Structural Analysis of Coxsackievirus A7 Reveals Conformational Changes Associated with Uncoating

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bCoxsackievirus A9 (CVA9) is an important pathogen of the Picornaviridae family. It utilizes cellular receptors from the integrin ␣ v family for binding to its host cells prior to entry and genome release. Among the integrins tested, it has the highest affinity for ␣ v ␤ 6 , which recognizes the arginine-glycine-aspartic acid (RGD) loop present on the C terminus of viral capsid protein, VP1. As the atomic model of CVA9 lacks the RGD loop, we used surface plasmon resonance, electron cryo-microscopy, and image reconstruction to characterize the capsid-integrin interactions and the conformational changes on genome release. We show that the integrin binds to the capsid with nanomolar affinity and that the binding of integrin to the virion does not induce uncoating, thereby implying that further steps are required for release of the genome. Electron cryo-tomography and single-particle image reconstruction revealed variation in the number and conformation of the integrins bound to the capsid, with the integrin footprint mapping close to the predicted site for the exposed RGD loop on VP1. Comparison of empty and RNA-filled capsid reconstructions showed that the capsid undergoes conformational changes when the genome is released, so that the RNA-capsid interactions in the N termini of VP1 and VP4 are lost, VP4 is removed, and the capsid becomes more porous, as has been reported for poliovirus 1, human rhinovirus 2, enterovirus 71, and coxsackievirus A7. These results are important for understanding the structural basis of integrin binding to CVA9 and the molecular events leading to CVA9 cell entry and uncoating. Picornaviruses that belong to the Human enterovirus B species (HEV-B) in the family Picornaviridae are the major cause of aseptic meningitis (1, 2). HEV-B types, including coxsackievirus A9 (CVA9), also exhibit a wide range of other clinical manifestations of acute disease, including respiratory infections, myocarditis, encephalitis, paralysis, rash, and severe generalized infections in newborns (3-7). Moreover, there is evidence that suggests that HEV-B, specifically, CVA9, CVB1, CVB3, and CVB5, may be involved in the pathogenesis of childhood diabetes, the rate of incidence of which is increasing in Western countries (8-11).The CVA9 particle is about 28 nm in diameter and consists of a nonenveloped capsid with icosahedral symmetry, surrounding a positive-sense RNA genome of 7,452 nucleotides (12, 13). The CVA9 Tϭ1 (pseudo-Tϭ3) capsid is made up of 60 copies each of the four viral capsid proteins (VP1 to VP4) as revealed by its atomic model (12). Each of VP1, VP2, and VP3 possesses an eightstranded ␤-barrel structure. VP1 mainly forms the vertices of the capsid, with VP2 and VP3 alternating on the 3-fold and 2-fold axes of symmetry (12). These are common characteristics that are shared by most picornaviruses (12,14). Depressions of about 15 Å form the canyon region and are also seen around the 2-fold axes (12). CVA9 interacts with cell surface ␣ v integrins during the early stages of infection via an arginine-g...

Section: Resultssupporting

confidence: 74%

Structural and Functional Analysis of Coxsackievirus A9 Integrin α _v β ₆ Binding and Uncoating

Shakeel

Seitsonen

Kajander

et al. 2013

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“…Measuring the dye accessibility at physiological temperature in HPeV1-AM28 complex for 12 h did not affect the fluorescence, which is in contrast to E18 MAb binding to EV71 at physiological temperature, where a significant increase in fluorescence was observed as a result of capsid destabilization (18). Significantly, increased porosity of picornavirus capsids for RNA release has been shown to be dependent on domain movements of the major capsid proteins opening up the interfaces at the 2-fold axes (24,43,(65)(66)(67)(68)(69). Our model of the HPeV1 capsid now includes identification of RNA stem-loops interacting directly with VP1 and VP3.…”

Section: Discussionmentioning

confidence: 86%

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

Shakeel

Westerhuis

Ora

et al. 2015

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Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCEHuman parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell. Human parechoviruses (HPeV) are single-stranded, positivesense RNA viruses in the Parechovirus genus within the Picornaviridae family (1). HPeV infections mainly cause mild gastrointestinal symptoms, although HPeV are also associated with more severe central nervous system symptoms, such as meningitis and neonatal sepsis (2-5). The HPeV genome is about 7,300 bases in length, enclose...

“…The altered particles still hold the genome inside the capsid shell, but recent evidence regarding rhinovirus and coxsackievirus B3 (CVB3) indicates that the RNA-capsid interaction is altered (10,11). Additionally, an opening forms at the 2-fold axis of the 135S particle, which facilitates the genome's egress from the capsid (12)(13)(14)(15)(16)(17). The generation of genome-free, empty (80S) particles occurs only after internalization to an endosome compartment, where the final step(s) of picornavirus uncoating is supposed to take place.…”

mentioning

confidence: 99%

A Novel Open and Infectious Form of Echovirus 1

Myllynen

Kazmertsuk

Marjomäki

2016

One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on previous studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to a CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR green II, it still contained most of its VP4, and it was able to bind to its receptor ␣ 2 ␤ 1 integrin and showed high infectivity. A thermal assay further showed that the ␣ 2 ␤ 1 integrin binding domain (I-domain) stabilized the virus particle. Finally, heating E1 particles to superphysiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate. IMPORTANCE In this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection.Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It was shown previously that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to superphysiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure, and it cannot be formed by heating. It still contains the VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses. Picornaviruses are a large family of pathogens infecting humans and animals across the globe. The vast disease range caused by these viruses spans from simple rashes to paralysis and meningitis. It has been estimated that approximately half of the seasonal common-cold cases reported are caused by rhinoviruses, members of the family subgroup Enterovirus (1). Increased emphasis on enterovirus research is...