MLN4924 Inhibits Defective Ribosomal Product Antigen Presentation Independently of Direct NEDDylation of Protein Antigens.

Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.

Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), the most severe pandemic in a century. The virus gains access to host cells when the viral spike protein (S-protein) binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interactions with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often, these cells only transiently express ACE2 proteins, and the levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bicistronic vector with an easy-to-quantify reporter protein, Thy1.1. We found that both the binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus are proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of Thy1.1. We also compared different ACE2 orthologs, which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein, while human ACE2 had the highest level detected, and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants' abilities to potentially infect different animals. IMPORTANCE SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here, we describe a method to generate cells stably expressing different orthologs of ACE2, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both the binding of the viral spike protein receptor-binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to the ACE2 levels at the cell surface. This method will allow the creation of a library of stably transfected cells expressing similar levels of different vertebrate ACE2 orthologs, which can be used repeatedly for identifying vertebrate species that may be susceptible to infection with SARS-CoV-2 and its many variants.

Population connectivity patterns of genetic diversity, immune responses and exposure to infectious pneumonia in a metapopulation of desert bighorn sheep.

Habitat fragmentation is an important driver of biodiversity loss and can be remediated through management actions aimed at maintenance of natural connectivity in metapopulations. Connectivity may protect populations from infectious diseases by preserving immunogenetic diversity and disease resistance. However, connectivity could exacerbate the risk of infectious disease spread across vulnerable populations. We tracked the spread of a novel strain of Mycoplasma ovipneumoniae in a metapopulation of desert bighorn sheep Ovis canadensis nelsoni in the Mojave Desert to investigate how variation in connectivity among populations influenced disease outcomes. M. ovipneumoniae was detected throughout the metapopulation, indicating that the relative isolation of many of these populations did not protect them from pathogen invasion. However, we show that connectivity among bighorn sheep populations was correlated with higher immunogenetic diversity, a protective immune response and lower disease prevalence. Variation in protective immunity predicted infection risk in individual bighorn sheep and was associated with heterozygosity at genetic loci linked to adaptive and innate immune signalling. Together, these findings may indicate that population connectivity maintains immunogenetic diversity in bighorn sheep populations in this system and has direct effects on immune responses in individual bighorn sheep and their susceptibility to infection by a deadly pathogen. Our study suggests that the genetic benefits of population connectivity could outweigh the risk of infectious disease spread and supports conservation management that maintains natural connectivity in metapopulations.

Chlamydia Lipooligosaccharide Has Varied Direct and Indirect Roles in Evading both Innate and Adaptive Host Immune Responses.

Chlamydia bacteria are obligate intracellular pathogens which can cause a variety of disease in humans and other vertebrate animals. To successfully complete its life cycle, Chlamydia must evade both intracellular innate immune responses and adaptive cytotoxic T cell responses. Here, we report on the role of the chlamydial lipooligosaccharide (LOS) in evading the immune response. Chlamydia infection is known to block the induction of apoptosis. However, when LOS synthesis was inhibited during Chlamydia trachomatis infection, HeLa cells regained susceptibility to apoptosis induction following staurosporine treatment. Additionally, the delivery of purified LOS to the cytosol of cells increased the levels of the antiapoptotic protein survivin. An increase in survivin levels was also detected following C. trachomatis infection, which was reversed by blocking LOS synthesis. Interestingly, while intracellular delivery of lipopolysaccharide (LPS) derived from Escherichia coli was toxic to cells, LOS from C. trachomatis did not induce any appreciable cell death, suggesting that it does not activate pyroptosis. Chlamydial LOS was also a poor stimulator of maturation of bone marrow-derived dendritic cells compared to E. coli LPS. Previous work from our group indicated that LOS synthesis during infection was necessary to alter host cell antigen presentation. However, direct delivery of LOS to cells in the absence of infection did not alter antigenic peptide presentation. Taken together, these data suggest that chlamydial LOS, which is remarkably conserved across the genus Chlamydia, may act both directly and indirectly to allow the pathogen to evade the innate and adaptive immune responses of the host.

Inhibition of the Deubiquitinase Usp14 Diminishes Direct MHC Class I Antigen Presentation.

