Viral Immune Evasion Research Articles

SOCS3 Promotes ALV-J Virus Replication via Inhibiting JAK2/STAT3 Phosphorylation During Infection.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes immunosuppression and neoplastic diseases in poultry. Cytokine signal-transduction inhibitor molecule 3 (SOCS3) is an important negative regulator of the JAK2/STAT3 signaling pathway and plays certain roles in ALV-J infection. It is of significance to confirm the roles of SOCS3 in ALV-J infection and study how this gene affects ALV-J infection. In this study, we assessed the expression of the SOCS3 gene in vivo and in vitro, and investigated the roles of SOCS3 in ALV-J infection using overexpressed or interfered assays with the SOCS3 in DF-1 cells. The results showed that the SOCS3 expression of ALV-J infected chickens was different from uninfected chickens in the spleen, thymus and cecal tonsil. Further, SOCS3 is mainly expressed in the nucleus as determined by immunofluorescence assay. Overexpression of SOCS3 in DF-1 cells promoted the replication of ALV-J virus, and the expression of interferons (IFNα and INFβ), inflammatory factors (IL-6 and TNFα) along with interferon-stimulating genes (CH25H, MX1, OASL, and ZAP). Conversely, interference of SOCS3 showed the opposite results. We also observed that SOCS3 promoted ALV-J virus replication by inhibiting JAK2/STAT3 phosphorylation. In conclusion, SOCS3 promotes ALV-J replication via inhibiting the phosphorylation of the JAK2/STAT3 signaling pathway. These results would advance further understanding of the persistent infection and the viral immune evasion of the ALV-J virus.

Viral Induced Protein Aggregation: A Mechanism of Immune Evasion.

Various intrinsic and extrinsic factors can interfere with the process of protein folding, resulting in protein aggregates. Usually, cells prevent the formation of aggregates or degrade them to prevent the cytotoxic effects they may cause. However, during viral infection, the formation of aggregates may serve as a cellular defense mechanism. On the other hand, some viruses are able to exploit the process of aggregate formation and removal to promote their replication or evade the immune response. This review article summarizes the process of cellular protein aggregation and gives examples of how different viruses exploit it. Particular emphasis is placed on the ribonucleotide reductases of herpesviruses and how their additional non-canonical functions in viral immune evasion are closely linked to protein aggregation.

Effect of SARS-CoV-2 B.1.1.7 mutations on spike protein structure and function.

The B.1.1.7 variant of SARS-CoV-2 first detected in the UK harbors amino-acid substitutions and deletions in the spike protein that potentially enhance host angiotensin conversion enzyme 2 (ACE2) receptor binding and viral immune evasion. Here we report cryo-EM structures of the spike protein of B.1.1.7 in the apo and ACE2-bound forms. The apo form showed one or two receptor-binding domains (RBDs) in the open conformation, without populating the fully closed state. All three RBDs were engaged in ACE2 binding. The B.1.1.7-specific A570D mutation introduces a molecular switch that could modulate the opening and closing of the RBD. The N501Y mutation introduces a π-π interaction that enhances RBD binding to ACE2 and abolishes binding of a potent neutralizing antibody (nAb). Cryo-EM also revealed how a cocktail of two nAbs simultaneously bind to all three RBDs, and demonstrated the potency of the nAb cocktail to neutralize different SARS-CoV-2 pseudovirus strains, including B.1.1.7.

Facing the wrath of enigmatic mutations: a review on the emergence of severe acute respiratory syndrome coronavirus 2 variants amid coronavirus disease-19 pandemic.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is an emerging respiratory virus responsible for the ongoing coronavirus disease 19 (COVID‐19) pandemic. More than a year into this pandemic, the COVID‐19 fatigue is still escalating and takes hold of the entire world population. Driven by the ongoing geographical expansion and upcoming mutations, the COVID‐19 pandemic has taken a new shape in the form of emerging SARS‐CoV‐2 variants. These mutations in the viral spike (S) protein enhance the virulence of SARS‐CoV‐2 variants by improving viral infectivity, transmissibility and immune evasion abilities. Such variants have resulted in cluster outbreaks and fresh infection waves in various parts of the world with increased disease severity and poor clinical outcomes. Hence, the variants of SARS‐CoV‐2 pose a threat to human health and public safety. This review enlists the most recent updates regarding the presently characterized variants of SARS‐CoV‐2 recognized by the global regulatory health authorities (WHO, CDC). Based on the slender literature on SARS‐CoV‐2 variants, we collate information on the biological implications of these mutations on virus pathology. We also shed light on the efficacy of therapeutics and COVID‐19 vaccines against the emerging SARS‐CoV‐2 variants.

