The 21st Annual Meeting of the Rocky Mountain Virology Association.

Nestled within the Rocky Mountain National Forest, 114 scientists and students gathered at Colorado State University's Mountain Campus for this year's 21st annual Rocky Mountain National Virology Association meeting. This 3-day retreat consisted of 31 talks and 30 poster presentations discussing advances in research pertaining to viral and prion diseases. The keynote address provided a timely discussion on zoonotic coronaviruses, lessons learned, and the path forward towards predicting, preparing, and preventing future viral disease outbreaks. Other invited speakers discussed advances in SARS-CoV-2 surveillance, molecular interactions involved in flavivirus genome assembly, evaluation of ethnomedicines for their efficacy against infectious diseases, multi-omic analyses to define risk factors associated with long COVID, the role that interferon lambda plays in control of viral pathogenesis, cell-fusion-dependent pathogenesis of varicella zoster virus, and advances in the development of a vaccine platform against prion diseases. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations.

Innate immunity to prions: anti-prion systems turn a tsunami of prions into a slow drip.

The yeast prions (infectious proteins) [URE3] and [PSI+] are essentially non-functional (or even toxic) amyloid forms of Ure2p and Sup35p, whose normal function is in nitrogen catabolite repression and translation termination, respectively. Yeast has an array of systems working in normal cells that largely block infection with prions, block most prion formation, cure most nascent prions and mitigate the toxic effects of those prions that escape the first three types of systems. Here we review recent progress in defining these anti-prion systems, how they work and how they are regulated. Polymorphisms of the prion domains partially block infection with prions. Ribosome-associated chaperones ensure proper folding of nascent proteins, thus reducing [PSI+] prion formation and curing many [PSI+] variants that do form. Btn2p is a sequestering protein which gathers [URE3] amyloid filaments to one place in the cells so that the prion is often lost by progeny cells. Proteasome impairment produces massive overexpression of Btn2p and paralog Cur1p, resulting in [URE3] curing. Inversely, increased proteasome activity, by derepression of proteasome component gene transcription or by 60S ribosomal subunit gene mutation, prevents prion curing by Btn2p or Cur1p. The nonsense-mediated decay proteins (Upf1,2,3) cure many nascent [PSI+] variants by associating with Sup35p directly. Normal levels of the disaggregating chaperone Hsp104 can also cure many [PSI+] prion variants. By keeping the cellular levels of certain inositol polyphosphates / pyrophosphates low, Siw14p cures certain [PSI+] variants. It is hoped that exploration of the yeast innate immunity to prions will lead to discovery of similar systems in humans.

Vaccination with Prion Peptide-Displaying Polyomavirus-Like Particles Prolongs Incubation Time in Scrapie-Infected Mice.

Prion diseases like scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jakob disease (CJD) in humans are fatal neurodegenerative diseases characterized by the conformational conversion of the normal, mainly α-helical cellular prion protein (PrPC) into the abnormal ß-sheet rich infectious isoform PrPSc. Various therapeutic or prophylactic approaches have been conducted, but no approved therapeutic treatment is available so far. Immunisation against prions is hampered by the self-tolerance to PrPC in mammalian species. One strategy to avoid this tolerance is presenting PrP variants in virus-like particles (VLPs). Therefore, we vaccinated C57/BL6 mice with nine prion peptide variants presented by hamster polyomavirus capsid protein VP1/VP2-derived VLPs. Mice were subsequently challenged intraperitoneally with the murine RML prion strain. Importantly, one group exhibited significantly increased mean survival time of 240 days post-inoculation compared with 202 days of the control group. These data show that immunisation with VLPs presenting PrP peptides may represent a promising strategy for an effective vaccination against transmissible spongiform encephalitis agents.

The Allosteric Effect in Antibody-Antigen Recognition.

