Increased receptor binding affinity may allow viruses to escape from Ab-mediated inhibition. However, how high-affinity receptor binding affects innate immune escape and T cell function is poorly understood. In this study, we used the lymphocytic choriomeningitis virus (LCMV) murine infection model system to create a mutated LCMV exhibiting higher affinity for the entry receptor α-dystroglycan (LCMV-GPH155Y). We show that high-affinity receptor binding results in increased viral entry, which is associated with type I IFN (IFN-I) resistance, whereas initial innate immune activation was not impaired during high-affinity virus infection in mice. Consequently, IFN-I resistance led to defective antiviral T cell immunity, reduced type II IFN, and prolonged viral replication in this murine model system. Taken together, we show that high-affinity receptor binding of viruses can trigger innate affinity escape including resistance to IFN-I resulting in prolonged viral replication.

Viruses must cross the cellular membrane to infect and replicate in host cells. Enveloped viruses accomplish this task by fusing their envelope with the target cell membrane, during which capsids are delivered into the cytosol, commencing the infection process. The entry can be initiated by binding of a virus to an appropriate receptor on the surface of the host cell, which is mediated by viral membrane proteins. In some enveloped viruses, such as influenza viruses, the receptor-binding functions may be executed by a single viral protein (1). Furthermore, a mutation at position D614G in the spike protein of the SARS-CoV-2 virus increased binding affinity to its host receptor ACE2, resulting in increased viral entry and consequently enhanced viral transmission (2, 3). This highlights the importance of correct expression, folding, and posttranslational modifications of viral membrane proteins for the success of viral invasive strategies. However, current knowledge about how high-affinity virus binding impacts the host’s innate and adaptive immunity is not well characterized.

RNA viruses undergo constant mutation and selection that alter replication efficacy and favor high-affinity receptor binding. To further explore the role of high-affinity mutations after infection and immune activation, we used the murine RNA ambisense lymphocytic choriomeningitis virus (LCMV) model system. LCMV virions are composed of a nucleocapsid surrounded by a lipid bilayer that presents spikes of glycoprotein (GP) (4). The initial step in LCMV infection involves interaction of GP with the cellular receptor on target cells. After internalization of the virions within vesicles, LCMV GP mediates fusion of the viral and cellular membranes, resulting in delivery of the nucleocapsid into the cytoplasm. The LCMV GP is glycosylated at conserved N-glycosylation sites that are important for protein folding and function, host cell entry, and infection (5, 6). LCMV is a member of the Old World Arenavirus family, which commonly uses α-dystroglycan (α-DG) as a cellular receptor for viral entry. However, members of the Tyro3/Axl/Mer family, DC-SIGN and LSECtin, were also identified as alternative receptors for Lassa virus and LCMV, respectively (7, 8). The use of alternative receptors for cell entry is particularly observed in the case of LCMV variants exhibiting low affinity to α-DG (9, 10). Furthermore, it has been shown that mutations in the GP of LCMV (including glycosylation sites) modulate the binding affinity to α-DG, influencing its virulence and cell tropism (5, 11). This resulted in a dichotomous classification of low- (e.g., WE2.2, Armstrong, HPI WT) and high-affinity (e.g., Clone 13, WE54) LCMV variants. Interestingly, mutations within the GP1 domain not only affect the cell tropism but can also influence pathogenicity and outcome of infection (12).

In this study, we used an attenuated system containing an LCMV WE S segment and a LCMV Clone 13 L segment (13) to compare infection of a high affinity (H155Y) and wild-type (WT; Y155H) LCMV-GP. Unexpectedly, innate immune activation, such as dendritic cell activation and type I IFN (IFN-I) production, was similar between both virus strains in vivo. However, infection with high-affinity virus resulted in increased viral replication and viral load. Mechanistically, high-affinity viruses escaped IFN-I–mediated inhibition. Consequently, increased virus replication led to impaired CD8+ T cell immunity and reduced type II IFN (IFN-II) production in high-affinity virus–infected mice, leading to prolonged viral infection.

All mice were maintained under specific pathogen-free conditions. CD45.1+P14+ carried the transgenic TCR (P14) recognizing the LCMV gp33 peptide (14) and the CD45.1 congenic marker (15). Experiments were performed under the authorization of Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen in accordance with the German law for animal protection.

α-DG knockout HEK cells were generated as previously described (16). L929 (mouse fibroblasts), A549 (human lung epithelial cells), HepG2 (human liver epithelial-like cells), JASWII (mouse bone marrow monocyte cells), LLC (Lewis Lung Carcinoma, mouse lung cells), and MC-57G (mouse fibrosarcoma cells) were purchased from American Type Culture Collection and maintained according to the suggested tissue culture handling protocols. Vero cells (ATCC-CCL-81) were purchased from LGC Standards. THP-1 cells (human monocyte cells) were kindly provided by Prof. Dr. Münk (Heinrich Heine University). Huh7 cells (human liver carcinoma cells) were kindly provided by Prof. Dr. Bode (Heinrich Heine University).