CARs—A New Perspective to HCMV Treatment

Human cytomegalovirus (HCMV), by primary infection or reactivation, represents a great risk for immune-suppressed or compromised patients. In immunocompetent humans, the immune system suppresses the spread of HCMV during an infection, resulting in a mostly asymptomatic or mild course of the disease, whereas in immune suppressed patients, the compromised host immune response cannot control the viral infection. Multiple viral immunomodulatory mechanisms additionally contribute to immune evasion. Use of chimeric antigen receptors (CARs), a treatment strategy adapted from cancer immunotherapy, is investigated for possible application to combat HCMV and other infections in immunocompromised patients. The administration of CAR+ T-cells directed against HCMV antigens can bypass viral immune evasion and may complement existing treatment methods. This review gives a short overview of HCMV, the obstacles of current treatment options as well as a brief introduction to CARs and the current research situation on CAR+ T-cells against HCMV.

When Viruses Cross Developmental Pathways.

Aberrant regulation of developmental pathways plays a key role in tumorigenesis. Tumor cells differ from normal cells in their sustained proliferation, replicative immortality, resistance to cell death and growth inhibition, angiogenesis, and metastatic behavior. Often they acquire these features as a consequence of dysregulated Hedgehog, Notch, or WNT signaling pathways. Human tumor viruses affect the cancer cell hallmarks by encoding oncogenic proteins, and/or by modifying the microenvironment, as well as by conveying genomic instability to accelerate cancer development. In addition, viral immune evasion mechanisms may compromise developmental pathways to accelerate tumor growth. Viruses achieve this by influencing both coding and non-coding gene regulatory pathways. Elucidating how oncogenic viruses intersect with and modulate developmental pathways is crucial to understanding viral tumorigenesis. Many currently available antiviral therapies target viral lytic cycle replication but with low efficacy and severe side effects. A greater understanding of the cross-signaling between oncogenic viruses and developmental pathways will improve the efficacy of next-generation inhibitors and pave the way to more targeted antiviral therapies.

Transcriptome sequencing analysis of porcine alveolar macrophages infected with PRRSV strains to elucidate virus pathogenicity and immune evasion strategies.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) causes a serious disease to the swine industry worldwide. To understand the mechanisms of HP-PRRSV infection, RNA-seq-based transcriptome analyses were performed on porcine alveolar macrophages (PAMs) infected with a HP-PRRSV strain (TJ), a less virulent strain of a classical lineage (CH-1a), and a vaccine strain TJM-F92. Gene ontology, Kyoto Encyclopedia of Genes and Genomes analyses indicate that TJM-F92 led to significant up-regulation of gene expression for proteins associated with membrane-bound organelles. The differentially expressed genes of HP-PRRSV TJ-infected PAM cells were up-regulated in the special G-protein coupled receptor. The six cytokines were tested by real time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The relative expression levels showed the same trend of expression difference. Significant up-regulation of TMEM173 plays an important role in the cytosolic DNA-sensing pathway and the RIG-I-like receptor signaling pathway in TJM-F92 infected PAM cells. These data provide new insight into PRRSV pathogenicity and immune evasion strategies.

Animal Models for the Study of Hepatitis B Virus Pathobiology and Immunity: Past, Present, and Future.

Hepatitis B virus (HBV) infection is a global public health problem that plagues approximately 240 million people. Chronic hepatitis B (CHB) often leads to liver inflammation and aberrant repair which results in diseases ranging from liver fibrosis, cirrhosis, to hepatocellular carcinoma. Despite its narrow species tropism, researchers have established various in vivo models for HBV or its related viruses which have provided a wealth of knowledge on viral lifecycle, pathogenesis, and immunity. Here we briefly revisit over five decades of endeavor in animal model development for HBV and summarize their advantages and limitations. We also suggest directions for further improvements that are crucial for elucidation of the viral immune-evasion strategies and for development of novel therapeutics for a functional cure.

