the inhibition of cellular innate antiviral responses. Mechanistically, the inhibition of type 1 interferon signaling by AXL and MER is essential for MuV replication in Sertoli and Leydig cells. Our findings provide novel insights into the mechanisms behind MuV infection and replication in the testis.


INTRODUCTION
The mumps virus (MuV) is a pathogen that causes epidemic mumps, a painful parotitis (Latner and Hickman, 2015). MuV also has tropism for the testis, thereby frequently inducing orchitis, which may result in male infertility (Litman and Baum, 2010). Although a large vaccination program has remarkably reduced the spread of the pathogen and declined the incidence of mumps, a global outbreak of mumps has been recently reported in the vaccinated population (Vandermeulen et al., 2004;Castilla et al., 2007;Cortese et al., 2008;Dayan et al., 2008;Marin et al., 2008;Bangor-Jones et al., 2009;Centers for Disease Control and Prevention, 2009;Patel et al., 2017;Willocks et al., 2017). Knowledge gaps in the basic biology of MuV hinder progress in eliminating mumps (Ramanathan et al., 2018). The mechanisms underlying MuV infection and replication in target cells need clarification, which may aid in the development of preventive and therapeutic strategies for MuV-induced pathogenesis.
MuV is an enveloped and non-segmented negative-sense RNA virus (Rubin et al., 2015). The MuV genomic RNA is encapsidated by a nucleoprotein (NP). MuV infection in children usually results in mumps, an acute inflammation of the parotid glands (Anderson et al., 1987). In addition to mumps, MuV infection may result in inflammation in multiple other organs, including the central nervous system, pancreas, kidney, ovary, and testis (Rubin and Sauder, 2013). Orchitis is the most common complication of mumps, occurring in about 20-30% of mumps cases (Masarani et al., 2006). While mumps orchitis in childhood is self-limiting and rarely leads to sterility, MuV infection in post-pubertal men frequently results in chronic orchitis, one important etiological factor of male infertility. The recovery of MuV from the testis of mumps orchitis patients confirms that MuV directly infects the testis (Bjorvatn, 1973). MuV can infect various testicular cells (Wu et al., 2017). MuV triggers innate immune responses in mouse Sertoli and Leydig cells, thereby inducing the expression of pro-inflammatory cytokines and impairing testosterone synthesis (Wu et al., 2016). In Sertoli cells, MuV-induced pro-inflammatory cytokines, such as TNFA and CXCL10, induce male germ cell apoptosis . MuV infection of Sertoli cells impairs the integrity of the blood-testis barrier through the induction of TNFA (Wu et al., 2019). While MuV infects testicular cells and impairs testicular functions, the mechanisms underlying MuV infection and replication in the testicular cells remain to be elucidated.
AXL and MER belong to a subfamily of receptor tyrosine kinases. AXL and MER are involved in the maintenance of immune homeostasis and play important roles in regulating the testicular immune privilege (Rothlin et al., 2015;Deng et al., 2016). Several studies have demonstrated that AXL facilitates infection of a broad spectrum of viruses, including Ebola (Shimojima et al., 2006), vaccinia (Morizono et al., 2011), dengue (Meertens et al., 2012), influenza (Shibata et al., 2014), and Zika (Richard et al., 2017) viruses (ZIKV). Both AXL and MER are abundantly expressed in Sertoli and Leydig cells and inhibit innate antiviral responses (Wang et al., 2005;Sun et al., 2010;Shang et al., 2011). The potential roles of AXL and MER in regulating MuV infection in Sertoli and Leydig cells have yet to be investigated. Sialic acid facilitates multiple viral infections that are of significant public concern, including avian influenza virus (Weis et al., 1988), picornavirus (Liu et al., 2015), and coronavirus (Li et al., 2017). Recent studies have demonstrated that sialic acid on the cell surface mediates MuV and ZIKV infection (Kubota et al., 2016;Tan et al., 2019). Whether sialic acid plays a role in regulating MuV infection in testicular cells is unknown. In the present study, we elucidate the roles of sialic acid, AXL, and MER in MuV infection and replication in mouse Sertoli and Leydig cells.
All of the mice were maintained in a specific pathogen-free facility with a 12/12 h light/dark cycle, and they were provided with food and water ad libitum.