WT and H155Y viruses were created using reverse genetics (13, 17) using the WT LCMV WE and H155Y LCMV WE S segment in combination with the L segment from LCMV Clone 13. Viruses were propagated in L929 cells as previously described (13, 18). Virus titers were determined using a plaque-forming assay, as described before (19). In brief, organs were extracted into HBSS and homogenized using a Tissue Lyser (QIAGEN). MC-57G cells were added to 24-well plates containing diluted viral samples; after 3 h, a medium with 1% methylcellulose (MC) was applied. Forty-eight hours later, the plates were fixed with 4% formalin (Sigma Aldrich), permeabilized with 1% Triton X (Sigma Aldrich) in HBSS medium (Sigma Aldrich), and stained with anti–LCMV-Nucleoprotein (Clone VL-4, generated in-house) Ab and peroxidase anti-rat secondary Ab (Jackson ImmunoResearch). LCMVs were delivered to animals i.v. in all experiments.

mRNA from cells was purified by TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. mRNA expression levels were analyzed using the iTaq Universal SYBR Green 1-Step Kit (Bio-Rad). RT-PCR primers were obtained from Eurofins Scientific; detailed sequences are listed in Supplemental Table I. For analysis, the expression levels of all targets were normalized to β-actin (ΔCt) or to untreated samples (ΔΔCt).

Following the manufacturer’s recommendations, single-cell suspended splenocytes were enriched with the mouse CD8 purification kit (Miltenyi Biotec).

Experiments were performed using a FACS Fortessa and analyzed using FlowJo software. For dendritic cell staining, singly suspended cells were incubated with Abs (anti-CD19, CD8a, CD11c, and MHC-II from Thermo Fisher Scientific; CD40, CD80, and CD86 from BD Biosciences) for 30 min at 4°C. Tetramer and intracellular cytokine staining were performed as described previously (13, 15). For tetramer staining, singly suspended cells were incubated with tetramer-gp33 (CD8) for 15 min at 37°C. After incubation, surface Abs (anti-CD8, CD19, 2B4, IL-7R, KLRG1, and TIM-3 from Thermo Fisher Scientific; CD44, CD62L, PD-1, and LAG-3 from BD Biosciences) were added for 30 min at 4°C. For intracellular cytokine restimulation, singly suspended cells were stimulated with LCMV-specific peptides gp33 for 1 h. Brefeldin A (Thermo Fisher Scientific) was added for another 5-h incubation at 37°C followed by staining with anti-CD8 and anti–IFN-γ (Thermo Fisher Scientific). For adoptive T cell transfer, splenocytes from adoptively transferred P14 T cells were stained with anti-CD45.1 and anti-CD8 Abs (Thermo Fisher Scientific), followed by intracellular labeling with anti-Ki67 Abs (Thermo Fisher Scientific). For surface staining for α-DG, cells were seeded at a density of 500,000 cells/well in a six-well plate. On the next day, cells were washed with Dulbecco’s Phosphate Buffered Saline (Merck Millipore) without calcium chloride and magnesium chloride and subjected to 1 ml Accutase solution treatment for cell detachment (A6964; Merck Millipore) at 37°C for 3 min. Subsequently, the cells underwent washing by adding 5 ml of DMEM (Pan Biotech) with 10% FBS and penicillin-streptomycin-L-glutamine through centrifugation. Next, cells were washed with PBS and stained with anti-dystroglycan Ab (Clone IIH6C4; Merck Millipore) for 30 min at 4°C followed by washing step with FACS buffer. Cells were stained with secondary FITC-conjugated anti-mouse IgM (Thermo Fisher Scientific) for 30 min at 4°C.

Cells were kept on ice together with LCMV at multiplicity of infection (MOI) of 0.5 for 1 h followed by incubation at 37°C. At 0.3, 1, 2.5, or 5 h postincubation, monensin (Thermo Fisher Scientific) was added. Twenty-four hours later, LCMV-infected cells were quantified by flow cytometry using anti-LCMV NP Ab (Clone VL-4, generated in-house).

Histological analysis of snap-frozen tissue was performed as previously described (20, 21). Abs against B220, F4/80, and CD31 (Thermo Fisher Scientific), donkey anti-rat secondary Ab (Jackson ImmunoResearch), and anti-LCMV mAb (Clone VL-4) were used. Images were acquired on a ZEISS Axio Observer Z1.