Peroxiredoxin 1 Interacts with TBK1/IKKε and Negatively Regulates Pseudorabies Virus Propagation by Promoting Innate Immunity.

Peroxiredoxin 1 (PRDX1) is a cellular antioxidant enzyme that is crucial for diverse fundamental biological processes, such as autophagy, inflammation, and carcinogenesis. However, molecular mechanisms underpinning its diverse roles are not well understood. Here, we report that PRDX1 positively regulates interferon (IFN) induction and that pseudorabies virus (PRV) targets PRDX1 to evade IFN induction. PRV UL13 encodes a serine/threonine kinase important for PRV infection, although its biological function remains obscure. We identified PRDX1 as a UL13-interacting protein. Virological and biochemical assays demonstrate that PRDX1 promotes IFN induction by interacting with TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε). Conversely, UL13 accelerates PRDX1 degradation via the ubiquitin-proteosome pathway in a kinase-dependent manner. In doing so, PRV inhibits IFN induction during productive infection, which requires PRDX1 expression. This study uncovers an essential role of PRDX1 in the innate immune response and reveals a new viral immune evasion strategy to counteract cellular defenses. IMPORTANCE PRV interacts with numerous cellular proteins during productive infection. Here, we demonstrated the interaction of viral protein UL13 with the antioxidant enzyme PRDX1, which functions in multiple signal transduction pathways. We found that PRDX1 participates in the type I IFN pathway by interacting with TBK1 and IKKε, thereby negatively regulating PRV propagation. However, UL13 ubiquitinates PRDX1, which routes PRDX1 into proteasomes for degradation and effectively reduces its expression. These results illuminate the fundamental role that PRDX1 plays in the IFN pathway, and they identify a potential target for the control of PRV infection.

Extracellular Vesicles as a Means of Viral Immune Evasion, CNS Invasion, and Glia-Induced Neurodegeneration.

Extracellular vesicles (EVs) are small, membrane-bound vesicles released by cells as a means of intercellular communication. EVs transfer proteins, nucleic acids, and other biologically relevant molecules from one cell to another. In the context of viral infections, EVs can also contain viruses, viral proteins, and viral nucleic acids. While there is some evidence that the inclusion of viral components within EVs may be part of the host defense, much of the research in this field supports a pro-viral role for EVs. Packaging of viruses within EVs has repeatedly been shown to protect viruses from antibody neutralization while also allowing for their integration into cells otherwise impervious to the virus. EVs also bidirectionally cross the blood-brain barrier (BBB), providing a potential route for peripheral viruses to enter the brain while exiting EVs may serve as valuable biomarkers of neurological disease burden. Within the brain, EVs can alter glial activity, increase neuroinflammation, and induce neurotoxicity. The purpose of this mini-review is to summarize research related to viral manipulation of EV-mediated intercellular communication and how such manipulation may lead to infection of the central nervous system, chronic neuroinflammation, and neurodegeneration.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease.

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

Battle Royale: Innate Recognition of Poxviruses and Viral Immune Evasion.

Host pattern recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs), which are molecular signatures shared by different pathogens. Recognition of PAMPs by PRRs initiate innate immune responses via diverse signaling pathways. Over recent decades, advances in our knowledge of innate immune sensing have enhanced our understanding of the host immune response to poxviruses. Multiple PRR families have been implicated in poxvirus detection, mediating the initiation of signaling cascades, activation of transcription factors, and, ultimately, the expression of antiviral effectors. To counteract the host immune defense, poxviruses have evolved a variety of immunomodulators that have diverse strategies to disrupt or circumvent host antiviral responses triggered by PRRs. These interactions influence the outcomes of poxvirus infections. This review focuses on our current knowledge of the roles of PRRs in the recognition of poxviruses, their elicited antiviral effector functions, and how poxviral immunomodulators antagonize PRR-mediated host immune responses.