Infected or transformed cells must present peptides derived from endogenous proteins on MHC class I molecules to be recognized and targeted for elimination by Ag-specific cytotoxic T cells. In the first step of peptide generation, proteins are degraded by the proteasome. In this study, we investigated the role of the ubiquitin-specific protease 14 (Usp14), a proteasome-associated deubiquitinase, in direct Ag presentation using a ligand-stabilized model protein expressed as a self-antigen. Chemical inhibition of Usp14 diminished direct presentation of the model antigenic peptide, and the effect was especially pronounced when presentation was restricted to the defective ribosomal product (DRiP) form of the protein. Additionally, presentation specifically from DRiP Ags was diminished by expression of a catalytically inactive form of Usp14. Usp14 inhibition did not appreciably alter protein synthesis and only partially delayed protein degradation as measured by a slight increase in the half-life of the model protein when its degradation was induced. Taken together, these data indicate that functional Usp14 enhances direct Ag presentation, preferentially of DRiP-derived peptides, suggesting that the processing of DRiPs is in some ways different from other forms of Ag.

Chlamydia spp. development is differentially altered by treatment with the LpxC inhibitor LPC-011.

BACKGROUND: Chlamydia species are obligate intracellular bacteria that infect a broad range of mammalian hosts. Members of related genera are pathogens of a variety of vertebrate and invertebrate species. Despite the diversity of Chlamydia, all species contain an outer membrane lipooligosaccharide (LOS) that is comprised of a genus-conserved, and genus-defining, trisaccharide 3-deoxy-D-manno-oct-2-ulosonic acid Kdo region. Recent studies with lipopolysaccharide inhibitors demonstrate that LOS is important for the C. trachomatis developmental cycle during RB- > EB differentiation. Here, we explore the effects of one of these inhibitors, LPC-011, on the developmental cycle of five chlamydial species. RESULTS: Sensitivity to the drug varied in some of the species and was conserved between others. We observed that inhibition of LOS biosynthesis in some chlamydial species induced formation of aberrant reticulate bodies, while in other species, no change was observed to the reticulate body. However, loss of LOS production prevented completion of the chlamydial reproductive cycle in all species tested. In previous studies we found that C. trachomatis and C. caviae infection enhances MHC class I antigen presentation of a model self-peptide. We find that treatment with LPC-011 prevents enhanced host-peptide presentation induced by infection with all chlamydial-species tested. CONCLUSIONS: The data demonstrate that LOS synthesis is necessary for production of infectious progeny and inhibition of LOS synthesis induces aberrancy in certain chlamydial species, which has important implications for the use of LOS synthesis inhibitors as potential antibiotics.

Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection.

The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8(+) cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8(+) T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8(+) killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins.

Innate and adaptive immune responses in migrating spring-run adult chinook salmon, Oncorhynchus tshawytscha.

Adult Chinook salmon (Oncorhynchus tshawytscha) migrate from salt water to freshwater streams to spawn. Immune responses in migrating adult salmon are thought to diminish in the run up to spawning, though the exact mechanisms for diminished immune responses remain unknown. Here we examine both adaptive and innate immune responses as well as pathogen burdens in migrating adult Chinook salmon in the Upper Willamette River basin. Messenger RNA transcripts encoding antibody heavy chain molecules slightly diminish as a function of time, but are still present even after fish have successfully spawned. In contrast, the innate anti-bacterial effector proteins present in fish plasma rapidly decrease as spawning approaches. Fish also were examined for the presence and severity of eight different pathogens in different organs. While pathogen burden tended to increase during the migration, no specific pathogen signature was associated with diminished immune responses. Transcript levels of the immunosuppressive cytokines IL-10 and TGF beta were measured and did not change during the migration. These results suggest that loss of immune functions in adult migrating salmon are not due to pathogen infection or cytokine-mediated immune suppression, but is rather part of the life history of Chinook salmon likely induced by diminished energy reserves or hormonal changes which accompany spawning.

Identification of B cells as a major site for cyprinid herpesvirus 3 latency.