Vero cells were seeded at 2.5 × 104 cells/well in 1 ml maintenance medium (DMEM; Thermo Fisher), 2% FBS (PAN Biotech), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies) 24 h prior to infection. Cells were then infected with a SARS-CoV-2 Wuhan-Hu-1 WT strain EPI_ISL_425126 or ο BA.5 EPI_ISL_14167576 strain at MOI 0.01 and treated with IFN-α2 after 4 h (PBL Assay Science). At 4 d postinfection (p.i.; dpi), 100 μl cell culture supernatant was recovered and incubated with 200 μl AVL buffer (Qiagen) for 10 min at room temperature and mixed with 100 μl 100% ethanol. RNA extraction was performed with 200 μl cell culture mix using the EZ1 Virus Mini Kit v2 (Qiagen). In the subsequent quantitative RT-PCR, a 113-bp amplicon was detected as described previously (22) with minor modifications (23). LightMix Modular SARS and Wuhan CoV E-gene (Cat. No. 53–0776-96) and the LightMix Modular EAV RNA Extraction Control were used with the AgPath-ID One-Step RT–PCR Kit (Cat. No. 4387391; Applied Biosystems) on an ABI 7500 FAST sequence detector system (PE Applied Biosystems). As a DNA standard, a plasmid (pEX-A128-nCoV2019-E-gene) to create a standard curve allowed calculation of RNA copies per milliliter.

Data are expressed as mean ± SEM. For analysis of statistical significance between two groups, a Student t test was used. For analysis of multiple time point experiments, two-way ANOVA with an additional Bonferroni posttest was used. The following p values were considered statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001.

To study the host immune response against high-affinity viruses, we first generated attenuated chimeric LCMV using the S segment from LCMV WE and L segment from LCMV Clone 13 (named WT viruses) (13). According to previous published results, tyrosine substitution of histidine in the LCMV GP position 155 increases its binding affinity to the entry receptor α-DG (11). Therefore, we generated a chimeric LCMV containing the S segment from LCMV WE strain with the high-affinity α-DG binding tyrosine mutation at position 155 (named H155Y viruses). As expected, when HEK cells were infected followed by a Golgi block in a time-dependent manner, a higher frequency of infected cells was observed p.i. with the H155Y LCMV, when compared with WT LCMV control (Fig. 1A). Furthermore, the increased rate of infection was dependent on α-DG expression because we did not observe a difference between WT and H155Y viruses in α-DG–deficient cells (Fig. 1A). Moreover, the virus titer in the supernatant was increased in H155Y virus–infected cells when compared with WT controls in an α-DG expression–dependent manner (Fig. 1B). Next, we infected several cell lines with differing surface α-DG expression (Supplemental Fig. 1A). Higher viral titers were detected in the supernatant of most, but not all, cells infected with H155Y in comparison with cells infected with the WT virus (Fig. 1C). Notably, viral entry did not solely depend on expression of α-DG (Supplemental Fig. 1B). However, we found that α-DG protein levels correlated positively with an increased difference between the viral entry of H155Y and WT virus, which is consistent with the hypothesis that H155Y facilitates high-affinity virus entry via α-DG (Supplemental Fig. 1C). Taken together, the high-affinity virus showed increased replication p.i., which was dependent on the expression of the corresponding entry receptor α-DG.

FIGURE 1.

H155Y LCMV exhibits enhanced cell infectivity in an α-DG expression–dependent manner. (A) WT or α-DG–deficient HEK cells were infected with LCMVs consisting of the WE S segment containing either the WT or the H155Y GP and the Clone 13 L segment at MOI 0.5. At the indicated time points p.i., monensin was added. Twenty-four hours later, LCMV-infected cells were quantified by anti–LCMV-NP staining using flow cytometry analysis (n = 6). (B) WT or α-DG–deficient HEK cells were infected with WT or H155Y viruses at MOI 0.01. At 12 or 24 h p.i., viral titers from infected cell culture supernatants were quantified (n = 6). (C) Indicated cell lines were infected with WT or H155Y viruses at MOI 0.1. Virus titer from infected cell supernatants was determined (A549, LLC 12 h p.i., Huh7, JAWS-II, L929, 24 h p.i.) (n = 6). Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

FIGURE 1.

