Pathogenic and Apathogenic Strains of Lymphocytic Choriomeningitis Virus Have Distinct Entry and Innate Immune Activation Pathways.

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.

High-Affinity-Mediated Viral Entry Triggers Innate Affinity Escape Resulting in Type I IFN Resistance and Impaired T Cell Immunity.

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.

Lymphocytic choriomeningitis arenavirus requires cellular COPI and AP-4 complexes for efficient virion production.

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harboring a FLAG-tagged glycoprotein (GP-1) envelope spike (rLCMV-GP1-FLAG), we showed infection resulted in marked co-localization of individual COPI and AP-4 components with both LCMV nucleoprotein (NP) and GP-1, consistent with their involvement in viral processes. To further investigate the role of both COPI and AP-4 complexes during LCMV infection, we utilized the ARF-I inhibitor brefeldin A (BFA) that prevents complex formation. Within a single 12-h cycle of virus multiplication, BFA pre-treatment caused no significant change in LCMV-specific RNA synthesis, alongside no significant change in LCMV NP expression, as measured by BFA time-of-addition experiments. In contrast, BFA addition resulted in a significant drop in released virus titers, approaching 50-fold over the same 12-h period, rising to over 600-fold over 24 h. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors required for the formation and release of infectious LCMV. IMPORTANCE: Arenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member lymphocytic choriomeningitis virus (LCMV) being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. A detailed understanding of how arenaviruses subvert host cell processes to complete their multiplication cycle is incomplete. Here, using a combination of gene ablation and pharmacological inhibition techniques, we showed that host cellular COPI and AP-4 complexes, with native roles in cellular vesicular transport, were required for efficient LCMV growth. We further showed these complexes acted on late stages of the multiplication cycle, post-gene expression, with a significant impact on infectious virus egress. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal critical cellular trafficking pathways required during infection.

Lymphocytic choriomeningitis virus injures the developing brain: effects and mechanisms.

Lymphocytic choriomeningitis virus (LCMV) is a prevalent pathogen, whose natural host and reservoir is the wild mouse. Humans can be infected when they contact the secretions of mice. Most infections of postnatal humans result in mild illness. However, the consequences can be severe when the infection occurs during pregnancy, as the virus crosses the placenta to infect the fetus. LCMV infection of the human fetus can lead to severe neuropathologic effects, including microencephaly, hydrocephalus, focal destructive lesions, and cerebellar hypoplasia. Outcomes among children with congenital LCMV are variable, but most are permanently and severely disabled. The neonatal rat inoculated with LCMV models human prenatal infection. The rat model has demonstrated that effects of LCMV depend on host age at the time of infection. Some effects, including encephalomalacia and neuronal migration disturbances, are immune-mediated and depend on the actions of T-lymphocytes. Other effects, including cerebellar hypoplasia, are virus-mediated and do not depend on T-lymphocytes. Cerebellar neuronal migration disturbances are caused by immune-mediated corruption of Bergmann glia structure. The rat pup inoculated with LCMV is a superb animal model for human congenital infection. All neuropathologic effects observed in human congenital LCMV infection can be recapitulated in the rat model. IMPACT: Lymphocytic choriomeningitis virus (LCMV) is a prevalent human pathogen that can cause serious neurologic birth defects when the infection occurs during pregnancy. The effects of the virus on the developing brain depend strongly on the age of the host at the time of infection. Some of the pathologic effects of LCMV are immune-mediated and are driven by T-lymphocytes, while other pathologic effects are due to the virus itself.

Differential kinetics of splenic CD169+ macrophage death is one underlying cause of virus infection fate regulation.

Acute infection and chronic infection are the two most common fates of pathogenic virus infections. While several factors that contribute to these fates are described, the critical control points and the mechanisms that underlie infection fate regulation are incompletely understood. Using the acute and chronic lymphocytic choriomeningitis virus (LCMV) infection model of mice, we find that the early dynamic pattern of the IFN-I response is a differentiating trait between both infection fates. Acute-infected mice generate a 2-wave IFN-I response while chronic-infected mice generate only a 1-wave response. The underlying cause is a temporal difference in CD8 T cell-mediated killing of splenic marginal zone CD169+ macrophages. It occurs later in acute infection and thus enables CD169+ marginal zone macrophages to produce the 2nd IFN-I wave. This is required for subsequent immune events including induction of inflammatory macrophages, generation of effector CD8+ T cells and virus clearance. Importantly, these benefits come at a cost for the host in the form of spleen fibrosis. Due to an earlier marginal zone destruction, these ordered immune events are deregulated in chronic infection. Our findings demonstrate the critical importance of kinetically well-coordinated sequential immune events for acute infection control and highlights that it may come at a cost for the host organism.