MuV Preparation
MuV (strain SP-A) was isolated from mumps patients (Liang et al., 2014), and provided by Prof. Qihan Li (The Institute of Medical Biology, Chinese Academy of Medical Science, Kunming, China). MuV was amplified and titrated in Vero cells. Briefly, Vero cells (5 × 10 6 ) were seeded in 100 mm culture dishes with 10 ml Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific). After 24 h, the cells were infected with MuV at a multiplicity of infection (MOI) of 1.0. Seven days after MuV infection, the culture media were collected, and the cells were lysed by freezing in liquid nitrogen and thawing at 37°C three times. After centrifugation at 2,000 rpm for 10 min, the supernatants were collected. MuV preparations were retained in PBS at a density of 1 × 10 9 plaque-forming unit (PFU)/ ml and stored at −80°C until further use.

Plaque Assay
Vero cells were seeded in 6-well plates at 2 × 10 5 cells/well. After 24 h, the cells were infected with a serial dilution of MuV for 1 h at 37°C. The medium was removed and replaced Frontiers in Microbiology | www.frontiersin.org with DMEM containing 2% FCS and 1.5% methylcellulose (Sigma-Aldrich). The cells were cultured in a humidified incubator containing 5% CO 2 at 37°C for 7 days. The cells were stained with 1% crystal violet solution (Sigma-Aldrich) according to the manufacturer's instructions. Plaques were counted and PFU was calculated.

MuV Binding, Internalization, and Replication
MuV binding to Sertoli and Leydig cells was determined after incubating the cells with 50 MOI MuV on ice for 1 h. After triple washes with PBS, cells were collected for virus detection. For MuV internalization, cells were incubated with 50 MOI MuV for 1 h at 37°C and the surface-bound MuV was removed by treating cells with 0.25% trypsin (Thermo Fisher Scientific) for 5 min. The cells were collected for MuV detection. For MuV replication, cells were infected with 1.0 MOI MuV. Two hours later, the cells were washed twice with PBS, and then were cultured in fresh medium at 32°C. The cells were harvested for MuV detection each 24 h.

Cell Isolation
Sertoli and Leydig cells were isolated from 3-week-old mice (n = 3 each isolation) following previously described procedures (Zhu et al., 2013). Briefly, the testes were decapsulated and incubated with 0.5 mg/ml collagenase type I (Sigma-Aldrich) in PBS at 37°C for 15 min. The interstitial cells were separated from the seminiferous tubules after filtration with 80 μm copper meshes. The interstitial cells were cultured at 32°C in F12/ DMEM (Thermo Fisher Scientific) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS. Twenty-four hours later, Leydig cells were detached by treatment with 0.25% trypsin for 5 min. The purity of Leydig cells was more than 95% based on immunostaining for 3β-HSD, a marker of Leydig cells (Klinefelter et al., 1987).
The seminiferous tubules were re-suspended in collagenase type I at room temperature for 15 min to remove peritubular myoid cells. The seminiferous tubules were cut into small pieces of approximately 1 mm and incubated with 1 mg/ml hyaluronidase (Sigma-Aldrich) at 37°C for 15 min with gentle pipetting to dissociate germ cells from Sertoli cells. The cell suspensions were cultured at 32°C for 24 h and then treated with a hypotonic solution (20 mM Tris, pH 7.4) for 3 min to remove germ cells adhering to Sertoli cells. The purity of Sertoli cells was >95% based on the immunostaining for WT1, a marker of Sertoli cells (Sharpe et al., 2003).

Immunofluorescence Staining
Cells were cultured on 35 mm culture dishes and fixed with pre-cold 4% methanol at −20°C for 3 min. After washing twice with PBS, the cells were permeabilized with 0.2% Triton X-100 (Zhongshan Biotechnology Co.) in PBS for 10 min and blocked with 5% normal goat serum (Zhongshan Biotechnology Co.) for 1 h at room temperature. The cells were subsequently incubated with primary antibodies at 4°C for 24 h. After washing twice with PBS, the cells were incubated with appropriate TRITC-or FITC-conjugated secondary antibodies for 1 h at room temperature. Nuclei were counterstained with DAPI according to the manufacturer's instructions. The cells were mounted with antifade mounting medium (Zhongshan Biotechnology Co.) and observed under a confocal laser scanning microscope FV1000 (Olympus, Tokyo, Japan) or fluorescence microscope BX-51 (Olympus).