H155Y LCMV exhibits enhanced cell infectivity in an α-DG expression–dependent manner. (A) WT or α-DG–deficient HEK cells were infected with LCMVs consisting of the WE S segment containing either the WT or the H155Y GP and the Clone 13 L segment at MOI 0.5. At the indicated time points p.i., monensin was added. Twenty-four hours later, LCMV-infected cells were quantified by anti–LCMV-NP staining using flow cytometry analysis (n = 6). (B) WT or α-DG–deficient HEK cells were infected with WT or H155Y viruses at MOI 0.01. At 12 or 24 h p.i., viral titers from infected cell culture supernatants were quantified (n = 6). (C) Indicated cell lines were infected with WT or H155Y viruses at MOI 0.1. Virus titer from infected cell supernatants was determined (A549, LLC 12 h p.i., Huh7, JAWS-II, L929, 24 h p.i.) (n = 6). Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

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IFN-I production can be detected shortly after viral infection and is one of the most important antiviral defense mechanisms (24). Host cells can detect LCMV RNA through pattern recognition receptors such as RIG-I and MAD5 and produce IFN-I (25). Consistent with the increased viral entry and replication, we observed increased RNA levels of LCMV in H155Y-infected cells when compared with WT-infected cells (Supplemental Fig. 1D). Consistently, a modest but significant increase in IFN-I mRNA levels and expression levels of mRNA encoding for the IFN-stimulated gene (ISG) Ifit2 was detected in H155Y LCMV–infected cells when compared with WT LCMV–infected cells, whereas for other ISGs we did not observe a significant difference (Supplemental Fig. 1E). To investigate whether innate immune activation was affected in vivo, we infected C57BL/6 mice with WT and H155Y viruses and monitored cytokine production and dendritic cell activation. Interestingly, we did not find a major difference in serum IFN-β and IFN-α levels between WT- and H155Y-infected hosts in this setting using WE/Clone13 chimeric viruses (Fig. 2A). Furthermore, the expression levels of ISGs in lung and spleen tissue were increased in both WT- and H155Y-infected mice (Fig. 2B). IFN-I production is facilitated by innate immune cells, including CD11c+ cells, following LCMV infection (26). Consistent with the previous data, we observed increased expression of the costimulatory molecules CD80, CD86, and CD40 in splenic conventional DC1 or total splenic conventional DCs p.i. with both WT and H155Y chimeric LCMV-infected animals (Fig. 2C). To investigate whether infection with a high-affinity virus affects activation of antiviral T cells, we transferred negatively sorted T cells from a mouse carrying the transgenic TCR (P14) recognizing the LCMV gp33 peptide (14) and CD45.1 congenic marker (15) into WT animals followed by infection with WT or H155Y viruses. Consistent with the increase in expression of costimulatory molecules, infection with both virus strains activated transferred T cells and resulted in their proliferation, expansion, and increased Ki-67 expression (Fig. 2D). Overall, our findings suggest that high-affinity receptor binding does not affect innate immune activation in this in vivo model system.

FIGURE 2.

H155Y LCMV infection induces normal IFN-I production and dendritic cell activation. (A–C) C57BL/6 mice were infected 106 PFUs of WT or H155Y viruses i.v. At days 1 and 3 p.i., (A) IFN-β and IFN-α levels were determined from serum using ELISA (n = 3). (B) mRNA expression levels for genes encoding for the ISGs Mx1, Rnasel, and Osal1 were determined by RT-PCR in lung (left panel) and spleen (right panel) tissue (n = 3). (C) CD80, CD86, and CD40 surface expression was measured on cDC1 (CD8a+CD11c+MHCII+) or total cDCs (CD11c+MHCII+) using flow cytometry analysis (n = 3). (D) A total of 106 negatively sorted CD8+ T cells from CD45.1+P14+ WT were transferred into C57BL/6 mice followed by infection of 106 PFUs of WT or H155Y viruses i.v. At day 2 p.i., transferred T cells in spleen tissue were determined by flow cytometry (left panel), and proliferation of transferred T cells was determined by staining for Ki67 expression using flow cytometry analysis (right panel) (n = 3).

FIGURE 2.

H155Y LCMV infection induces normal IFN-I production and dendritic cell activation. (A–C) C57BL/6 mice were infected 106 PFUs of WT or H155Y viruses i.v. At days 1 and 3 p.i., (A) IFN-β and IFN-α levels were determined from serum using ELISA (n = 3). (B) mRNA expression levels for genes encoding for the ISGs Mx1, Rnasel, and Osal1 were determined by RT-PCR in lung (left panel) and spleen (right panel) tissue (n = 3). (C) CD80, CD86, and CD40 surface expression was measured on cDC1 (CD8a+CD11c+MHCII+) or total cDCs (CD11c+MHCII+) using flow cytometry analysis (n = 3). (D) A total of 106 negatively sorted CD8+ T cells from CD45.1+P14+ WT were transferred into C57BL/6 mice followed by infection of 106 PFUs of WT or H155Y viruses i.v. At day 2 p.i., transferred T cells in spleen tissue were determined by flow cytometry (left panel), and proliferation of transferred T cells was determined by staining for Ki67 expression using flow cytometry analysis (right panel) (n = 3).

