A molecular understanding of alphavirus entry

Alphaviruses cause severe human illnesses including persistent arthritis and fatal encephalitis. As alphavirus entry into target cells is the first step in infection, intensive research efforts have focused on elucidating aspects of this pathway, including attachment, internalization, and fusion. Herein, we review recent developments in the molecular understanding of alphavirus entry both in vitro and in vivo and how these advances might enable the design of therapeutics targeting this critical step in the alphavirus life cycle

Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV

Structural insights into host-pathogen interactions of alphaviruses

Alphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses of the Togaviridae family that infect various vertebrates worldwide in tropical and temperate areas, causing emerging and reemerging diseases in humans. Mature virions are 70 nm in diameter and contain a ~11-kilobase genome encapsidated within a nucleocapsid core, a host-derived lipid bilayer, and an envelope comprised of heterodimers of the glycoproteins E1 and E2 arranged into trimeric spikes with T=4 icosahedral symmetry. Alphaviruses are categorized into two groups based on their clinical symptoms: the arthritogenic alphaviruses, such as chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), Semliki Forest (SFV), and O’nyong-nyong (ONNV) viruses, which induce arthritis, polyarthralgia, and musculoskeletal-associated diseases, and the encephalitic alphaviruses, including Venezuelan (VEEV), Eastern (EEEV), and Western (WEEV) equine encephalitic viruses, which lead to meningitis, encephalitis, and long-term neurological sequelae in survivors. The global distribution of alphaviruses has increased in recent decades due to international travel, expansion of mosquito vectors, deforestation, and urbanization. Currently, there are no approved vaccines or treatments available to mitigate alphavirus infection and disease. Further understanding of host-alphavirus interactions may inform the development of such therapies for multiple members of this family. The studies encompassed in this dissertation describe how alphaviruses engage two different entry receptors and how a panel neutralizing anti-MAYV monoclonal antibodies protect against infection.Mxra8 is a receptor for multiple arthritogenic alphaviruses such as CHIKV, MAYV, RRV, and ONNV. We determined 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 CHIKV virus-like particles (VLPs) and infectious virus. Our structures revealed that the Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement, and that Mxra8 binds CHIKV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike while also engaging a neighboring spike. Furthermore, we observed two binding modes with the fully mature VLP, with one Mxra8 binding with unique additional contacts. This high- and low- binding-site model was supported by our surface plasmon resonance measurements. Lastly, we found that the low-affinity binding sites were sterically obscured by the retention of the E3 glycoprotein on infectious CHIKV, suggesting that viral maturation and E3 occupancy influences receptor binding-site usage. In later studies, we also determined near-atomic resolution cryo-electron microscopy reconstructions of VEEV VLPs alone and complexed with its entry receptor, LDLRAD3. We showed that domain 1 (D1) of LDLRAD3, a low-density lipoprotein receptor type-A (LA) module, binds VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, specifically engaging domains A and B of E2 and the fusion loop in E1. Our atomic modeling of this interface was supported by mutagenesis and anti-VEEV antibody binding competition assays. These studies demonstrated that VEEV engages LDLRAD3 in a manner that is remarkably similar to CHIKV with the Mxra8 receptor, but with an exceptionally smaller interface. We speculate that the common positioning of these receptors near the fusion loop might serve to modulate viral fusion during endocytosis. Our studies are among the first to structurally characterize alphavirus-receptor complexes. Additionally, we generated a panel of neutralizing monoclonal antibodies (mAbs) against MAYV, over half of which had “elite” activity that inhibited infection with EC50 values of \u3c10 ng/ml. We demonstrated that antibodies with the greatest inhibitory capacity in vitro mapped to epitopes near the fusion peptide of E1 and in domain B of E2. Unexpectedly, many of the elite neutralizing mAbs failed to prevent MAYV infection and disease in vivo. Instead, protection required fragment crystallizable (Fc) effector functions, as isotype-switched or aglycosyl variants with less or no capacity to interact with the complement component C1q or activating Fc-γ receptors lost protective activity in vivo. These results demonstrated that an optimally protective antibody response to MAYV and possibly other alphaviruses may require tandem optimization of virus neutralization by the Fab moiety and effector functions of the Fc region. Altogether, these studies establish how alphaviruses interact with the entry receptors and humoral responses of their hosts, which may inform the basis of future therapies and improved vaccine designs