Real-Time Quantitative RT-PCR
Total RNA was extracted from cells using Trizol reagents (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was treated with RNase-free DNase I (Thermo Fisher Scientific) to remove genomic DNA. Total RNA (1 μg) was reverse transcribed into cDNA in 20 μl reaction mixture containing random hexamers at 2.5 μM, 2 μM of deoxynucleotide triphosphates, and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). The PCR was performed in 20 μl reaction mixture containing 0.2 μl of cDNA, forward and reverse primers at 0.5 μM, and 10 μl of Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7300 real-time cycler (Applied Biosystems). Relative mRNA levels were determined using the 2 −ΔΔCt method as described in the Applied Biosystems User Bulletin No. 2 (P/N 4303859). The primer sequences for PCR are listed in Table 1.

Western Blot
The cells were lysed in a lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich). The protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Proteins (20 μg/well) were separated by 10% SDS-PAGE and then electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with Tris-buffered saline (pH 7.4) containing 5% non-fat milk for 1 h at room temperature and then incubated with the primary antibodies for 24 h at 4°C. After washing twice with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized with an enhanced Chemiluminescence Detection Kit (Zhongshan Biotechnology Co.). β-Actin was used as the loading control.

TCID 50 Assay
Vero cells were cultured in 96-well plates (6 × 10 3 /well) in 100 μl of culture medium. A serial dilution of MuV was added. After 1 week, TCID 50 values were calculated according to the Reed-Muench method.

Fluorescence Staining for Sialic Acid
The cells were fixed with 4% paraformaldehyde for 5 min. After washing twice with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min. Biotinylated M. amurensis lectin II (20 μg/ml) was then added to the cells for 1 h at room temperature. FITC-conjugated streptavidin (Solarbio, Beijing, China) was added to the cells at a dilution of 1:200 for 1 h at room temperature. The nuclei were stained with DAPI. The cells were mounted with antifade mounting medium and observed under a BX-51 microscope.

Enzyme-Linked Immunosorbent Assay
The cell media were collected at 48 h after MuV infection. The cytokine levels in the media were measured using ELISA kits in accordance with the manufacturer's instructions. ELISA kits for mouse IFNA (BMS6027) and IFNB (42400) were purchased from eBioscience (San Diego, CA, USA) and R&D Systems (Minneapolis, MN, USA), respectively.

MuV Infection in vivo
For MuV infection of the testis in vivo, 8-week-old mice were anesthetized with pentobarbital sodium at a dose of 50 mg/kg. The testes were surgically exposed and each testis was injected with 10 μl PBS containing 1 × 10 7 PFU of MuV using 30-gauge needle. Three mice in each group were injected.

Statistical Analysis
Statistical difference between two comparisons was determined using Student's t-test. One-way ANOVA with Bonferroni's (selected pairs) post hoc test was used for multiple comparisons.
The calculations were performed with SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Data were presented as the mean ± SEM of at least three experiments. A p < 0.05 was considered to be statistically significant.

MuV Binding and Internalization
To assess the MuV infection of testicular cells, viral binding and internalization to Sertoli and Leydig cells were examined. Sertoli ( Figure 1A, upper panels) and Leydig (lower panels) cells were identified by immunofluorescence staining for WT-1 and 3β-HSD, and the purity of each cell type was above 95%.

MuV Replication and Secretion
To examine MuV replication in Sertoli and Leydig cells, the cells were infected with 1.0 MOI MuV. Real-time qRT-PCR results showed that the MuV-NP RNA level dramatically increased in Sertoli and Leydig cells in a time-dependent manner (Figure 2A). Western blot results demonstrated that MuV-NP protein levels were remarkably increased in Sertoli and Leydig cells 48 and 72 h after infection ( Figure 2B). Both MuV-NP RNA and protein levels were higher in Sertoli cells than Leydig cells, suggesting that MuV replicated at relatively high efficiency in Sertoli cells compared to Leydig cells. Immunofluorescence staining confirmed that MuV-NP was increased in Sertoli ( Figure 2C, left panels) and Leydig (right panels) cells 48 h after infection. TCID 50 assay showed that MuV loads in the culture media of Sertoli and Leydig cells were significantly increased in a time-dependent manner ( Figure 2D), suggesting that MuV particles can be secreted from Sertoli and Leydig cells. The CCK-8 assay indicated that MuV infection insignificantly impaired cell viability ( Figure 2E).