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Next, we wondered how high-affinity viruses are distributed in the host p.i. Because α-DG expression varies in naive organs (Fig. 3A) we hypothesized that high-affinity virus strains will preferentially replicate within α-DG–expressing tissue. Hence C57BL/6 mice were infected with WT or H155Y viruses, and organs were collected at the indicated time points followed by determination of the virus titer by plaque-forming assay. As expected, both viruses infected α-DG–expressing organs, but viral titers were not detectable in kidney tissue, where the levels of α-DG are low, 1 dpi (Fig. 3B). However, 3 and 4 dpi, we observed increased viral replication of high-affinity viruses when compared with WT viruses in α-DG–expressing tissue (Fig. 3C, Supplemental Fig. 1F). Next, we wondered whether infection with a high-affinity virus causes infection in niches promoting viral replication (27). When we analyzed snap-frozen tissue sections for virus-infected cells by coimmunolabeling for LCMV-NP, B220 and F4/80, we observed an increased frequency of infected cells in animals receiving high-affinity virus particles, predominantly located in the splenic marginal and red pulp zones (Fig. 3D and 3E). Notably, these differences were modest in this setting using attenuated chimeric viruses and likely appear more pronounced in a WT virus setting (13, 27). Taken together, these data suggest that although high-affinity receptor binding is dispensable for innate immune activation, increased viral replication was observed in vivo.

FIGURE 3.

High-affinity LCMV particles exhibit increased virus replications in vivo. (A) mRNA expression levels encoding for α-DG in indicated tissue samples harvested from naive C57BL/6 mice were determined (n = 5). (B–E) C57BL/6 mice were infected with 106 PFUs of WT or H155Y viruses i.v. At days 1 (B) and 3 (C) p.i., virus titers were determined in spleen, lung, liver, and kidney tissues of infected animals (n = 6 for day 1 and n = 3 for day 3). Sections of snap-frozen spleen tissue harvested from mice infected with virus as indicated at either day 1 p.i. (D) or day 3 p.i. (E) were stained for LCMV-NP, F4/80, and B220 (representative images of n = 3 are shown; scale bars, 100 μm). Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

FIGURE 3.

High-affinity LCMV particles exhibit increased virus replications in vivo. (A) mRNA expression levels encoding for α-DG in indicated tissue samples harvested from naive C57BL/6 mice were determined (n = 5). (B–E) C57BL/6 mice were infected with 106 PFUs of WT or H155Y viruses i.v. At days 1 (B) and 3 (C) p.i., virus titers were determined in spleen, lung, liver, and kidney tissues of infected animals (n = 6 for day 1 and n = 3 for day 3). Sections of snap-frozen spleen tissue harvested from mice infected with virus as indicated at either day 1 p.i. (D) or day 3 p.i. (E) were stained for LCMV-NP, F4/80, and B220 (representative images of n = 3 are shown; scale bars, 100 μm). Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

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Given that we did not observe a difference in innate immune activation in vivo but observed an increase in viral replication in high-affinity virus–infected hosts relative to WT virus–infected hosts, we hypothesized that high-affinity viruses may be resistant to IFN-I–mediated inhibition. Accordingly, we wondered whether high-affinity viral replication could be resistant to IFN-α–mediated suppression. Hence MC-57G cells were infected with WT or H155Y viruses at an MOI of 0.01 and 0.1, followed by the addition of different concentrations of rIFN2-α or rIFN4-α (28). First, we added rIFN-α after 4 h, because in a physiological setting virus infection precedes IFN-I production. Although replication of the WT virus was highly reduced after treatment with rIFN-α, we observed only a modest reduction in the presence of high-affinity virus particles (Fig. 4A). Next, we wondered whether uninfected cells pretreated with rIFN-α would still be susceptible toward high-affinity virus infection. Accordingly, we pretreated cells for 4 h with rIFN-α followed by infection with WT and H155Y virus particles. We still observed a high inhibition of WT virus replication, whereas replication of H155Y particles showed only limited effects (Fig. 4B). To further characterize whether high-affinity virus particles are completely resistant toward IFN-I effects, we preincubated MC-57G cells with rIFN-α for 24 h followed by infection with WT and H155Y virus particles. Although we still observed a reduced inhibitory effect of IFN-I in high-affinity virus–infected cells compared with WT virus–infected cells, pretreatment of IFN-I for 24 h was able to limit virus replication also with the high-affinity mutation (Fig. 4C). Consistently, when we performed viral cell entry assays in the presence of rIFN2-α, H155Y virus entry was resistant to IFN2-α treatment, whereas WT virus entry was significantly inhibited (Supplemental Fig. 1G). Furthermore, to monitor the replication speed of a single virus particle, we infected cells with an MOI of 0.0001, in the presence of MC, which is commonly used to inhibit viral spread in tissue culture. Following infection of MC57G cells, we observed an increased LCMV spread of viral particles with the H155Y virus when compared with the WT controls (Fig. 4D). Notably, consistent with published results, LCMV-NP Ag was predominantly observed in the cytoplasm (Fig. 4D) (29, 30). As for many other viruses, SARS-CoV-2 infection and the resulting clinical outcome are also determined by IFN-I–driven immunity (31, 32). Accordingly, to investigate whether high-affinity–mediated IFN-I resistance applies to SARS-CoV-2, we infected Vero cells with a WT (Wuhan-Hu-1) and ο strain (ο BA.5). The receptor-binding domain from the ο strain was shown to have reduced ACE2 binding activity when compared with the prototype receptor-binding domain (33). Interestingly, the Wuhan-Hu-1–infected cells readily produced more viral copies than ο BA.5–infected cells in the presence of rIFN-α (Supplemental Fig. 2). Taken together, these data indicate that high-affinity receptor binding is associated with IFN-I resistance.

