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Crystal Structure of the Human Astrovirus Capsid Protein.

ABSTRACT: UNLABELLED:Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ?34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP90(71-415) (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP90(71-415) encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP90(71-415) is comprised of two domains: an S domain, which adopts the typical jelly-roll ?-barrel fold, and a P1 domain, which forms a squashed ?-barrel consisting of six antiparallel ?-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP90(71-415) structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation. IMPORTANCE:Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus, HAstV exhibits an intriguing feature in that its maturation requires extensive proteolytic processing of the astrovirus capsid protein (CP) both inside and outside the host cell. Mature HAstV contains three predominant protein species, but the mechanism for acquired infectivity upon maturation is unclear. We have solved the crystal structure of VP90(71-415) of human astrovirus serotype 8. VP90(71-415) encompasses the conserved N-terminal domain of the viral CP. Fitting of the VP90(71-415) structure into the cryo-EM maps of HAstV produced an atomic model for the T=3 icosahedral capsid. Our model of the HAstV capsid provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation. Such information has potential applications in the development of a VLP vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation.

SUBMITTER: Toh Y

PROVIDER: S-EPMC5044835 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Crystal Structure of the Human Astrovirus Capsid Protein.

Toh Yukimatsu Y Harper Justin J Dryden Kelly A KA Yeager Mark M Arias Carlos F CF Méndez Ernesto E Tao Yizhi J YJ

Journal of virology 20160929 20

<h4>Unlabelled</h4>Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside ...[more]

PMID: 27466429

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Project description:The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus-host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro. IMPORTANCE Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.

Project description:UnlabelledHuman astroviruses (HAstVs) are nonenveloped, positive-sense, single-stranded RNA viruses that are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we report the crystal structures of the two main structural domains of the HAstV CP: the core domain at 2.60-Å resolution and the spike domain at 0.95-Å resolution. Fitting of these structures into the previously determined 25-Å-resolution electron cryomicroscopy density maps of HAstV allowed us to characterize the molecular features on the surfaces of immature and mature T=3 HAstV particles. The highly electropositive inner surface of HAstV supports a model in which interaction of the HAstV CP core with viral RNA is a driving force in T=3 HAstV particle formation. Additionally, mapping of conserved residues onto the HAstV CP core and spike domains in the context of the immature and mature HAstV particles revealed dramatic changes to the exposure of conserved residues during virus maturation. Indeed, we show that antibodies raised against mature HAstV have reactivity to both the HAstV CP core and spike domains, revealing for the first time that the CP core domain is antigenic. Together, these data provide new molecular insights into HAstV that have practical applications for the development of vaccines and antiviral therapies.ImportanceAstroviruses are a leading cause of viral diarrhea in young children, immunocompromised individuals, and the elderly. Despite the prevalence of astroviruses, little is known at the molecular level about how the astrovirus particle assembles and is converted into an infectious, mature virus. In this paper, we describe the high-resolution structures of the two main astrovirus capsid proteins. Fitting these structures into previously determined low-resolution maps of astrovirus allowed us to characterize the molecular surfaces of immature and mature astroviruses. Our studies provide the first evidence that astroviruses undergo viral RNA-dependent assembly. We also provide new insight into the molecular mechanisms that lead to astrovirus maturation and infectivity. Finally, we show that both capsid proteins contribute to the adaptive immune response against astrovirus. Together, these studies will help to guide the development of vaccines and antiviral drugs targeting astrovirus.

Dataset Information

Crystal Structure of the Human Astrovirus Capsid Protein.

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Crystal Structure of the Human Astrovirus Capsid Protein.

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