Role of Sialic Acid in MuV Internalization
Given that trisaccharide with α2,3-linked sialic acid is a MuV receptor (Kubota et al., 2016), we examined the role of sialic  β-Actin was used as internal control for qRT-PCR and loading control for Western blot. (E) Intracellular MuV-NP were determined by IF staining with antibodies against MuV-NP (red). Cellular plasma and nuclei were visualized using IF for α-tubulin (green) and DAPI (blue) counterstaining, respectively. Cells, incubated with MuV-free PBS served as the controls (Ctrl). Images represent at least three independent experiments. Data are presented as the mean ± SEM of three experiments. Scale bars, 50 μm. ns, not significant.
α2,3-Sialidase ( Figure 3C, right panel). However, the internalized MuV-NP RNA (Figure 3D, left panel) and protein (right panel) levels in both Sertoli and Leydig cells were significantly decreased by the presence of α2,3-Sialidase. The decreased MuV internalization by α2,3-Sialidase was confirmed using immunofluorescence staining for MuV-NP ( Figure 3E). These results suggest that sialic acid facilitates MuV internalization into Sertoli and Leydig cells, but does not affect MuV binding to these cells.

Role of AXL and MER in MuV Replication
Given that AXL and MER inhibit innate antiviral responses in Sertoli and Leydig cells (

Inhibition of IFN Expression by AXL and MER
Since virus-induced production of type 1 IFNs is the universal mechanism behind the cellular innate antiviral responses that limit virus replication, we

Expression of Antiviral Proteins
Given that IFN-induced antiviral proteins limit virus replication (Sadler and Williams, 2008), we examined the expression of major antiviral proteins, including 2'-5'-oligoadenylate synthetase 1

Role of IFN Signaling in MuV Replication
To  Figure 7E, left panels) and protein (right panels) levels in WT cells. Consistently, MuV loads were significantly higher in the culture media of Ifnar1 −/− cells than in WT cells ( Figure 7F). LDC1267 reduced the MuV loads in the media of WT cells, but did not affect the MuV loads in the media of Ifnar1 −/− Sertoli and Leydig cells.