FIGURE 4.

High-affinity receptor-binding virus triggers resistance to IFN-I–mediated viral inhibition. (A–C) MC-57G cells were infected by WT or H155Y viruses at MOI 0.01 and 0.1. Cells were treated with mouse rIFN4-α and rIFN2-α at the indicated concentrations (A) 4 h p.i., (B) 4 h prior to infection, and (C) 24 h prior to infection. At 24 h p.i., virus titer was determined from cell culture supernatant (A, n = 6; B and C, n = 3). (D) MC-57G cells were infected with WT or H155Y viruses at MOI 0.0001. At 4 h p.i., MC was added. At 36 h p.i., infected cells were visualized by staining with rat anti–LCMV-NP Ab using immunofluorescence. Representative images are shown (n = 4). Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

FIGURE 4.

High-affinity receptor-binding virus triggers resistance to IFN-I–mediated viral inhibition. (A–C) MC-57G cells were infected by WT or H155Y viruses at MOI 0.01 and 0.1. Cells were treated with mouse rIFN4-α and rIFN2-α at the indicated concentrations (A) 4 h p.i., (B) 4 h prior to infection, and (C) 24 h prior to infection. At 24 h p.i., virus titer was determined from cell culture supernatant (A, n = 6; B and C, n = 3). (D) MC-57G cells were infected with WT or H155Y viruses at MOI 0.0001. At 4 h p.i., MC was added. At 36 h p.i., infected cells were visualized by staining with rat anti–LCMV-NP Ab using immunofluorescence. Representative images are shown (n = 4). Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

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LCMV infection is controlled by antiviral CD8+ T cell immunity (14, 34). Although we found that T cell activation was similar between WT and high-affinity virus, we wondered whether increased virus replication might cause T cell dysfunction (35). We therefore characterized CD8+ T cell responses after WT or H155Y virus infection. We observed a decreased frequency of antiviral T cells specific for the immune-dominant epitope gp33 in animals infected with the high-affinity virus when compared with the WT virus–infected mice (Fig. 5A). Furthermore, the effector T cell subset defined as KLRG1+IL7R was reduced in splenic tet-gp33+ cells in H155Y-infected mice when compared with WT virus–infected counterparts (Fig. 5B). Consistently, we observed increased expression of T cell activation markers KLRG1 and CD44 in WT virus–infected mice when compared with H155Y-infected mice (Fig. 5C). Additional staining for T cell exhaustion makers revealed that LCMV-specific T cells expressed increased PD-1 molecules in H155Y-infected mice compared with controls (Fig. 5C). Notably, other exhaustion makers, including TIM3, 2B4, and Lag3, were similar between WT- and H155Y-infected mice (Fig. 5C). These modest differences in exhausted T cells were likely observed because of the attenuated chimeric virus model system (13). To test the functionality of the CD8+ T cells, we restimulated the single-cell suspension from spleen, liver, and lung tissues ex vivo with LCMV gp33–41 peptides. We detected a decreased frequency of IFN-γ–producing CD8+ T cells in organs from H155Y-infected mice compared with WT virus–infected animals (Fig. 5D). Consistently, reduced IFN-γ levels were detected in serum samples from H155Y-infected mice compared with samples from WT virus–infected animals (Fig. 6A). Accordingly, we found the expression levels of genes encoding for Ido1, Icam1, and Mov10, which are specifically stimulated by IFN-γ (36) to be downregulated in lung and liver tissues of mice p.i. with H155Y compared with WT virus particles (Fig. 6B). In addition, expression levels of other ISGs, including Mx1, Rnasel, and Osal1, were also downregulated in lung, but not in liver, tissues harvested from H155Y-infected mice compared with controls (Fig. 6B). Consistent with the reduced effector T cell function and IFN-II responses, we observed prolonged virus replication in H155Y-infected animals when compared with WT-infected animals (Fig. 6C and 6D). Moreover, we observed increased viral Ags in the H155Y-infected spleen tissue when compared with WT LCMV–infected mice (Fig. 6E). In liver tissue, both WT- and H155Y-infected mice showed infected Kupffer cells (Fig. 6F), whereas infection of CD31+ endothelial cells was more frequent in mice infected with H155Y virus particles (Fig. 6F). Notably, both viruses were attenuated compared with the WT LCMV strains because we used recombinant chimeric LCMV containing the WE S segment and the Clone 13 L segment (13). The attenuated nature of these recombinant chimeric virus strains resulted in detection of LCMV in H155Y-infected animals in only a fraction of samples measured at day 12 (Supplemental Fig. 3). Collectively, we observed reduced CD8+ T cell immunity and prolonged viral replication after high-affinity virus infection.