Chemical constituents from coconut waste and their in silico evaluation as potential antiviral agents against SARS-CoV-2

Eleven compounds were isolated from the ethyl acetate extract of Cocos nucifera L endocarp, jezonofol 1, scirpusin A 2, cassigarol G 3, maackin A 4, threoguiacyl glycerol-8’-vanillic acid ether 5, erythroguiacyl glycerol-8’-vanillic acid ether 6, apigenin-7-O-β-D-glucoside 7, piceatannol 8, p-hydroxy-benzoic acid 9, protocatechuic acid 10 and vanillic acid 11. Compounds 1-7 were isolated for the first time from the plant. The isolated compounds were virtually screened against four critical components of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), the main protease (Mpro), papain-like protease (PLpro), nonstructural protein 13 (nsp13) and RNA dependent RNA polymerase (RdRp). Stilbene dimers 1-4 showed remarkable binding affinities towards the investigated targets (binding energy <−7.6 kcal/mol). Compounds 1, 3 and 4 interacted with the catalytic dyad (Cys145-His41) at the active pocket of Mpro which is essential for achieving good inhibitory activity. Compounds 1-3 showed molecular interaction with the conserved ubiquitin-specific protease residues of PLpro, responsible for binding ability at different active sites of nsp13, which are crucial for decreasing the resistance caused by viral immune evasion. Compounds 2 and 3 showed the ability to bind at different active sites of nsp13, which is a key binding site for reducing antiviral resistance. Finally, compounds 1-3 showed the ability to bind with RdRp before and after RNA binding. Our findings suggested that the dimeric stilbene skeleton is a promising candidate for developing anti-COVID-19 drugs. Particularly, 1, 2 and 3, showed a promiscuity pattern binding to multiple targets of SARS-CoV-2 replication. Herein, 20 ns molecular dynamics (MD) simulations combined with molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations were performed to estimate the binding affinity of the most potent three compounds against the viral SARS-CoV-2 targets. MM-GBSA calculations unveiled the outshine potency of compound 1 towards PLpro with a binding energy of −60.7 kcal/mol. Structural and energetic analyses over 20 ns MD simulation displayed the high stability of compound 1 in complex with PLpro. The list of the compounds was considered herein forms a primer for clinical investigation in COVID-19 patients and directing for further antiviral examinations. Drug likeness properties of compounds 1-4 were evaluated.

Ceramide and Related Molecules in Viral Infections.

Ceramide is a lipid messenger at the heart of sphingolipid metabolism. In concert with its metabolizing enzymes, particularly sphingomyelinases, it has key roles in regulating the physical properties of biological membranes, including the formation of membrane microdomains. Thus, ceramide and its related molecules have been attributed significant roles in nearly all steps of the viral life cycle: they may serve directly as receptors or co-receptors for viral entry, form microdomains that cluster entry receptors and/or enable them to adopt the required conformation or regulate their cell surface expression. Sphingolipids can regulate all forms of viral uptake, often through sphingomyelinase activation, and mediate endosomal escape and intracellular trafficking. Ceramide can be key for the formation of viral replication sites. Sphingomyelinases often mediate the release of new virions from infected cells. Moreover, sphingolipids can contribute to viral-induced apoptosis and morbidity in viral diseases, as well as virus immune evasion. Alpha-galactosylceramide, in particular, also plays a significant role in immune modulation in response to viral infections. This review will discuss the roles of ceramide and its related molecules in the different steps of the viral life cycle. We will also discuss how novel strategies could exploit these for therapeutic benefit.

