Entry receptors - The gateway to alphavirus infection

Alphaviruses are enveloped, insect-transmitted, positive-sense RNA viruses that infect humans and other animals and cause a range of clinical manifestations, including arthritis, musculoskeletal disease, meningitis, encephalitis, and death. Over the past four years, aided by CRISPR/Cas9-based genetic screening approaches, intensive research efforts have focused on identifying entry receptors for alphaviruses to better understand the basis for cellular and species tropism. Herein, we review approaches to alphavirus receptor identification and how these were used for discovery. The identification of new receptors advances our understanding of viral pathogenesis, tropism, and evolution and is expected to contribute to the development of novel strategies for prevention and treatment of alphavirus infection

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

Neutralizing antibodies protect mice against Venezuelan equine encephalitis virus aerosol challenge

Venezuelan equine encephalitis virus (VEEV) remains a risk for epidemic emergence or use as an aerosolized bioweapon. To develop possible countermeasures, we isolated VEEV-specific neutralizing monoclonal antibodies (mAbs) from mice and a human immunized with attenuated VEEV strains. Functional assays and epitope mapping established that potently inhibitory anti-VEEV mAbs bind distinct antigenic sites in the A or B domains of the E2 glycoprotein and block multiple steps in the viral replication cycle including attachment, fusion, and egress. A 3.2-Å cryo-electron microscopy reconstruction of VEEV virus-like particles bound by a human Fab suggests that antibody engagement of the B domain may result in cross-linking of neighboring spikes to prevent conformational requirements for viral fusion. Prophylaxis or postexposure therapy with these mAbs protected mice against lethal aerosol challenge with VEEV. Our study defines functional and structural mechanisms of mAb protection and suggests that multiple antigenic determinants on VEEV can be targeted for vaccine or antibody-based therapeutic development

Broadly neutralizing monoclonal antibodies protect against multiple tick-borne flaviviruses

Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type-specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses

Potently neutralizing and protective human antibodies against SARS-CoV-2

The COVID-19 pandemic is a major threat to global health1 for which there are limited medical countermeasures2,3. Moreover, we currently lack a thorough understanding of mechanisms of humoral immunity4. From a larger panel of human monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein5, we identified several that exhibited potent neutralizing activity and fully blocked the receptor-binding domain of S (SRBD) from interacting with human ACE2 (hACE2). Competition-binding, structural, and functional studies allowed clustering of the mAbs into classes recognizing distinct epitopes on the SRBD as well as distinct conformational states of the S trimer. Potent neutralizing mAbs recognizing non-overlapping sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two mouse models of SARS-CoV-2 infection, passive transfer of either COV2-2196 or COV2-2130 alone or a combination of both mAbs protected mice from weight loss and reduced viral burden and inflammation in the lung. In addition, passive transfer of each of two of the most potently ACE2 blocking mAbs (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutics

A streamlined method for transposon mutagenesis of Rickettsia parkeri yields numerous mutations that impact infection.

The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, rickettsiae are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improved mariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group species Rickettsia parkeri. Transposon insertions disrupted genes whose products are implicated in a variety of pathways, including bacterial replication and metabolism, the type IV secretion system, factors with previously established roles in host cell interactions and pathogenesis, or are of unknown function. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis

Transposon insertion sites in <i>R</i>. <i>parkeri</i>.

<p>Transposon insertion sites in <i>R</i>. <i>parkeri</i>.</p

Mapping the transposon insertion sites.

<p>(A) Diagram showing the insertion of the transposon cassette into a chromosomal region (in grey). Primers specific to the transposon ends were paired with universal primers to amplify the chromosome- transposon junctions (red triangles), using semi-random nested PCR. Two nested PCR reactions were done to improve amplification of the chromosome-transposon junction directly from boiled bacteria. (B) <i>R</i>. <i>parkeri</i> chromosomal map showing all transposon insertion sites (see red lines) identified in this screen.</p

Transposon mutagenesis of <i>R</i>. <i>parkeri</i>.

<p>(A) Map of the pMW1650 plasmid used in this study for transposon mutagenesis (IR, inverted repeats). (B) Experimental scheme for transposon mutagenesis and isolation of individual mutants.</p