FIGURE 5.

Infection with H155Y LCMV results in impaired CD8+ T cell immunity. C57BL/6 mice were infected with 106 PFUs of WT or H155Y viruses i.v. At day 6 p.i., (A) LCMV-specific T cells tet-gp33+CD8+ were determined in spleen, liver (n = 12), and lung tissues (n = 11). (B) Short-lived effector cells (SLECs; KLRG1+, IL-7R) and memory precursor cells (MEPCs; KLRG1, IL-7R+) were shown from splenic tet-gp33+ cells (n = 12). (C) Representative histograms showing surface molecule expression on splenic tet-gp33+ cells are shown (n = 6). (D) Singly suspended cells from spleen, liver (n = 12), or lung tissues (n = 6) were restimulated with LCMV-specific CD8+ T cell epitopes gp33 or left untreated (negative control [n.c.]) followed by staining for IFN-γ as ascertained by FACS analysis. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

FIGURE 5.

Infection with H155Y LCMV results in impaired CD8+ T cell immunity. C57BL/6 mice were infected with 106 PFUs of WT or H155Y viruses i.v. At day 6 p.i., (A) LCMV-specific T cells tet-gp33+CD8+ were determined in spleen, liver (n = 12), and lung tissues (n = 11). (B) Short-lived effector cells (SLECs; KLRG1+, IL-7R) and memory precursor cells (MEPCs; KLRG1, IL-7R+) were shown from splenic tet-gp33+ cells (n = 12). (C) Representative histograms showing surface molecule expression on splenic tet-gp33+ cells are shown (n = 6). (D) Singly suspended cells from spleen, liver (n = 12), or lung tissues (n = 6) were restimulated with LCMV-specific CD8+ T cell epitopes gp33 or left untreated (negative control [n.c.]) followed by staining for IFN-γ as ascertained by FACS analysis. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, statistically not significant between the indicated groups.

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FIGURE 6.

Impaired CD8+ T cell immunity induced by H155Y virus leads to prolonged virus replication. C57BL/6 mice were infected with 106 PFUs of WT or H155Y viruses i.v. At day 6 p.i., (A) IFN-γ concentration was determined from infected mouse serum (n = 9). (B) mRNA levels of genes encoding for ISG were determined by RT-PCR in organs harvested from infected animals as indicated (n = 6). (C) Virus titers were determined in spleen, lung, liver, and kidney tissues of infected animals (n = 9). (D) Viral NP mRNA levels were determined in organ tissue samples as indicated by RT-PCR (n = 12 for spleen tissues, n = 6 for liver and lung tissues). (E) Sections of snap-frozen spleen tissue were stained for LCMV-NP, F4/80, and B220. (F) Sections of snap-frozen liver tissue harvested from infected animals as indicated were stained for LCMV-NP, F4/80, and CD31. Representative picture is shown (n = 6; scale bars, 100 μm). Error bars show SEM. *p < 0.05, ***p < 0.001. ns, statistically not significant between the indicated groups.

FIGURE 6.

Impaired CD8+ T cell immunity induced by H155Y virus leads to prolonged virus replication. C57BL/6 mice were infected with 106 PFUs of WT or H155Y viruses i.v. At day 6 p.i., (A) IFN-γ concentration was determined from infected mouse serum (n = 9). (B) mRNA levels of genes encoding for ISG were determined by RT-PCR in organs harvested from infected animals as indicated (n = 6). (C) Virus titers were determined in spleen, lung, liver, and kidney tissues of infected animals (n = 9). (D) Viral NP mRNA levels were determined in organ tissue samples as indicated by RT-PCR (n = 12 for spleen tissues, n = 6 for liver and lung tissues). (E) Sections of snap-frozen spleen tissue were stained for LCMV-NP, F4/80, and B220. (F) Sections of snap-frozen liver tissue harvested from infected animals as indicated were stained for LCMV-NP, F4/80, and CD31. Representative picture is shown (n = 6; scale bars, 100 μm). Error bars show SEM. *p < 0.05, ***p < 0.001. ns, statistically not significant between the indicated groups.