STAT3 determines IL-4 signalling outcomes in na\xefve T cells

IL-4 production is associated with low-avidity, poorly cytotoxic T cell induction that contributes to viral immune evasion and the failure of T cell-based vaccines. Yet, the precise mechanisms that regulate IL-4 signalling in T cells remain elusive. Mounting evidence indicates that cells can dynamically alter their IL-4/IL-13 receptor signature to modulate downstream immune outcomes upon pathogen encounter. Here, we describe how naïve (CD62L+CD44lo–mid) CD4 and CD8 T cells distinctly engage both STAT6 and STAT3 in response to IL-4. We further show that IL-4R⍺ expression is both time- and IL-4 concentration-dependent. Remarkably, our findings reveal that STAT3 inhibition can ablate IL-4R⍺ and affect transcriptional expression of other Stat and Jak family members. By extension, the loss of STAT3 lead to aberrant STAT6 phosphorylation, revealing an inter-regulatory relationship between the two transcription factors. Moreover, IL-4 stimulation down-regulated TGF-β1 and IFN-γR1 expression on naïve T cells, possibly signifying the broad regulatory implications of IL-4 in conditioning lineage commitment decisions during early infection. Surprisingly, naïve T cells were unresponsive to IL-13 stimulation, unlike dendritic cells. Collectively, these findings could be exploited to inform more efficacious vaccines, as well as design treatments against IL-4/IL-13-associated disease conditions.

Emerging roles of extracellular vesicles in COVID-19, a double-edged sword?

The sudden outbreak of SARS‐CoV‐2‐infected disease (COVID‐19), initiated from Wuhan, China, has rapidly grown into a global pandemic. Emerging evidence has implicated extracellular vesicles (EVs), a key intercellular communicator, in the pathogenesis and treatment of COVID‐19. In the pathogenesis of COVID‐19, cells that express ACE2 and CD9 can transfer these viral receptors to other cells via EVs, making recipient cells more susceptible for SARS‐CoV‐2 infection. Once infected, cells release EVs packaged with viral particles that further facilitate viral spreading and immune evasion, aggravating COVID‐19 and its complications. In contrast, EVs derived from stem cells, especially mesenchymal stromal/stem cells, alleviate severe inflammation (cytokine storm) and repair damaged lung cells in COVID‐19 by delivery of anti‐inflammatory molecules. These therapeutic beneficial EVs can also be engineered into drug delivery platforms or vaccines to fight against COVID‐19. Therefore, EVs from diverse sources exhibit distinct effects in regulating viral infection, immune response, and tissue damage/repair, functioning as a double‐edged sword in COVID‐19. Here, we summarize the recent progress in understanding the pathological roles of EVs in COVID‐19. A comprehensive discussion of the therapeutic effects/potentials of EVs is also provided.

Defects in the CD8+ T cell response to arthritogenic alphaviruses

Abstract Arthritogenic alphaviruses have infected millions of people globally. Following acute infection, symptoms of joint pain, swelling, and stiffness can last for months or even years, likely due to persistence of viral RNA and antigen, which also is seen in mice. Intriguingly, wild-type and CD8−/− mice show a similar lack of clearance of viral RNA. Despite this, a mechanism for this CD8+ T cell evasion has not been identified. We hypothesized that there are defects in the generation, function, and migration of the CD8+ T cells after infection with Ross River virus (RRV), a prototype, arthritogenic alphavirus. To study the antigen-specific response, we used a genetically modified RRV expressing the lymphocytic choriomeningitis virus (LCMV)-derived gp33 peptide. Compared to LCMV infection, in which CD8+ T cells are required for viral clearance, RRV infection generates fewer gp33-specific CD8+ T cells in the spleen and draining lymph node, especially at early times after infection. Antigen-specific CD8+ T cells from the draining lymph node at 5 days after RRV infection express much less granzyme B than in LCMV-infected mice. In vivo cytolysis assays showed a qualitative difference in CD8+ T cell killing of targets especially when variant peptides were used for pulsing. Finally, intravital imaging revealed that while both RRV and LCMV infection recruit CD8+ T cells and retain them in an antigen specific manner near infected cells, CD8+ T cells appear to have less contact with RRV than LCMV-infected cells. Identification of a specific defect in the CD8+ T cell response to arthritogenic alphaviruses would increase our understanding of viral immune evasion and provide a target for therapy of these debilitating infections.