Cellular and transcriptional impacts of Janus kinase and/or IFN-gamma inhibition in a mouse model of primary hemophagocytic lymphohistiocytosis.

Background: Primary hemophagocytic lymphohistiocytosis (pHLH) is an inherited inflammatory syndrome driven by the exuberant activation of interferon-gamma (IFNg)-producing CD8 T cells. Towards this end, ruxolitinib treatment or IFNg neutralization (aIFNg) lessens immunopathology in a model of pHLH in which perforin-deficient mice (Prf1-/-) are infected with Lymphocytic Choriomeningitis virus (LCMV). However, neither agent completely eradicates inflammation. Two studies combining ruxolitinib with aIFNg report conflicting results with one demonstrating improvement and the other worsening of disease manifestations. As these studies used differing doses of drugs and varying LCMV strains, it remained unclear whether combination therapy is safe and effective. Methods: We previously showed that a ruxolitinib dose of 90 mg/kg lessens inflammation in Prf1-/- mice infected with LCMV-Armstrong. To determine whether this dose controls inflammation induced by a different LCMV strain, we administered ruxolitinib at 90mg/kg to Prf1-/- mice infected with LCMV-WE. To elucidate the impacts of single agent versus combination therapy, Prf1-/- animals were infected with LCMV, treated or not with ruxolitinib, aIFNg or both agents, and analyzed for disease features and the transcriptional impacts of therapy within purified CD8 T cells. Results: Ruxolitinib is well-tolerated and controls disease regardless of the viral strain used. aIFNg, administered alone or with ruxolitinib, is most effective at reversing anemia and reducing serum IFNg levels. In contrast, ruxolitinib appears better than aIFNg, and equally or more effective than combination therapy, at lessening immune cell expansion and cytokine production. Each treatment targets distinct gene expression pathways with aIFNg downregulating IFNg, IFNa, and IL-6-STAT3 pathways, and ruxolitinib downregulating IL-6-STAT3, glycolysis, and reactive oxygen species pathways. Unexpectedly, combination therapy is associated with upregulation of genes driving cell survival and proliferation. Conclusions: Ruxolitinib is tolerated and curtails inflammation regardless of the inciting viral strain and whether it is given alone or in combination with aIFNg. When administered at the doses used in this study, the combination of ruxolitinb and aIFNg appears no better than treatment with either drug alone in lessening inflammation. Further studies are warranted to elucidate the optimal doses, schedules, and combinations of these agents for the treatment of patients with pHLH.

Exhausted CD8+ T cells face a developmental fork in the road.

Reinvigorating the function of exhausted CD8+ T cells during chronic viral infection and cancer is a major goal of current immunotherapy regimens. Here, we discuss recent advances in our understanding of exhausted CD8+ T cell heterogeneity as well as the potential differentiation trajectories that exhausted T cells follow during chronic infection and/or cancer. We highlight surmounting evidence suggesting that some T cell clones are divergent in nature and can develop into either terminally differentiated effector or exhausted CD8+ T cells. Lastly, we consider the potential therapeutic implications of such a bifurcation model of CD8+ T cell differentiation, including the intriguing hypothesis that redirecting progenitor CD8+ T cell differentiation along an effector pathway may serve as a novel approach to mitigate T cell exhaustion.

Meningeal macrophages protect against viral neuroinfection.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.

TGF-ß regulates the stem-like state of PD-1+ TCF-1+ virus-specific CD8 T cells during chronic infection.

Recent studies have defined a novel population of PD-1+ TCF-1+ stem-like CD8 T cells in chronic infections and cancer. These quiescent cells reside in lymphoid tissues, are critical for maintaining the CD8 T cell response under conditions of persistent antigen, and provide the proliferative burst after PD-1 blockade. Here we examined the role of TGF-ß in regulating the differentiation of virus-specific CD8 T cells during chronic LCMV infection of mice. We found that TGF-ß signaling was not essential for the generation of the stem-like CD8 T cells but was critical for maintaining the stem-like state and quiescence of these cells. TGF-ß regulated the unique transcriptional program of the stem-like subset, including upregulation of inhibitory receptors specifically expressed on these cells. TGF-ß also promoted the terminal differentiation of exhausted CD8 T cells by suppressing the effector-associated program. Together, the absence of TGF-ß signaling resulted in significantly increased accumulation of effector-like CD8 T cells. These findings have implications for immunotherapies in general and especially for T cell therapy against chronic infections and cancer.