Close modal

In this study, we analyzed the impact of the virus-binding affinity on innate and adaptive immune activation and antiviral defenses. Although innate immune activation was intact p.i. with high receptor-binding affinity virus, we observed increased viral replication. High receptor-binding affinity of virus particles exhibited resistance towards IFN-I–mediated inhibition of viral entry and replication. During in vivo infection, high-affinity virus particles replicated faster than WT LCMV particles in the presence of innate immunity, eventually leading to impaired T cell immunity and prolonged virus load.

IFN-I is an important antiviral factor during the course of LCMV infections (37). Hence arenaviruses acquired evasion mechanisms to curb IFN-I production. The Z protein of arenaviruses can block the interaction between cytosolic RNA receptors and mitochondrial antiviral signaling protein (38). Moreover, the C-terminal part of the Lassa NP exhibits an exonuclease activity targeting dsRNA, thus degrading potential ligands for cytosolic RNA receptors (39, 40). Hence the nucleoprotein of most arenaviruses can inhibit IFN-I production by blocking IRF-3 and NF-κB translocation into the nucleus (41–43). In addition, a recent study suggested that an impaired exonuclease activity mutant in Tacaribe virus NP was also able to block IFN-β production, suggesting additional IFN antagonism functions of the arenavirus NP (44). Indeed, it has been shown that NP binds to the kinase domain of IκB kinase–related kinase IκB kinase ε, blocking its ability to phosphorylate IRF3 (45). Moreover, slow viral replication can prevent immune sensing and consequently IFN-I production as exemplified by LCMV Docile to later result in excessive viral replication and prolonged infection (13). All these studies concluded that production of IFN-I can be inhibited by Arenavirus infection, thereby limiting innate immunity. In this study, we suggest that high-affinity binding to entry receptors can limit antiviral effects of IFN-I.

The LCMV strains Clone 13, Traub, and Docile are relatively more resistant to IFN-I and IFN-II and are more likely to establish a persistent infection in immunocompetent mice (46). Indeed, previous reports demonstrated that LCMV Clone 13 exhibited higher α-DG receptor-binding affinity than its counterpart LCMV Armstrong, possibly contributing to establishment of a chronic Clone 13 infection (27). Our data show that although high- and low-affinity viruses can induce similar IFN-I production in vivo, the antiviral effects of IFN-I were curbed by high-affinity receptor binding. Consistently, the consequent increased viral replication reduced T cell immunity over the course of infection, exacerbating the effects of increased viral replication (13, 35). Notably, our study was performed in an attenuated virus model system using chimeric viruses to investigate the binding affinity in a defined setting (13). Interestingly, excess production of IFN-I, specifically IFN-β, can contribute to PD-1L production and T cell dysfunction (47–49). Based on our findings, we propose that high-affinity binding to entry receptors prevents antiviral effects of IFN-I without compromising IFN-I production, which might contribute to T cell dysfunction.

Taken together, our data demonstrated that viruses can undergo innate affinity escape resulting in increased viral entry and replication, T cell dysfunction, and prolonged viral infection.

H.C.X., P.P., K.S.L., and P.A.L. are involved and listed as inventors of patents in the development of LCMV for clinical application in oncology in cooperation with or as advisors to Abalos Therapeutics GmbH.

We thank the National Institutes of Health tetramer facility for providing Ag-specific monomers.

This work was supported by Deutsche Forschungsgemeinschaft (German Research Council; LA2558/8-1, RTG1949), Forschungskommission of the Faculty of Medicine of Heinrich Heine University Düsseldorf (Project 9772816, “The role of polysaccharides in arenavirus infection”), Jürgen Manchot Foundation (MOI IV), Christiane and Claudia Hempel Foundation, and Volkswagen Foundation.

The online version of this article contains supplemental material.

cDC

conventional dendritic cell

α-DG

α-dystroglycan

dpi

days postinfection

GP

glycoprotein

IFN-I

type I IFN

IFN-II

type II IFN

ISG

IFN-stimulated gene

LCMV

lymphocytic choriomeningitis virus

MC

methylcellulose

MOI

multiplicity of infection

NP

nucleoprotein

p.i.

postinfection

WT

wild-type

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