Risk of rapid evolutionary escape from biomedical interventions targeting SARS-CoV-2 spike protein.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is the molecular target for many vaccines and antibody-based prophylactics aimed at bringing COVID-19 under control. Such a narrow molecular focus raises the specter of viral immune evasion as a potential failure mode for these biomedical interventions. With the emergence of new strains of SARS-CoV-2 with altered transmissibility and immune evasion potential, a critical question is this: how easily can the virus escape neutralizing antibodies (nAbs) targeting the spike RBD? To answer this question, we combined an analysis of the RBD structure-function with an evolutionary modeling framework. Our structure-function analysis revealed that epitopes for RBD-targeting nAbs overlap one another substantially and can be evaded by escape mutants with ACE2 affinities comparable to the wild type, that are observed in sequence surveillance data and infect cells in vitro. This suggests that the fitness cost of nAb-evading mutations is low. We then used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to vaccines or other prophylactics that rely on one or two antibodies for protection can develop quickly -and repeatedly- under positive selection. Predicted resistance timelines are comparable to those of the decay kinetics of nAbs raised against vaccinal or natural antigens, raising a second potential mechanism for loss of immunity in the population. Strategies for viral elimination should therefore be diversified across molecular targets and therapeutic modalities.

Cleavage of TANK-Binding Kinase 1 by HIV-1 Protease Triggers Viral Innate Immune Evasion.

Type-I interferons (IFN-I) are the innate immune system’s principal defense against viral infections. Human immunodeficiency virus-1 (HIV-1) has evolved several ways to suppress or evade the host’s innate immunity in order to survive and replicate to sustain infection. Suppression of IFN-I is one among the multiple escape strategies used by HIV-1 to prevent its clearance. HIV-1 protease which helps in viral maturation has also been observed to cleave host cellular protein kinases. In this study we performed a comprehensive screening of a human kinase library using AlphaScreen assay and identified that TANK binding kinase-1 (TBK1) was cleaved by HIV-1 protease (PR). We demonstrate that PR cleaved TBK1 fails to phosphorylate IFN regulatory factor 3 (IRF3), thereby reducing the IFN-I promoter activity and further reveal that the PR mediated suppression of IFN-I could be counteracted by protease inhibitors (PI) in vitro. We have also revealed that mutations of HIV-1 PR that confer drug resistance to PIs reduce the enzyme’s ability to cleave TBK1. The findings of this study unearth a direct link between HIV-1 PR activity and evasion of innate immunity by the virus, the possible physiological relevance of which warrants to be determined.

Direct Priming of CD8+ T Cells Persists in the Face of Cowpox Virus Inhibitors of Antigen Presentation.

Vaccinia virus (VACV) was the vaccine used to eradicate smallpox and is being repurposed as a vaccine vector. CD8+ T cells are key anti-viral mediators, but require priming to become effector or memory cells. Priming requires an interaction with dendritic cells that are either infected (direct priming), or that have acquired virus proteins but remain uninfected (cross priming). To investigate CD8+ T cell priming pathways for VACV, we engineered the virus to express CPXV12 and CPXV203, two inhibitors of antigen presentation encoded by cowpox virus. These intracellular proteins would be expected to block direct but not cross priming. The inhibitors had diverse impacts on the size of anti-VACV CD8+ T cell responses across epitopes and by different infection routes in mice, superficially suggesting variable use of direct and cross priming. However, when we then tested a form of antigen that requires direct priming, we found surprisingly that CD8+ T cell responses were not diminished by co-expression with CPXV12 and CPXV203. We then directly quantified the impact of CPXV12 and CPXV203 on viral antigen presentation using mass spectrometry, which revealed strong, but incomplete inhibition of antigen presentation by the CPXV proteins. Therefore, direct priming of CD8+ T cells by poxviruses is robust enough to withstand highly potent viral inhibitors of antigen presentation. This is a reminder of the limits of viral immune evasion and shows that viral inhibitors of antigen presentation cannot be assumed to dissect cleanly direct and cross priming of anti-viral CD8+ T cells.ImportanceCD8+ T cells are key to anti-viral immunity, so it is important to understand how they are activated. Many viruses have proteins that protect infected cells from T cell attack by interfering with the process that allows virus infection to be recognised by CD8+ T cells. It is thought that these proteins would also stop infected cells from activating T cells in the first place. However, we show here that this is not the case for two very powerful inhibitory proteins from cowpox virus. This demonstrates the flexibility and robustness of immune processes that turn on the immune responses required to fight infection.