CD164 is a host factor for lymphocytic choriomeningitis virus entry.

Lymphocytic choriomeningitis virus (LCMV) is a rodent-borne zoonotic arenavirus that causes congenital abnormalities and can be fatal for transplant recipients. Using a genome-wide loss-of-function screen, we identify host factors required for LCMV entry into cells. We identify the lysosomal mucin CD164, glycosylation factors, the heparan sulfate biosynthesis machinery, and the known receptor alpha-dystroglycan (α-DG). Biochemical analysis revealed that the LCMV glycoprotein binds CD164 at acidic pH and requires a sialylated glycan at residue N104. We demonstrate that LCMV entry proceeds by the virus switching binding from heparan sulfate or α-DG at the plasma membrane to CD164 prior to membrane fusion, thus identifying additional potential targets for therapeutic intervention.

DExD/H-box helicase 9 intrinsically controls CD8+ T cell-mediated antiviral response through noncanonical mechanisms.

Upon virus infection, CD8+ T cell accumulation is tightly controlled by simultaneous proliferation and apoptosis. However, it remains unclear how TCR signal coordinates these events to achieve expansion and effector cell differentiation. We found that T cell-specific deletion of nuclear helicase Dhx9 led to impaired CD8+ T cell survival, effector differentiation, and viral clearance. Mechanistically, Dhx9 acts as the key regulator to ensure LCK- and CD3ε-mediated ZAP70 phosphorylation and ERK activation to protect CD8+ T cells from apoptosis before proliferative burst. Dhx9 directly regulates Id2 transcription to control effector CD8+ T cell differentiation. The DSRM and OB_Fold domains are required for LCK binding and Id2 transcription, respectively. Dhx9 expression is predominantly increased in effector CD8+ T cells of COVID-19 patients. Therefore, we revealed a previously unknown regulatory mechanism that Dhx9 protects activated CD8+ T cells from apoptosis and ensures effector differentiation to promote antiviral immunity independent of nuclear sensor function.

CXCL10 chemokine regulates heterogeneity of the CD8+ T cell response and viral set point during chronic infection.

CD8+ T cells responding to chronic infection adapt an altered differentiation program that provides some restraint on pathogen replication yet limits immunopathology. This adaptation is imprinted in stem-like cells and propagated to their progeny. Understanding the molecular control of CD8+ T cell differentiation in chronic infection has important therapeutic implications. Here, we find that the chemokine receptor CXCR3 is highly expressed on viral-specific stem-like CD8+ T cells and that one of its ligands, CXCL10, regulates the persistence and heterogeneity of responding CD8+ T cells in spleens of mice chronically infected with lymphocytic choriomeningitis virus. CXCL10 is produced by inflammatory monocytes and fibroblasts of the splenic red pulp, where it grants stem-like cells access to signals promoting differentiation and limits their exposure to pro-survival niches in the white pulp. Consequently, functional CD8+ T cell responses are greater in Cxcl10-/- mice and are associated with a lower viral set point.

NFAT-dependent and -independent exhaustion circuits program maternal CD8 T cell hypofunction in pregnancy.

Pregnancy is a common immunization event, but the molecular mechanisms and immunological consequences provoked by pregnancy remain largely unknown. We used mouse models and human transplant registry data to reveal that pregnancy induced exhausted CD8 T cells (Preg-TEX), which associated with prolonged allograft survival. Maternal CD8 T cells shared features of exhaustion with CD8 T cells from cancer and chronic infection, including transcriptional down-regulation of ribosomal proteins and up-regulation of TOX and inhibitory receptors. Similar to other models of T cell exhaustion, NFAT-dependent elements of the exhaustion program were induced by fetal antigen in pregnancy, whereas NFAT-independent elements did not require fetal antigen. Despite using conserved molecular circuitry, Preg-TEX cells differed from TEX cells in chronic viral infection with respect to magnitude and dependency of T cell hypofunction on NFAT-independent signals. Altogether, these data reveal the molecular mechanisms and clinical consequences of maternal CD8 T cell hypofunction and identify pregnancy as a previously unappreciated context in which T cell exhaustion may occur.