UNLABELLED: Cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV), is a member of the Alloherpesviridae, and is a recently discovered emerging herpesvirus that is highly pathogenic for koi and common carp. Our previous study demonstrated that CyHV-3 becomes latent in peripheral white blood cells (WBC). In this study, CyHV-3 latency was further investigated in IgM(+) WBC. The presence of the CyHV-3 genome in IgM(+) WBC was about 20-fold greater than in IgM(-) WBC. To determine whether CyHV-3 expressed genes during latency, transcription from all eight open reading frames (ORFs) in the terminal repeat was investigated in IgM(+) WBC from koi with latent CyHV-3 infection. Only a spliced ORF6 transcript was found to be abundantly expressed in IgM(+) WBC from CyHV-3 latently infected koi. The spliced ORF6 transcript was also detected in vitro during productive infection as early as 1 day postinfection. The ORF6 transcript from in vitro infection begins at -127 bp upstream of the ATG codon and ends +188 bp downstream of the stop codon, +20 bp downstream of the polyadenylation signal. The hypothetical protein of ORF6 contains a consensus sequence with homology to a conserved domain of EBNA-3B and ICP4 from Epstein-Barr virus and herpes simplex virus 1, respectively, both members of the Herpesviridae. This is the first report of latent CyHV-3 in B cells and identification of gene transcription during latency for a member of the Alloherpesviridae. IMPORTANCE: This is the first demonstration that a member of the Alloherpesviridae, cyprinid herpesvirus 3 (CyHV-3), establishes a latent infection in the B cells of its host, Cyprinus carpio. In addition, this is the first report of identification of gene transcription during latency for a member of Herpesvirales outside Herpesviridae. This is also the first report that the hypothetical protein of latent transcript of CyHV-3 contains a consensus sequence with homology to a conserved domain of EBNA-3B from Epstein-Barr virus and ICP4 from herpes simplex virus 1, which are genes important for latency. These strongly suggest that latency is evolutionally conserved across vertebrates.

MHC class I antigen processing distinguishes endogenous antigens based on their translation from cellular vs. viral mRNA.

To better understand the generation of MHC class I-associated peptides, we used a model antigenic protein whose proteasome-mediated degradation is rapidly and reversibly controlled by Shield-1, a cell-permeant drug. When expressed from a stably transfected gene, the efficiency of antigen presentation is ~2%, that is, one cell-surface MHC class I-peptide complex is generated for every 50 folded source proteins degraded upon Shield-1 withdrawal. By contrast, when the same protein is expressed by vaccinia virus, its antigen presentation efficiency is reduced ~10-fold to values similar to those reported for other vaccinia virus-encoded model antigens. Virus infection per se does not modify the efficiency of antigen processing. Rather, the efficiency difference between cellular and virus-encoded antigens is based on whether the antigen is synthesized from transgene- vs. virus-encoded mRNA. Thus, class I antigen-processing machinery can distinguish folded proteins based on the precise details of their synthesis to modulate antigen presentation efficiency.

Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.

Sensitivity is essential in CD8+ T-cell killing of virus-infected cells and tumor cells. Although the affinity of the T-cell receptor (TCR) for antigen is relatively low, the avidity of T cell-antigen-presenting cell interactions is greatly enhanced by increasing the valence of the interaction. It is known that TCRs cluster into protein islands after engaging their cognate antigen (peptides bound to MHC molecules). Here, we show that mouse K(b) class I molecules segregate into preformed, long-lasting (hours) clusters on the antigen-presenting cell surface based on their bound viral peptide. Peptide-specific K(b) clustering occurs when source antigens are expressed by vaccinia or vesicular stomatitis virus, either as proteasome-liberated precursors or free intracellular peptides. By contrast, K(b)-peptide complexes generated by incubating cells with synthetic peptides are extensively intermingled on the cell surface. Peptide-specific complex sorting is first detected in the Golgi complex, and compromised by removing the K(b) cytoplasmic tail. Peptide-specific clustering is associated with increased T-cell sensitivity: on a per-complex basis, endogenous SIINFEKL activates T cells more efficiently than synthetic SIINFEKL, and wild-type K(b) presents endogenous SIINFEKL more efficiently than tailless K(b). We propose that endogenous processing generates peptide-specific clusters of class I molecules to maximize the sensitivity and speed of T-cell immunosurveillance.

Distinct pathways generate peptides from defective ribosomal products for CD8+ T cell immunosurveillance.