We studied the molecular details of the recognition of antigens by the variable domain of their cognate antibodies in as well as those elicited by the constant domains, which do not directly interact with antigens. Such effects are difficult to study experimentally; however, molecular dynamics simulations and subsequent residue interaction network analysis provide insight into the allosteric communication between the antigen-binding CDR region and the constant domain. We performed MD simulations of the complex of Fab and prion-associated peptide in the apo and bound forms and follow the conformational changes in the antibody and cross-talk between its subunits and with antigens. These protocols could be generally applied for studies of other antigens-antibody recognition systems.

The Effects of Immune System Modulation on Prion Disease Susceptibility and Pathogenesis.

Prion diseases are a unique group of infectious chronic neurodegenerative disorders to which there are no cures. Although prion infections do not stimulate adaptive immune responses in infected individuals, the actions of certain immune cell populations can have a significant impact on disease pathogenesis. After infection, the targeting of peripherally-acquired prions to specific immune cells in the secondary lymphoid organs (SLO), such as the lymph nodes and spleen, is essential for the efficient transmission of disease to the brain. Once the prions reach the brain, interactions with other immune cell populations can provide either host protection or accelerate the neurodegeneration. In this review, we provide a detailed account of how factors such as inflammation, ageing and pathogen co-infection can affect prion disease pathogenesis and susceptibility. For example, we discuss how changes to the abundance, function and activation status of specific immune cell populations can affect the transmission of prion diseases by peripheral routes. We also describe how the effects of systemic inflammation on certain glial cell subsets in the brains of infected individuals can accelerate the neurodegeneration. A detailed understanding of the factors that affect prion disease transmission and pathogenesis is essential for the development of novel intervention strategies.

Prions and Prion-like assemblies in neurodegeneration and immunity: The emergence of universal mechanisms across health and disease.

Prion-like behaviour is an abrupt process, an "all-or-nothing" transition between a monomeric species and an "infinite" fibrillated form. Once a nucleation point is formed, the process is unstoppable as fibrils self-propagate by recruiting and converting all monomers into the amyloid fold. After the "mad cow" episode, prion diseases have made the headlines, but more and more prion-like behaviours have emerged in neurodegenerative diseases, where formation of fibrils and large conglomerates of proteins deeply disrupt the cell homeostasis. More interestingly, in the last decade, examples emerged to suggest that prion-like conversion can be used as a positive gain of function, for memory storage or structural scaffolding. More recent experiments show that we are only seeing the tip of the iceberg and that, for example, prion-like amplification is found in many pathways of the immune response. In innate immunity, receptors on the cellular surface or within the cells 'sense' danger and propagate this information as signal, through protein-protein interactions (PPIs) between 'receptor', 'adaptor' and 'effector' proteins. In innate immunity, the smallest signal of a foreign element or pathogen needs to trigger a macroscopic signal output, and it was found that adaptor polymerize to create an extreme signal amplification. Interestingly, our body uses multiple structural motifs to create large signalling platform; a few innate proteins use amyloid scaffolds but most of the polymers discovered are composed by self-assembly in helical filaments. Some of these helical assemblies even have intercellular "contamination" in a "true" prion action, as demonstrated for ASC specks and MyD88 filaments. Here, we will describe the current knowledge in neurodegenerative diseases and innate immunity and show how these two very different fields can cross-seed discoveries.

Unaltered intravenous prion disease pathogenesis in the temporary absence of marginal zone B cells.

