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.

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.

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.

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.

Potent Antibody Protection against an Emerging Alphavirus Threat.

Chikungunya virus recently caused large outbreaks world-wide. In this issue of Cell, Fox et al. describe several potently neutralizing antibodies against multiple alphaviruses. The structure of the virus in complex with one of the antibodies reveals the antibody-induced rearrangement and crosslinking of the viral surface proteins that result in neutralization.

Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

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.

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores.

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

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.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

Molecular Epidemiology of Mayaro Virus among Febrile Patients, Roraima State, Brazil, 2018-2021.

We detected Mayaro virus (MAYV) in 3.4% (28/822) of febrile patients tested during 2018-2021 from Roraima State, Brazil. We also isolated MAYV strains and confirmed that these cases were caused by genotype D. Improved surveillance is needed to better determine the burden of MAYV in the Amazon Region.

Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque.

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.

Culex tarsalis Is a Competent Host of the Insect-Specific Alphavirus Eilat Virus (EILV).

Eilat virus (EILV) is an insect-specific alphavirus that has the potential to be developed into a tool to combat mosquito-borne pathogens. However, its mosquito host range and transmission routes are not well understood. Here, we fill this gap by investigating EILV's host competence and tissue tropism in five mosquito species: Aedes aegypti, Culex tarsalis, Anopheles gambiae, Anopheles stephensi, and Anopheles albimanus. Of the tested species, C. tarsalis was the most competent host for EILV. The virus was found in C. tarsalis ovaries, but no vertical or venereal transmission was observed. Culex tarsalis also transmitted EILV via saliva, suggesting the potential for horizontal transmission between an unknown vertebrate or invertebrate host. We found that reptile (turtle and snake) cell lines were not competent for EILV infection. We tested a potential invertebrate host (Manduca sexta caterpillars) but found they were not susceptible to EILV infection. Together, our results suggest that EILV could be developed as a tool to target pathogenic viruses that use Culex tarsalis as a vector. Our work sheds light on the infection and transmission dynamics of a poorly understood insect-specific virus and reveals it may infect a broader range of mosquito species than previously recognized. IMPORTANCE The recent discovery of insect-specific alphaviruses presents opportunities both to study the biology of virus host range and to develop them into tools against pathogenic arboviruses. Here, we characterize the host range and transmission of Eilat virus in five mosquito species. We find that Culex tarsalis-a vector of harmful human pathogens, including West Nile virus-is a competent host of Eilat virus. However, how this virus is transmitted between mosquitoes remains unclear. We find that Eilat virus infects the tissues necessary for both vertical and horizontal transmission-a crucial step in discerning how Eilat virus maintains itself in nature.

Dehydration stress and Mayaro virus vector competence in Aedes aegypti.

IMPORTANCE: Relative humidity (RH) is an environmental variable that affects mosquito physiology and can impact pathogen transmission. Low RH can induce dehydration in mosquitoes, leading to alterations in physiological and behavioral responses such as blood-feeding and host-seeking behavior. We evaluated the effects of a temporal drop in RH (RH shock) on mortality and Mayaro virus vector competence in Ae. aegypti. While dehydration induced by humidity shock did not impact virus infection, we detected a significant effect of dehydration on mosquito mortality and blood-feeding frequency, which could significantly impact transmission dynamics.