|Journal of Virology Structure and Assembly|
Human infection with highly pathogenic avian influenza A viruses causes severe disease and fatalities. We previously identified a potent and broadly neutralizing antibody (bnAb), 13D4, against the H5N1 virus. Here, we report the co-crystal structure of 13D4 in complex with the hemagglutinin (HA) of A/Vietnam/1194/2004 (H5N1). We show that heavy-chain complementarity-determining region 3 (HCDR3) of 13D4 confers broad yet specific neutralization against H5N1, undergoing conformational rearrangement to bind to the receptor binding site (RBS). Further, we show that mutating four critical residues within the RBSmmdash;Trp153, Lys156, Lys193, and Leu194mmdash;disrupts the binding between 13D4 and HA. Viruses bearing Asn193 instead of Lys/Arg can evade 13D4 neutralization, indicating that Lys193 polymorphism might be, at least in part, involved in the antigenicity of recent H5 genotypes (such as H5N6 and H5N8) as distinguished from H5N1. BnAb 13D4 may offers a template for therapeutic RBS inhibitor design and serve as an indicator of antigenic change for current H5 viruses.
IMPORTANCE Infection by highly pathogenic avian influenza A virus remains a threat to public health. Our broadly neutralizing antibody, 13D4, is capable of neutralizing all representative H5N1 viruses and protecting mice against lethal challenge. Structural analysis revealed that 13D4 uses heavy-chain complementarity-determining region 3 (HCDR3) to fit the receptor binding site (RBS) via conformational rearrangement. Four conserved residues within the RBS are critical for the broad potency of 13D4. Importantly, polymorphism of Lys193 on the RBS may be associated with the antigenicity shift from H5N1 to other newly emerging viruses, such as H5N6 and H5N8. Our findings may pave the way for highly pathogenic avian influenza virus vaccine development and therapeutic RBS inhibitor design.
Coronaviruses pose serious health threats to humans and other animals. Understanding the mechanisms of their replication has important implications for global health and economic stability. Nonstructural protein 9 (nsp9) is an essential RNA binding protein for coronavirus replication. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. Here, we report four crystal structures, including wild-type porcine delta coronavirus (PDCoV) nsp9, PDCoV nsp9-N7 (N-terminal 7 amino acids deleted), wild-type porcine epidemic diarrhea virus (PEDV) nsp9, and PEDV nsp9-C59A mutant. These structures reveal the diverse dimerization forms of coronavirus nsp9. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. Interestingly, size exclusion chromatography and analytical ultracentrifugation analyses indicate that the PDCoV nsp9-N7 and PEDV nsp9-C59A mutants are monomeric in solution. In addition, electrophoretic mobility shift assays and microscale thermophoresis analysis indicate that the monomeric forms of PDCoV nsp9 and PEDV nsp9 still have nucleic acid binding affinity, although it is lower than that of the wild type. Our results show that the diverse dimerization forms of coronavirus nsp9 proteins enhance their nucleic acid binding affinity.
IMPORTANCE Coronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and causing economic loss. Coronavirus nsp9, a member of the replication complex, is an important RNA binding subunit in the RNA-synthesizing machinery of all coronaviruses. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. In this study we determined the nsp9 crystal structures of PDCoV and PEDV. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. This study provides a structural and functional basis for understanding the mechanism of dimerization and shows that the diverse dimerization modes of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Importantly, these findings may provide a new insight for antiviral drug development.
Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of various amounts of 20 or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein, a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized, and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36, whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid.
IMPORTANCE Neuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument, and with it we begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.