The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies

Although neutralizing monoclonal antibodies (mAbs) against epitopes within the alphavirus E2 protein can protect against infection, the functional significance of non-neutralizing mAbs is poorly understood. Here, we evaluate the activity of 13 non-neutralizing mAbs against Mayaro virus (MAYV), an emerging arthritogenic alphavirus. These mAbs bind to the MAYV virion and surface of infected cells but fail to neutralize infection in cell culture. Mapping studies identify six mAb binding groups that localize to discrete epitopes within or adjacent to the A domain of the E2 glycoprotein. Remarkably, passive transfer of non-neutralizingmAbs protects against MAYV infection and disease in mice, and their efficacy requires Fc effector functions. Monocytes mediate the protection of non-neutralizing mAbs in vivo, as Fcg-receptor-expressing myeloid cells facilitate the binding, uptake, and clearance of MAYV without antibody-dependent enhancement of infection. Humoral protection against alphaviruses likely reflects contributions from non-neutralizing antibodies through Fc-dependent mechanisms that accelerate viral clearance

Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors

<div><p>Malaria parasites increase their host erythrocyte’s permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against <i>in vitro</i> parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel’s structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing.</p></div

A PRT-1 derivative with improved potency and specificity.

<p>(A) Structure of PRT1-20. A longer alkoxy side chain distinguishes this compound from PRT-1 (red highlight). (B) Mean ± S.E.M. inhibitor <i>K</i>_{<i>0</i>.<i>5</i>} values in PhTMA⁺ + 200 μM furosemide (red bars) and sorbitol (black). When compared to PRT-1, PRT1-20 has improved greater potency against residual transport and reduced activity against the primary mechanism.</p

Synergistic killing by combinations of primary and residual inhibitors, but not by combinations from one class only.

<p>(A) Isobologram showing effect of fixed ratio combinations of the primary component inhibitor cpd <b>1</b> and the residual transport inhibitor PRT1-20. Each symbol represented the <i>IC</i>_<i>50</i> for a fixed ratio of the two inhibitors, determined from a full dose response experiment with replicates. Error bars, shown for single compound <i>IC</i>_<i>50</i> values (symbols on the <i>x</i> and <i>y</i> axes), represent S.E.M. values. Solid line connecting these intercept values is the expected profile for additive parasite killing. Strong synergistic killing was found for this drug combination as the symbols are markedly below the additive line. (B) Isobologram for two primary component inhibitors, showing additive interaction. (C) Isobologram for two residual component inhibitors, showing additive interaction.</p

Effects of external nutrient levels on inhibitor efficacy against parasite growth.

<p>(A, B) Dose responses for growth inhibition by ISG-21 and PRT-1 in standard medium and PGIM (black and red symbols, respectively). While ISG-21 has significantly improved activity in PGIM, the efficacy of PRT-1 is unchanged. Solid lines represent best fits to a logistic decay with a Hill coefficient. (C) Ratio of <i>IC</i>_<i>50</i> values for parasite killing in standard RPMI 1640-based medium to PGIM for indicated inhibitors. Bars represent mean ± S.E.M. of replicates from up to 7 independent trials.</p

<i>In vitro</i> interactions between primary and residual PSAC inhibitors.

<p>Combination growth inhibition experiments, tallied as mean ± S.E.M. sum of FIC values for 50% growth inhibition (∑FIC₅₀). Combinations are grouped under headings to indicate experiments performed with a primary and a residual inhibitor, two primary inhibitors, or two residual inhibitors.</p