Distinct Cellular Tropism and Immune Responses to Alphavirus Infection.

Alphaviruses are emerging and reemerging viruses that cause disease syndromes ranging from incapacitating arthritis to potentially fatal encephalitis. While infection by arthritogenic and encephalitic alphaviruses results in distinct clinical manifestations, both virus groups induce robust innate and adaptive immune responses. However, differences in cellular tropism, type I interferon induction, immune cell recruitment, and B and T cell responses result in differential disease progression and outcome. In this review, we discuss aspects of immune responses that contribute to protective or pathogenic outcomes after alphavirus infection.

Structural and functional basis of VLDLR usage by Eastern equine encephalitis virus.

The very-low-density lipoprotein receptor (VLDLR) comprises eight LDLR type A (LA) domains and supports entry of distantly related alphaviruses, including Eastern equine encephalitis virus (EEEV) and Semliki Forest virus (SFV). Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage more than one LA domain simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection. Whereas all EEEV strains show conservation of two VLDLR-binding sites, the EEEV PE-6 strain and a few other EEE complex members feature a single amino acid substitution that enables binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.

Vertebrate-class-specific binding modes of the alphavirus receptor MXRA8.

MXRA8 is a receptor for chikungunya (CHIKV) and other arthritogenic alphaviruses with mammalian hosts. However, mammalian MXRA8 does not bind to alphaviruses that infect humans and have avian reservoirs. Here, we show that avian, but not mammalian, MXRA8 can act as a receptor for Sindbis, western equine encephalitis (WEEV), and related alphaviruses with avian reservoirs. Structural analysis of duck MXRA8 complexed with WEEV reveals an inverted binding mode compared with mammalian MXRA8 bound to CHIKV. Whereas both domains of mammalian MXRA8 bind CHIKV E1 and E2, only domain 1 of avian MXRA8 engages WEEV E1, and no appreciable contacts are made with WEEV E2. Using these results, we generated a chimeric avian-mammalian MXRA8 decoy-receptor that neutralizes infection of multiple alphaviruses from distinct antigenic groups in vitro and in vivo. Thus, different alphaviruses can bind MXRA8 encoded by different vertebrate classes with distinct engagement modes, which enables development of broad-spectrum inhibitors.

Vaccine elicitation and structural basis for antibody protection against alphaviruses.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

Structure of Semliki Forest virus in complex with its receptor VLDLR.

Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378 Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions at multiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.

The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type I IFN Signaling Axis.

Chikungunya virus (CHIKV), an emerging alphavirus, has infected millions of people. However, the factors modulating disease outcome remain poorly understood. Here, we show in germ-free mice or in oral antibiotic-treated conventionally housed mice with depleted intestinal microbiomes that greater CHIKV infection and spread occurs within 1 day of virus inoculation. Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN). Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, can restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission.

Molecular Basis of Arthritogenic Alphavirus Receptor MXRA8 Binding to Chikungunya Virus Envelope Protein.

Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaviruses.

Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor.

Mxra8 is a receptor for multiple arthritogenic alphaviruses that cause debilitating acute and chronic musculoskeletal disease in humans. Herein, we present a 2.2 Å resolution X-ray crystal structure of Mxra8 and 4 to 5 Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to chikungunya (CHIKV) virus-like particles and infectious virus. The Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement. Mxra8 binds by wedging into a cleft created by two adjacent CHIKV E2-E1 heterodimers in one trimeric spike and engaging a neighboring spike. Two binding modes are observed with the fully mature VLP, with one Mxra8 binding with unique contacts. Only the high-affinity binding mode was observed in the complex with infectious CHIKV, as viral maturation and E3 occupancy appear to influence receptor binding-site usage. Our studies provide insight into how Mxra8 binds CHIKV and creates a path for developing alphavirus entry inhibitors.

The RNA helicase DDX39A binds a conserved structure in chikungunya virus RNA to control infection.