The Inhibitory Receptor GPR56 (Adgrg1) Is Specifically Expressed by Tissue-Resident Memory T Cells in Mice But Dispensable for Their Differentiation and Function In Vivo.

Tissue-resident memory T (TRM) cells with potent antiviral and antibacterial functions protect the epithelial and mucosal surfaces of our bodies against infection with pathogens. The strong proinflammatory activities of TRM cells suggest requirement for expression of inhibitory molecules to restrain these memory T cells under steady state conditions. We previously identified the adhesion G protein-coupled receptor GPR56 as an inhibitory receptor of human cytotoxic lymphocytes that regulates their cytotoxic effector functions. Here, we explored the expression pattern, expression regulation, and function of GPR56 on pathogen-specific CD8+ T cells using two infection models. We observed that GPR56 is expressed on TRM cells during acute infection and is upregulated by the TRM cell-inducing cytokine TGF-ß and the TRM cell-associated transcription factor Hobit. However, GPR56 appeared dispensable for CD8+ T-cell differentiation and function upon acute infection with LCMV as well as Listeria monocytogenes. Thus, TRM cells specifically acquire the inhibitory receptor GPR56, but the impact of this receptor on TRM cells after acute infection does not appear essential to regulate effector functions of TRM cells.

Severity of Sepsis Determines the Degree of Impairment Observed in Circulatory and Tissue-Resident Memory CD8 T Cell Populations.

Sepsis reduces the number and function of memory CD8 T cells within the host, contributing to the long-lasting state of immunoparalysis. Interestingly, the relative susceptibility of memory CD8 T cell subsets to quantitative/qualitative changes differ after cecal ligation and puncture (CLP)-induced sepsis. Compared with circulatory memory CD8 T cells (TCIRCM), moderate sepsis (0-10% mortality) does not result in numerical decline of CD8 tissue-resident memory T cells (TRM), which retain their "sensing and alarm" IFN-γ-mediated effector function. To interrogate this biologically important dichotomy, vaccinia virus-immune C57BL/6 (B6) mice containing CD8 TCIRCM and skin TRM underwent moderate or severe (∼50% mortality) sepsis. Severe sepsis led to increased morbidity and mortality characterized by increased inflammation compared with moderate CLP or sham controls. Severe CLP mice also displayed increased vascular permeability in the ears. Interestingly, skin CD103+ CD8 TRM, detected by i.v. exclusion or two-photon microscopy, underwent apoptosis and subsequent numerical loss following severe sepsis, which was not observed in mice that experienced moderate CLP or sham surgeries. Consequently, severe septic mice showed diminished CD8 T cell-mediated protection to localized skin reinfection. Finally, the relationship between severity of sepsis and demise in circulatory versus tissue-embedded memory CD8 T cell populations was confirmed by examining tumor-infiltrating and nonspecific CD8 T cells in B16 melanoma tumors. Thus, sepsis can differentially affect the presence and function of Ag-specific CD8 T cells that reside inside tissues/tumors depending on the severity of the insult, a notion with direct relevance to sepsis survivors and their ability to mount protective memory CD8 T cell-dependent responses to localized Ag re-encounter.

Protein Immunization Induces Memory CD4+ T Cells That Lack Th Lineage Commitment.

Acute viral infection generates lineage-committed Th1 and T follicular helper (Tfh) memory cells that recall their lineage-specific functions following secondary challenge with virus. However, the lineage commitment of effector and memory Th cells in vivo following protein vaccination is poorly understood. In this study, we analyzed effector and memory CD4+ T cell differentiation in mice (Mus musculus) following adjuvanted glycoprotein immunization compared with acute lymphocytic choriomeningitis virus infection. Glycoprotein immunization induced CXCR5- non-Tfh effector and memory CD4+ T cells that surprisingly had not undergone polarization toward any particular Th cell lineage but had undergone memory differentiation. However, upon challenge with virus, these Th lineage-nonpolarized memory CD4+ T cells were able to generate Th1 secondary effector cells, demonstrating their lineage plasticity. In addition, Tfh and memory Tfh cells were generated in response to protein immunization, and these cells differed from infection-induced Tfh cells by their lack of the transcription factor Tbet. Rechallenge experiments demonstrated that viral infection, but not protein immunization, during either the primary or secondary immune response, restricts the recall of Bcl6 expression and the generation of germinal center Tfh cells. Together, these data demonstrate that protein immunization generates a combination of nonpolarized memory cells that are highly plastic and memory Tfh cells that can undergo further Th1-like modulation during a secondary response to viral infection.

