Locking and Blocking the Viral Landscape of an Alphavirus with Neutralizing Antibodies

Porta, Jason; Jose, Joyce; Roehrig, John T.; Blair, Carol D.; Kühn, Richard; Rossmann, Michael G.

doi:10.1128/jvi.01286-14

Cited by 51 publications

(48 citation statements)

References 35 publications

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“…The structures of CHIK-VLPs and also of other alphaviruses had shown that the B domain of glycoprotein E2 covers and protects the fusion loop in domain II of E1 (6,7,12). However, the B domain is exceedingly flexible (8,15) as observed in the crystal structure of the E1E2 heterodimer (14) or the (E1E2) 3 trimer (15).…”

Section: Fitting the Fab Fragments Into The Cryo-em Densitiesmentioning

confidence: 95%

“…Most antibodies against alphaviruses target the E2 glycoprotein, since it covers much of E1 and is therefore far more exposed. The two most common mechanisms for neutralizing alphaviruses are by binding to the A domain of E2 and therefore blocking receptor attachment (9)(10)(11)(12) and by preventing exposure of the E1 fusion loop by stabilizing the E2 B domain to cover and protect the fusion loop in the mature virus (9,12). This can be achieved by binding the flexible B domain to the rigid A domain via a Fab fragment (8,12).…”

mentioning

confidence: 99%

“…The E1 glycoprotein of CHIKV consists of 442 amino acids that form three ␤-barrel domains (I, II, and III). The CHIKV E2 glycoprotein consists of 423 amino acids arranged into three immunoglobulin-like domains, A, B, and C. Domain A contains the receptor binding site (9)(10)(11)(12), domain B is at the distal end of each spike protecting the fusion loop on DII of E1, and domain C is situated closest to the viral membrane. Domain B is connected to domains A and C by a ␤ ribbon (Fig.…”

mentioning

confidence: 99%

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Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Porta

Prasad

Wang

et al. 2016

J Virol

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Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. IMPORTANCE Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the OldWorld that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments. C hikungunya virus (CHIKV) is the causative agent of an emerging disease. The first outbreaks occurred in the 1950s in Tanzania (1). The virus later reemerged in late 2005 on the Reunion islands and, subsequently, also in several Southeast Asian countries (2). However, by 2013 large epizootics/epidemics of CHIKV had spread into the Caribbean and by 2014 into South America as well as the United States (3). CHIKV is a member of the Togaviridae family, Alphavirus genus. Some alphaviruses, such as Sindbis virus (SINV), generally cause only mild disease symptoms in humans (4). Although mortality rates from CHIKV infections are low, CHIKV and Venezuelan equine encephalitis virus (VEEV) can be lethal or permanently disabling (2, 3). Furthermore, there are currently no treatments or vaccines for alphaviral infections.The structures of a number of alphaviruses have been determined to resolutions better than 10 Å, including the structures of chikungunya virus-like particles (CHIK-VLPs), which have been determined to 5.3-Å resolution (5-7), and of VEEV, determined to 4.6-Å resolution (6). Alphaviruses have an external diameter of about 700 Å and are icosahedral with quasi-Tϭ4 symmetry. They have a nucleocapsid core that is completely surrounded by a lipid envelope, derived from a host membrane, into which is embedded an icosahedral array of glycoproteins (8). A single virus...

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Section: Fitting the Fab Fragments Into The Cryo-em Densitiesmentioning

confidence: 95%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Porta

Prasad

Wang

et al. 2016

J Virol

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“…Three of these antibodies (m10, m242, and CHK-9) bound to the putative receptor binding sites, whereas one, CHK-152, inhibited the exposure of the fusion loop by immobilizing the E2-B domain (22). Analogous structural studies have been reported for other alphaviruses, such as Ross River virus (27), Sindbis virus (33), and VEEV (34). However, there are no published structures of CHIKV in complex with human antibodies.…”

Section: Significancementioning

confidence: 84%

Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity

Long

Fong

Austin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. Here, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain's β-ribbon connector of the viral glycoprotein E2. The footprints of these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. This finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes. chikungunya virus-antibody complexes | cryo-electron microscopy structure | neutralizing mechanism | viral fusion inhibition C hikungunya virus (CHIKV) is an enveloped, positive-stranded RNA virus that belongs to the alphavirus genus of the Togaviridae family (1, 2). CHIKV is transmitted to humans by Aedes species mosquitoes and causes a debilitating febrile illness associated with acute and chronic arthritis (3, 4). Since the first human CHIKV infection was reported in East Africa in 1952 (5), epidemics of CHIKV have occurred in Africa, Asia, and Europe (6, 7). A CHIKV outbreak in the Caribbean area in late 2013 spread through the Americas and caused about 1.4 million infections (8). Despite its global disease burden and risk of spread, there is no available vaccine or effective antiviral drug for CHIKV.The genome of CHIKV is ∼11.8 kb long and encodes nine viral proteins, five of which are structural (capsid, E3, E2, 6K, and E1) (2). These structural proteins are translated as a single polyprotein, which is then cleaved into the capsid, p62, 6K, and E1 proteins by cellular and viral proteases. During maturation, p62 is cleaved to release E3, which protects the fusion loop in the immature virus. The virus consists of a central core with diameter of ∼400 Å with the icosahedrally organized capsid proteins surrounding the viral genome. The nucleocapsid core is enveloped by a lipid membrane into which the E1 and E2 glycoproteins are inserted (9). The mature CHIKV particle has a diameter of 700 Å. The E2 glycoprotein binds to uncharacterized cellular receptors to initiate virus entry into host cells, whereas E1 glycoprotein participates in virus-host cell membrane fusion (10, 11). Although the E3 and 6K proteins contribute to virus assembly and maturation, they are released during the format...

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“…The 4UOM structure is described as the F5 Fab bound to the alphavirus Venezuelan equine encephalitis virus (VEEV) and is described in Porta et al (2014).…”

Section: Analysis Of the Blast Resultsmentioning

confidence: 99%

Extracting human antibody sequences from public databases for antibody humanization: high frequency of species assignment errors

Martin

Rees²

2016

Protein Engineering, Design and Selection

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In antibody humanization, CDRs from a 'donor' antibody are often grafted onto a human framework selected by high sequence identity with the donor. In our own humanization experiments, we have found that species information is often incorrect.Here we take three mouse antibodies and perform BLAST searches against sequences annotated as being human. We find that the first genuine human hits for the six chains appear at positions 30, 4, 11, 24, 18 and 29 in the hit lists. This illustrates both the need for caution in performing humanization and for improvements in annotation.

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Locking and Blocking the Viral Landscape of an Alphavirus with Neutralizing Antibodies

Cited by 51 publications

References 35 publications

Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity

Extracting human antibody sequences from public databases for antibody humanization: high frequency of species assignment errors

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