DISCUSSION
The mammalian testis adopts an immunoprivileged environment for the protection of immunogenic male germ cells from adverse immune responses. The immunoprivileged environment may also provide a sanctuary for microbial pathogens such as viruses to escape immune surveillance (Dejucq and Jegou, 2001;Li et al., 2012). Various virus types have tropism for the testis and impair male fertility (Zhao et al., 2014;Liu et al., 2018). A typical example is MuV, which frequently causes orchitis and may lead to male infertility. The recent outbreak of MuV orchitis is a resurgent threat to male fertility (Philip et al., 2006). Understanding the mechanisms behind MuV infection and replication in the testis can help in the development of preventive and therapeutic strategies for MuV orchitis. In the present study,  MuV and produce pro-inflammatory cytokines and chemokines, which could contribute to the pathogenesis of orchitis (Wu et al., 2016;Jiang et al., 2017).
MuV is a member of the Paramyxoviridae family. Sialic acid plays a role in mediating infection of different paramyxoviruses (Lamb and Parks, 2013). A recent study demonstrated that a trisaccharide with α2,3-linked sialic acid is a receptor mediating MuV infection (Kubota et al., 2016). In the present study, we showed that sialic acid was exposed on the surface of both Sertoli and Leydig cells, and the treatment of these cells with α2,3-Sialidase efficiently removed sialic acid. Coincidently, the sialidase treatment impaired MuV internalization into Sertoli and Leydig cells while MuV binding was not affected by removing sialic acid. These observations suggest that sialic acid plays a role in mediating MuV internalization into Sertoli and Leydig cells, but it is not involved in MuV binding to these cells. The mechanism by which sialic acid facilitates MuV internalization into Sertoli and Leydig cells requires further investigation. In this regard, we should focus on the potential role of sialic acid in promoting the fusion between MuV and target cells through the interplay with viral fusion proteins. Further understanding of the mechanism underlying sialic acid-mediated MuV internalization may be helpful to discover targets for the prevention of MuV infection. It was surprising to find that sialic acid did not affect MuV binding to Sertoli and Leydig cells because sialic acid was supposed to bind MuV as a receptor (Kubota et al., 2016). However, our results are in agreement with recent observations for the role of sialic acid in ZIKV infection, in which sialic acid facilitates ZIKV internalization into target cells but does not affect ZIKV binding (Tan and Huan Hor, 2019). The identification of the specific molecules that mediate MuV binding to testicular cells may also aid in the prevention of MuV infection in the testis. AXL and MER facilitate infection of a broad spectrum of viruses (Morizono et al., 2011;Meertens et al., 2012;Shibata et al., 2014;Richard et al., 2017). Both AXL and MER are expressed in mouse Sertoli and Leydig cells (Wang et al., 2005). Therefore, we explored the role of AXL and MER in regulating MuV infection in testicular cells. By using Axl and Mer gene knockout cells, we demonstrated that AXL and MER redundantly facilitated MuV replication in Sertoli and Leydig cells. However, AXL and MER did not affect MuV binding and internalization into the testicular cells. These observations are also helpful in explaining the recent debate regarding to the role of AXL in ZIKV infection. Several studies showed that AXL facilitated ZIKV infection (Fleming, 2016;Meertens et al., 2017;Richard et al., 2017). On the contrary, other studies failed to detect significant differences in ZIKV infection between WT and Axl −/− mice (Wells et al., 2016;Hastings et al., 2017;Wang et al., 2017). The debate that these previous studies raised could have been caused by the use of different models. The studies used in vitro cells that express only Axl support Axl-dependent ZIKV infection (Fleming, 2016;Meertens et al., 2017;Richard et al., 2017). In contrast, the studies using an in vivo model show that Axl is not indispensable for ZIKV infection (Wells et al., 2016;Hastings et al., 2017;Wang et al., 2017). The discrepancy can be explained by the fact that AXL and MER function in a redundant manner. Our present study confirmed that single knockout of either Axl or Mer insignificantly affected MuV replication, whereas double knockout of both Axl and Mer remarkably decreased MuV replication. Therefore, AXL and MER could redundantly facilitate viral replication by dampening the cellular innate antiviral response, rather than mediating viral infection by serving as receptors.
This speculation is also supported by our previous studies showing that AXL and MER redundantly inhibit innate antiviral responses in mouse Sertoli and Leydig cells (Sun et al., 2010;Shang et al., 2011). Given that the expression of type 1 IFNs and subsequently IFN-inducible antiviral proteins in host cells upon viral infection is a key mechanism underlying the restriction of virus replication (Sadler and Williams, 2008;Diamond and Farzan, 2013), we examined the role of IFN signaling in limiting MuV replication in Sertoli and Leydig cells. We demonstrated that the double knockout of Axl and Mer significantly increased the expression of IFNA and IFNB, as well as major antiviral proteins, including Isg15, Oas1, and Mx1, in Sertoli and Leydig cells in response to MuV infection. Isg15, Oas1, and Mx1 limit viral replication by amplifying antiviral signaling, degrading viral RNA, and inhibiting viral gene transcription, respectively (Schoggins and Rice, 2011). Accordingly, disabled IFN signaling using a neutralizing antibody against IFNAR significantly increased MuV replication in both Axl −/− Mer −/− , and WT cells. Moreover, MuV replication was significantly increased in Ifnar1 −/− Sertoli and Leydig cells. Notably, LDC1267, an inhibitor of AXL and MER did not reduce MuV replication in Ifnar1 −/− cells, while it significantly decreased MuV replication in WT Sertoli and Leydig cells. These results suggest that AXL and MER facilitate MuV replication in Sertoli and Leydig cells by inhibiting the antiviral IFN signaling.
The roles of AXL and MER in MuV replication and type 1 IFN induction would not be a specificity of MuV within the paramyxoviridae family and the attenuated strains of MuV are based on following observations: (1) AXL and MER redundantly inhibit IFN expression induced by synthetic RNA analog poly (I:C) that can be produced by various viruses in Sertoli and Leydig cells (Sun et al., 2010;Shang et al., 2011), (2) AXL and MER indeed inhibit the infection of various viruses among different virus families (Morizono et al., 2011;Meertens et al., 2012;Shibata et al., 2014;Richard et al., 2017), (3) AXL and MER could not function as a specific receptor of MuV because they do not affect MuV binding and entry. These observations suggest that AXL and MER facilitate replication of different viruses by inhibiting type 1 IFN production.
In summary, the present study elucidated the mechanisms behind MuV infection and replication in mouse Sertoli and Leydig cells. An α2,3-linked sialic acid mediates MuV internalization into Sertoli and Leydig cells, whereas AXL and MER redundantly facilitate MuV replication within these testicular cells through the inhibition of IFN signaling. These results should aid in the development of preventive and therapeutic approaches for MuV infection in the testis.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/ supplementary material.