Prion diseases are a unique, infectious, neurodegenerative disorders that can affect animals and humans. Data from mouse transmissions show that efficient infection of the host after intravenous (IV) prion exposure is dependent upon the early accumulation and amplification of the prions on stromal follicular dendritic cells (FDC) in the B cell follicles. How infectious prions are initially conveyed from the blood-stream to the FDC in the spleen is uncertain. Addressing this issue is important as susceptibility to peripheral prion infections can be reduced by treatments that prevent the early accumulation of prions upon FDC. The marginal zone (MZ) in the spleen contains specialized subsets of B cells and macrophages that are positioned to continuously monitor the blood-stream and remove pathogens, toxins and apoptotic cells. The continual shuttling of MZ B cells between the MZ and the B-cell follicle enables them to efficiently capture and deliver blood-borne antigens and antigen-containing immune complexes to splenic FDC. We tested the hypothesis that MZ B cells also play a role in the initial shuttling of prions from the blood-stream to FDC. MZ B cells were temporarily depleted from the MZ by antibody-mediated blocking of integrin function. We show that depletion of MZ B cells around the time of IV prion exposure did not affect the early accumulation of blood-borne prions upon splenic FDC or reduce susceptibility to IV prion infection. In conclusion, our data suggest that the initial delivery of blood-borne prions to FDC in the spleen occurs independently of MZ B cells.

Pro-Inflammatory S100A9 Protein Aggregation Promoted by NCAM1 Peptide Constructs.

Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from Aß peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid self-assembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.

Unaltered prion disease in mice lacking developmental endothelial locus-1.

Progression of prion diseases is driven by the accumulation of prions in the brain. Ablation of microglia or deletion of the eat-me-signal, milk-fat globule epidermal growth factor VIII (Mfge8), accelerates prion pathogenesis, suggesting that microglia defend the brain by phagocytosing prions. Similar to Mfge8, developmental endothelial locus-1 (Del-1) is a secreted protein that acts as an opsonin bridging phagocytes and apoptotic cells to facilitate phagocytosis. We therefore asked whether Del-1 might play a role in controlling prion pathogenesis. We assessed the anti-inflammatory and phagocytosis-promoting functions of Del-1 in prion disease and determined whether Del-1 complements Mfge8 in prion clearance in mice with a C57BL/6J genetic background. We found that Del-1 deficiency did not change prion disease progression or lesion patterns. In addition, prion clearance and scrapie prion protein deposition were unaltered in Del-1-deficient mice. In addition, prion-induced neuroinflammation was not affected by Del-1 deficiency. We conclude that Del-1 is not a major determinant of prion pathogenesis in this context.

Neuroinflammation, Microglia, and Cell-Association during Prion Disease.

Prion disorders are transmissible diseases caused by a proteinaceous infectious agent that can infect the lymphatic and nervous systems. The clinical features of prion diseases can vary, but common hallmarks in the central nervous system (CNS) are deposition of abnormally folded protease-resistant prion protein (PrPres or PrPSc), astrogliosis, microgliosis, and neurodegeneration. Numerous proinflammatory effectors expressed by astrocytes and microglia are increased in the brain during prion infection, with many of them potentially damaging to neurons when chronically upregulated. Microglia are important first responders to foreign agents and damaged cells in the CNS, but these immune-like cells also serve many essential functions in the healthy CNS. Our current understanding is that microglia are beneficial during prion infection and critical to host defense against prion disease. Studies indicate that reduction of the microglial population accelerates disease and increases PrPSc burden in the CNS. Thus, microglia are unlikely to be a foci of prion propagation in the brain. In contrast, neurons and astrocytes are known to be involved in prion replication and spread. Moreover, certain astrocytes, such as A1 reactive astrocytes, have proven neurotoxic in other neurodegenerative diseases, and thus might also influence the progression of prion-associated neurodegeneration.

A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity.

Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy.

Prolonged follicular helper T cell responses in ME7 scrapie-infected mice.

We previously reported that mice intracerebrally inoculated with the mouse-adapted scrapie strain ME7 have markedly diminished T zones in the spleen due to the decreased expression of CCL19 and CCL21. In addition, follicular dendritic cell networks in germinal centers were larger in ME7-infected spleens compared to uninfected spleens. As an extension of that study, we set out to determine how ME7 infection affects spleen structure and follicular helper T (Tfh) cell responses in mice. For this study, mice were intraperitoneally inoculated with brain homogenate of the ME7 inoculum and spleens were analyzed 50, 130, and 200 days after inoculation and compared with those from uninfected mice. The result showed that ME7- infected mice had increased Tfh cell responses which were maintained until end-stage prion disease. Although CD4 T cells decreased in white pulps, they increased in germinal centers, and expressed higher levels of the Tfh-related genes, such as Bcl6, Il21, Cxcr5, Icos, and Pdcd1. In addition, ME7-infected spleens had increased numbers of CD4 memory T cells. These data indicate that although ME7 infection led to impaired splenic white pulp structure, CD4 memory T cells were increased and Tfh cell responses were required and prolonged to provide help for the replication and accumulation of pathogenic prion protein in germinal centers.