Single-Cell Transcriptomics Reveals Core Regulatory Programs That Determine the Heterogeneity of Circulating and Tissue-Resident Memory CD8+ T Cells.

During acute infections, CD8+ T cells form various memory subpopulations to provide long-lasting protection against reinfection. T central memory (TCM), T effector memory (TEM), and long-lived effector (LLE) cells are circulating memory populations with distinct plasticity, migration patterns, and effector functions. Tissue-resident memory (TRM) cells permanently reside in the frontline sites of pathogen entry and provide tissue-specific protection upon reinfection. Here, using single-cell RNA-sequencing (scRNA-seq) and bulk RNA-seq, we examined the different and shared transcriptomes and regulators of TRM cells with other circulating memory populations. Furthermore, we identified heterogeneity within the TRM pool from small intestine and novel transcriptional regulators that may control the phenotypic and functional heterogeneity of TRM cells during acute infection. Our findings provide a resource for future studies to identify novel pathways for enhancing vaccination and immunotherapeutic approaches.

T Cell-Intrinsic CDK6 Is Dispensable for Anti-Viral and Anti-Tumor Responses In Vivo.

The cyclin-dependent kinase 6 (CDK6) regulates the transition through the G1-phase of the cell cycle, but also acts as a transcriptional regulator. As such CDK6 regulates cell survival or cytokine secretion together with STATs, AP-1 or NF-κB. In the hematopoietic system, CDK6 regulates T cell development and promotes leukemia and lymphoma. CDK4/6 kinase inhibitors are FDA approved for treatment of breast cancer patients and have been reported to enhance T cell-mediated anti-tumor immunity. The involvement of CDK6 in T cell functions remains enigmatic. We here investigated the role of CDK6 in CD8+ T cells, using previously generated CDK6 knockout (Cdk6-/-) and kinase-dead mutant CDK6 (Cdk6K43M) knock-in mice. RNA-seq analysis indicated a role of CDK6 in T cell metabolism and interferon (IFN) signaling. To investigate whether these CDK6 functions are T cell-intrinsic, we generated a T cell-specific CDK6 knockout mouse model (Cdk6fl/fl CD4-Cre). T cell-intrinsic loss of CDK6 enhanced mitochondrial respiration in CD8+ T cells, but did not impact on cytotoxicity and production of the effector cytokines IFN-γ and TNF-α by CD8+ T cells in vitro. Loss of CDK6 in peripheral T cells did not affect tumor surveillance of MC38 tumors in vivo. Similarly, while we observed an impaired induction of early responses to type I IFN in CDK6-deficient CD8+ T cells, we failed to observe any differences in the response to LCMV infection upon T cell-intrinsic loss of CDK6 in vivo. This apparent contradiction might at least partially be explained by the reduced expression of Socs1, a negative regulator of IFN signaling, in CDK6-deficient CD8+ T cells. Therefore, our data are in line with a dual role of CDK6 in IFN signaling; while CDK6 promotes early IFN responses, it is also involved in the induction of a negative feedback loop. These data assign CDK6 a role in the fine-tuning of cytokine responses.

Characterization of CD8 T Cell-Mediated Mutations in the Immunodominant Epitope GP33-41 of Lymphocytic Choriomeningitis Virus.

Cytotoxic T lymphocytes (CTLs) represent key immune effectors of the host response against chronic viruses, due to their cytotoxic response to virus-infected cells. In response to this selection pressure, viruses may accumulate escape mutations that evade CTL-mediated control. To study the emergence of CTL escape mutations, we employed the murine chronic infection model of lymphocytic choriomeningitis virus (LCMV). We developed an amplicon-based next-generation sequencing pipeline to detect low frequency mutations in the viral genome and identified non-synonymous mutations in the immunodominant LCMV CTL epitope, GP33-41, in infected wildtype mice. Infected Rag2-deficient mice lacking CTLs did not contain such viral mutations. By using transgenic mice with T cell receptors specific to GP33-41, we characterized the emergence of viral mutations in this epitope under varying selection pressure. We investigated the two most abundant viral mutations by employing reverse genetically engineered viral mutants encoding the respective mutations. These experiments provided evidence that these mutations prevent activation and expansion of epitope-specific CD8 T cells. Our findings on the mutational dynamics of CTL escape mutations in a widely-studied viral infection model contributes to our understanding of how chronic viruses interact with their host and evade the immune response. This may guide the development of future treatments and vaccines against chronic infections.