To understand better the endogenous sources of MHC class I peptide ligands, we generated an antigenic reporter protein whose degradation is rapidly and reversibly controlled with Shield-1, a cell-permeant drug. Using this system, we demonstrate that defective ribosomal products (DRiPs) represent a major and highly efficient source of peptides and are completely resistant to our attempts to stabilize the protein. Although peptides also derive from nascent Shield-1-sensitive proteins and "retirees" created by Shield-1 withdrawal, these are much less efficient sources on a molar basis. We use this system to identify two drugs--each known to inhibit polyubiquitin chain disassembly--that selectively inhibit presentation of Shield-1-resistant DRiPs. These findings provide the initial evidence for distinct biochemical pathways for presentation of DRiPs versus retirees and implicate polyubiquitin chain disassembly or the actions of deubiquitylating enzymes as playing an important role in DRiP presentation.

Compartmentalized MHC class I antigen processing enhances immunosurveillance by circumventing the law of mass action.

MHC class I molecules function to display peptides generated from cellular and pathogen gene products for immune surveillance by CD8(+) T cells. Cells typically express approximately 100,000 class I molecules, or approximately 1 per 30,000 cellular proteins. Given "one protein, one peptide" representation, immunosurveillance would be heavily biased toward the most abundant cell proteins. Cells use several mechanisms to prevent this, including the predominant use of defective ribosomal products (DRiPs) to generate peptides from nascent proteins and, as we show here, compartmentalization of DRiP peptide generation to prevent competition from abundant cytosolic peptides. This provides an explanation for the exquisite ability of T cells to recognize peptides generated from otherwise undetected gene products.

Potential Promises and Perils of Human Biological Treatments for Immunotherapy in Veterinary Oncology.

The emergence of immunotherapy for the treatment of human cancers has heralded a new era in oncology, one that is making its way into the veterinary clinic. As the immune system of many animal species commonly seen by veterinarians is similar to humans, there is great hope for the translation of human therapies into veterinary oncology. The simplest approach for veterinarians would be to adopt existing reagents that have been developed for human medicine, due to the potential of reduced cost and the time it takes to develop a new drug. However, this strategy may not always prove to be effective and safe with regard to certain drug platforms. Here, we review current therapeutic strategies that could exploit human reagents in veterinary medicine and also those therapies which may prove detrimental when human-specific biological molecules are used in veterinary oncology. In keeping with a One Health framework, we also discuss the potential use of single-domain antibodies (sdAbs) derived from camelid species (also known as Nanobodies™) for therapies targeting multiple veterinary animal patients without the need for species-specific reformulation. Such reagents would not only benefit the health of our veterinary species but could also guide human medicine by studying the effects of outbred animals that develop spontaneous tumors, a more relevant model of human diseases compared to traditional laboratory rodent models.

RNA polymerase II inhibitors dissociate antigenic peptide generation from normal viral protein synthesis: a role for nuclear translation in defective ribosomal product synthesis?

Following viral infection, cells rapidly present peptides from newly synthesized viral proteins on MHC class I molecules, likely from rapidly degraded forms of nascent proteins. The nature of these defective ribosomal products (DRiPs) remains largely undefined. Using inhibitors of RNA polymerase II that block influenza A virus neuraminidase (NA) mRNA export from the nucleus and inhibit cytoplasmic NA translation, we demonstrate a surprising disconnect between levels of NA translation and generation of SIINFEKL peptide genetically inserted into the NA stalk. A 33-fold reduction in NA expression is accompanied by only a 5-fold reduction in K(b)-SIINFEKL complex cell-surface expression, resulting in a net 6-fold increase in the overall efficiency of Ag presentation. Although the proteasome inhibitor MG132 completely blocked K(b)-SIINFEKL complex generation, we were unable to biochemically detect a MG132-dependent cohort of NA DRiPs relevant for Ag processing, suggesting that a minute population of DRiPs is a highly efficient source of antigenic peptides. These data support the idea that Ag processing uses compartmentalized translation, perhaps even in the nucleus itself, to increase the efficiency of the generation of class I peptide ligands.

Defective ribosomal products are the major source of antigenic peptides endogenously generated from influenza A virus neuraminidase.