Enhanced detection of infectious prions by direct ELISA from the brains of asymptomatic animals using DRM2-118 monoclonal antibody and Gdn-HCl.

In this report we describe the use of a novel anti-prion monoclonal antibody (DRM2-118) for the direct detection of infectious prions by ELISA. Epitope mapping using overlapping hamster (SHa) prion peptides indicates DRM2-118 binding occurs between residues 93-100 and at the 310-helix (residues 163-170) between alpha helix-A and -B. This antibody shows broad species binding to endogenous prions from brain homogenates and corresponding recombinant prion proteins. To evaluate the performance of this MAb for the detection of prion proteins we performed an animal time course and evaluated prion detection from both crude brain homogenates and lipid raft fractions (DRM) by direct ELISA. Prion detection was significantly enhanced by the addition of the chaotropic guanidine-HCl (Gdn-HCl) during protein immobilization with detection of PK-resistant prion from asymptomatic animal brains at (45-DPI) and from lipid rafts at (24-DPI). Our data demonstrates enhanced prion detection from brain lipid rafts of asymptomatic animals by a simple direct ELISA using the DRM2-118 MAb combined with Gdn-HCl.

Induction of PrPSc-specific systemic and mucosal immune responses in white-tailed deer with an oral vaccine for chronic wasting disease.

The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on disease-associated misfolding of PrPC into PrPSc. Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrPSc-specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrPSc-specific oral vaccine for CWD as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines.

Complement Regulatory Protein Factor H Is a Soluble Prion Receptor That Potentiates Peripheral Prion Pathogenesis.

Several complement proteins exacerbate prion disease, including C3, C1q, and CD21/35. These proteins of the complement cascade likely increase uptake, trafficking, and retention of prions in the lymphoreticular system, hallmark sites of early prion propagation. Complement regulatory protein factor H (fH) binds modified host proteins and lipids to prevent C3b deposition and, thus, autoimmune cell lysis. Previous reports show that fH binds various conformations of the cellular prion protein, leading us to question the role of fH in prion disease. In this article, we report that transgenic mice lacking Cfh alleles exhibit delayed peripheral prion accumulation, replication, and pathogenesis and onset of terminal disease in a gene-dose manner. We also report a biophysical interaction between purified fH and prion rods enriched from prion-diseased brain. fH also influences prion deposition in brains of infected mice. We conclude from these data and previous findings that the interplay between complement and prions likely involves a complex balance of prion sequestration and destruction via local tissue macrophages, prion trafficking by B and dendritic cells within the lymphoreticular system, intranodal prion replication by B and follicular dendritic cells, and potential prion strain selection by CD21/35 and fH. These findings reveal a novel role for complement-regulatory proteins in prion disease.

The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.

The signaling adaptor MAVS forms prion-like aggregates to activate an innate antiviral immune response after viral infection. However, the molecular mechanisms that regulate MAVS aggregation are poorly understood. Here we identified TRIM31, an E3 ubiquitin ligase of the TRIM family of proteins, as a regulator of MAVS aggregation. TRIM31 was recruited to mitochondria after viral infection and specifically regulated antiviral signaling mediated by RLR pattern-recognition receptors. TRIM31-deficient mice were more susceptible to infection with RNA virus than were wild-type mice. TRIM31 interacted with MAVS and catalyzed the Lys63 (K63)-linked polyubiquitination of Lys10, Lys311 and Lys461 on MAVS. This modification promoted the formation of prion-like aggregates of MAVS after viral infection. Our findings reveal new insights in the molecular regulation of MAVS aggregation and the cellular antiviral response through TRIM31-mediated K63-linked polyubiquitination of MAVS.