Alphaviruses are a large group of re-emerging arthropod-borne RNA viruses. The compact viral RNA genomes harbor diverse structures that facilitate replication. These structures can be recognized by antiviral cellular RNA-binding proteins, including DExD-box (DDX) helicases, that bind viral RNAs to control infection. The full spectrum of antiviral DDXs and the structures that are recognized remain unclear. Genetic screening identified DDX39A as antiviral against the alphavirus chikungunya virus (CHIKV) and other medically relevant alphaviruses. Upon infection, the predominantly nuclear DDX39A accumulates in the cytoplasm inhibiting alphavirus replication, independent of the canonical interferon pathway. Biochemically, DDX39A binds to CHIKV genomic RNA, interacting with the 5' conserved sequence element (5'CSE), which is essential for the antiviral activity of DDX39A. Altogether, DDX39A relocalization and binding to a conserved structural element in the alphavirus genomic RNA attenuates infection, revealing a previously unknown layer to the cellular control of infection.

The lncRNA ALPHA specifically targets chikungunya virus to control infection.

Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O'nyong'nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.

Interaction of chikungunya virus glycoproteins with macrophage factors controls virion production.

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.

Positive-strand RNA virus genome replication organelles: structure, assembly, control.

Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.

The many ways in which alphaviruses bind to cells.

Only a subset of viruses can productively infect many different host species. Some arthropod-transmitted viruses, such as alphaviruses, can infect invertebrate and vertebrate species including insects, reptiles, birds, and mammals. This broad tropism may be explained by their ability to engage receptors that are conserved across vertebrate and invertebrate classes. Through several genome-wide loss-of-function screens, new alphavirus receptors have been identified, some of which bind to multiple related viruses in different antigenic complexes. Structural analysis has revealed that distinct sites on the alphavirus glycoprotein can mediate receptor binding, which opposes the idea that a single receptor-binding site mediates viral entry. Here, we discuss how different paradigms of receptor engagement on cells might explain the promiscuity of alphaviruses for multiple hosts.

System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies.

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

Changes in the chikungunya virus E1 glycoprotein domain II and hinge influence E2 conformation, infectivity, and virus-receptor interactions.

In a previous study to understand how the chikungunya virus (CHIKV) E1 glycoprotein ß-strand c functions, we identified several attenuating variants at E1 residue V80 and the emergence of second-site mutations in the fusion loop (E1-M88L) and hinge region (E1-N20Y) with the V80 variants in vivo. The emergence of these mutations led us to question how changes in E1 may contribute to CHIKV infection at the molecular level. Here, we use molecular dynamics to understand how changes in the E1 glycoprotein may influence the CHIKV glycoprotein E1-E2 complex. We found that E1 domain II variants lead to E2 conformational changes, allowing us to hypothesize that emerging variants E1-M88L and E1-N20Y could also change E2 conformation and function. We characterized CHIKV E1-M88L and E1-N20Y in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. We found that CHIKV E1-N20Y enhanced infectivity in mosquito cells, while the CHIKV E1-M88L variant enhanced infectivity in both BHK-21 and C6/36 cells and led to changes in viral cholesterol-dependence. Moreover, we found that E1-M88L and E1-N20Y changed E2 conformation, heparin binding, and interactions with the receptor Mxra8. Interestingly, the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, yet was attenuated in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type-dependent entry. Taken together, these studies show that key residues in the CHIKV E1 domain II and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry.IMPORTANCEArboviruses are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo to address these gaps in our knowledge. Here, we show that changes in the CHIKV E1 domain II and hinge alter E2 conformations leading to changes in virus-receptor and -glycosaminoglycan interactions and cell-specific infection. These results highlight that adaptive changes in E1 can have a major effect on virus attachment and entry, furthering our knowledge of how alphaviruses infect mammals and insects.

Chikungunya virus release is reduced by TIM-1 receptors through binding of envelope phosphatidylserine.

T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production. IMPORTANCE: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.