The defective ribosomal product (DRiP) hypothesis of endogenous Ag processing posits that rapidly degraded forms of nascent proteins are a major source of peptide ligands for MHC class I molecules. Although there is broad experimental support for the DRiP hypothesis, careful kinetic analysis of the generation of defined peptide class I complexes has been limited to studies of recombinant vaccinia viruses expressing genes derived from other organisms. In this study, we show that insertion of the SIINFEKL peptide into the stalk of influenza A virus neuraminidase (NA) does not detectably modify NA folding, degradation, transport, or sp. act. when expressed in its natural context of influenza A virus infection. Using the 25-D1.16 mAb specific for K(b)-SIINFEKL to precisely quantitate cell surface complexes by flow cytometry, we demonstrate that SIINFEKL is generated in complete lockstep with initiation and abrogation of NA biosynthesis in both L-K(b) fibroblast cells and DC2.4 dendritic/monocyte cells. SIINFEKL presentation requires active proteasomes and TAP, consistent with its generation from a cytosolic DRiP pool. From the difference in the shutoff kinetics of K(b)-SIINFEKL complex expression following protein synthesis versus proteasome inhibition, we estimate that the t(1/2) of the biosynthetic source of NA peptide is approximately 5 min. These observations extend the relevance of the DRiP hypothesis to viral proteins generated in their natural context.

Unexpected role for the immunoproteasome subunit LMP2 in antiviral humoral and innate immune responses.

Proteasomes are multisubunit proteases that initiate degradation of many Ags presented by MHC class I molecules. Vertebrates express alternate forms of each of the three catalytic proteasome subunits: standard subunits, and immunosubunits, which are constitutively expressed by APCs and are induced in other cell types by exposure to cytokines. The assembly of mixed proteasomes containing standard subunits and immunosubunits is regulated in a tissue specific manner. In this study, we report that the presence of mixed proteasomes in immune cells in LMP2(-/-) mice compromises multiple components that contribute to the generation of antiviral Ab responses, including splenic B cell numbers, survival and function of adoptively transferred B cells, Th cell function, and dendritic cell secretion of IL-6, TNF-alpha, IL-1beta, and type I IFNs. These defects did not result from compromised overall protein degradation, rather they were associated with altered NF-kappaB activity. These findings demonstrate an important role for immunoproteasomes in immune cell function beyond their contribution to Ag processing.

Translating DRiPs: progress in understanding viral and cellular sources of MHC class I peptide ligands.

It has been 15 years since we proposed the defective ribosomal product (DRiP) hypothesis to explain the rapid presentation of viral peptides by MHC class I molecules on the surface of infected cells. Here, we review the evidence for the contribution of DRiPs to antigen processing, pointing to the uncertainties regarding the physical nature of DRiPs, and emphasizing recent findings suggesting that peptide generation is a specialized process involving compartmentalized translation.

The Many Potential Fates of Non-Canonical Protein Substrates Subject to NEDDylation.

Neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) is a ubiquitin-like protein (UBL) whose canonical function involves binding to, and thus, activating Cullin-Ring finger Ligases (CRLs), one of the largest family of ubiquitin ligases in the eukaryotic cell. However, in recent years, several non-canonical protein substrates of NEDD8 have been identified. Here we attempt to review the recent literature regarding non-canonical NEDDylation of substrates with a particular focus on how the covalent modification of NEDD8 alters the protein substrate. Like much in the study of ubiquitin and UBLs, there are no clear and all-encompassing explanations to satisfy the textbooks. In some instances, NEDD8 modification appears to alter the substrates localization, particularly during times of stress. NEDDylation may also have conflicting impacts upon a protein's stability: some reports indicate NEDDylation may protect against degradation whereas others show NEDDylation can promote degradation. We also examine how many of the in vitro studies measuring non-canonical NEDDylation were conducted and compare those conditions to those which may occur in vivo, such as cancer progression. It is likely that the conditions used to study non-canonical NEDDylation are similar to some types of cancers, such as glioblastoma, colon and rectal cancers, and lung adenocarcinomas. Although the full outcomes of non-canonical NEDDylation remain unknown, our review of the literature suggests that researchers keep an open mind to the situations where this modification occurs and determine the functional impacts of NEDD8-modification to the specific substrates which they study.