Activation of microglia by retroviral infection correlates with transient clearance of prions from the brain but does not change incubation time.

Prion diseases are fatal transmissible diseases, where conversion of the endogenous prion protein (PrPC ) into a misfolded isoform (PrPSc ) leads to neurodegeneration. Microglia, the immune cells of the brain, are activated in neurodegenerative disorders including prion diseases; however, their impact on prion disease pathophysiology is unclear with both beneficial PrPSc -clearing and detrimental potentially neurotoxic effects. Moreover, monocytes entering the brain from the periphery during disease course might add to disease pathophysiology. Here, the degree of microglia activation in the brain of prion infected mice with and without an additional intraperitoneal retrovirus infection was studied. Peripheral murine retrovirus infection leads to activation of parenchymal microglia without recruitment of monocytes. This activation correlated with transient clearance or delay in accumulation of infectious prions specifically from the brain at early time points in the diseases course. Microglia expression profiling showed upregulation of genes involved in protein degradation coinciding with prion clearance. This enforces a concept where microglia act beneficial in prion disease if adequately activated. Once microglia activation has ceased, prion disease reemerges leading to disease kinetics undistinguishable from the situation in prion-only infected mice. This might be caused by the loss of microglial homeostatic function at clinical prion disease.

The Anti-Prion Antibody 15B3 Detects Toxic Amyloid-ß Oligomers.

15B3 is a monoclonal IgM antibody that selectively detects pathological aggregates of the prion protein (PrP). We report the unexpected finding that 15B3 also recognizes oligomeric but not monomeric forms of amyloid-ß (Aß)42, an aggregating peptide implicated in the pathogenesis of Alzheimer's disease (AD). The 15B3 antibody: i) inhibits the binding of synthetic Aß42 oligomers to recombinant PrP and neuronal membranes; ii) prevents oligomer-induced membrane depolarization; iii) antagonizes the inhibitory effects of oligomers on the physiological pharyngeal contractions of the nematode Caenorhabditis elegans; and iv) counteracts the memory deficits induced by intracerebroventricular injection of Aß42 oligomers in mice. Thus this antibody binds to pathologically relevant forms of Aß, and offers a potential research, diagnostic, and therapeutic tool for AD.

Complement factors alter the amount of PrP(Sc) in primary-cultured mouse cortical neurons associated with increased membrane permeability.

We examined the effects of complement factors on primary-cultured neurons infected with prions. The amount of protease K (PK)-resistant abnormal form of prion protein (PrP(Sc)) reached a maximum level at 12 and 16 days post exposure (dpe) in 22L- and Chandler-infected neurons, respectively. In Chandler-infected neurons, the reaction of complement factors C1q, C3 and C9 significantly increased membrane permeability. This was followed by a decrease of PK-resistant PrP(Sc) at 16 and 20dpe. In contrast, in 22L-infected neurons, the effects of complement factors were observed at 12 and 16dpe, but not at 20dpe. Membrane permeability also increased in 22L-infected neurons by reaction of complement factor C3, but interestingly, the amount of PK-resistant PrP(Sc) initially decreased, and then increased. These results suggest that the reactivity of complement factors in prion-infected neurons depends on the amount of PrP(Sc) and the prion strain.

TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion.

Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. Misfolded human wild-type (HuWt) SOD1 has been detected in both familial and sporadic ALS patients, despite mutations in SOD1 accounting for only 2% of total cases. We previously showed that accumulation of pathological TDP-43 or FUS coexist with misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells. Here, we used immunocytochemistry and immunoprecipitation to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. In this system, intercellular spread of SOD1 misfolding is not accompanied by transmission of TDP-43 or FUS pathology. Our findings argue that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of propagated misfolding of SOD1.