|Journal of Virology Structure and Assembly|
Despite very low sequence homology, the major capsid proteins of double-stranded DNA (dsDNA) bacteriophages, some archaeal viruses, and the herpesviruses share a structural motif, the HK97 fold. Bacteriophage P22, a paradigm for this class of viruses, belongs to a phage gene cluster that contains three homology groups: P22-like, CUS-3-like, and Sf6-like. The coat protein of each phage has an inserted domain (I-domain) that is more conserved than the rest of the coat protein. In P22, loops in the I-domain are critical for stabilizing intra- and intersubunit contacts that guide proper capsid assembly. The nuclear magnetic resonance (NMR) structures of the P22, CUS-3, and Sf6 I-domains reveal that they are all six-stranded, anti-parallel bbeta;-barrels. Nevertheless, significant structural differences occur in loops connecting the bbeta;-strands, in surface electrostatics used to dock the I-domains with their respective coat protein core partners, and in sequence motifs displayed on the capsid surfaces. Our data highlight the structural diversity of I-domains that could lead to variations in capsid assembly mechanisms and capsid surfaces adapted for specific phage functions.
IMPORTANCE Comparative studies of protein structures often provide insights into their evolution. The HK97 fold is a structural motif used to form the coat protein shells that encapsidate the genomes of many dsDNA phages and viruses. The structure and function of coat proteins based on the HK97 fold are often embellished by the incorporation of I-domains. In the present work we compare I-domains from three phages representative of highly divergent P22-like homology groups. While the three I-domains share a six-stranded bbeta;-barrel skeleton, there are differences (i) in structure elements at the periphery of the conserved fold, (ii) in the locations of disordered loops important in capsid assembly and conformational transitions, (iii) in surfaces charges, and (iv) in sequence motifs that are potential ligand-binding sites. These structural modifications on the rudimentary I-domain fold suggest that considerable structural adaptability was needed to fulfill the versatile range of functional requirements for distinct phages.
Tailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, 29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed an in vitro P22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.
IMPORTANCE The existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assembly in vivo indicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).
The environment represents a significant barrier to infection. Physical stressors (heat) or chemical agents (ethanol) can render virions noninfectious. As such, discrete proteins are necessary to stabilize the dual-layered structure of mammalian orthoreovirus (reovirus). The outer capsid participates in cell entry: (i) 3 is degraded to generate the infectious subviral particle, and (ii) mmu;1 facilitates membrane penetration and subsequent core delivery. mmu;1-3 interactions also prevent inactivation; however, this activity is not fully characterized. Using forward and reverse genetic approaches, we identified two mutations (mmu;1 M258I and 3 S344P) within heat-resistant strains. 3 S344P was sufficient to enhance capsid integrity and to reduce protease sensitivity. Moreover, these changes impaired replicative fitness in a reassortant background. This work reveals new details regarding the determinants of reovirus stability.
IMPORTANCE Nonenveloped viruses rely on protein-protein interactions to shield their genomes from the environment. The capsid, or protective shell, must also disassemble during cell entry. In this work, we identified a determinant within mammalian orthoreovirus that regulates heat resistance, disassembly kinetics, and replicative fitness. Together, these findings show capsid function is balanced for optimal replication and for spread to a new host.
Parvovirus B19, one of the most common human pathogens, is a small DNA virus that belongs to the Parvoviridae. As a result of previous infections, antibodies to B19 are present in most adults. B19 has a strong tropism to erythroid progenitor cells and is able to cause a series of medical conditions, including fifth disease, arthritis, myocarditis, hydrops fetalis, and aplastic crisis. No approved vaccine is currently available for B19, and there is a lack of structural characterization of any B19 epitopes. Here we present the first cryo-electron microscopy (cryo-EM) structure of a B19 virus-like particle (VLP) complexed with the antigen-binding fragment (Fab) of a human neutralizing antibody, 860-55D. A model was built into the 3.2-AAring;-resolution map, and the antigenic residues on the surface of the B19 capsid were identified. Antibody 860-55D bridges the capsid of B19 by binding to a quaternary structure epitope formed by residues from three neighboring VP2 capsid proteins.
IMPORTANCE Parvovirus B19 is a common human pathogen and a particular threat to children, pregnant women, and patients with sickle cell disease or AIDS. Currently, neutralizing antibody is the most efficient treatment for acute B19 infections. Research on the antigenic properties of B19 will guide the usage of these antibodies and facilitate vaccine development. We have determined and report here the high-resolution structure of B19 virus-like particles (VLPs) complexed with the Fab of a human neutralizing antibody. The structure shows a quaternary structure epitope formed by three VP2 proteins and provides details on host recognition of human B19 virus.
Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227nndash;2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255nndash;12260, 2017,
IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227nndash;2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255nndash;12260, 2017,
The complete genome sequence of an RNA virus was assembled from RNA sequencing of virus particles purified from threespine stickleback intestine tissue samples. This new virus is most closely related to the Eel picornavirus and can be assigned to the genus Potamipivirus in the family Picornaviridae. Its unique genetic properties are enough to establish a new species, dubbed the Threespine Stickleback picornavirus (TSPV). Due to their broad geographic distribution throughout the Northern Hemisphere and parallel adaptation to freshwater, threespine sticklebacks have become a model in evolutionary ecology. Further analysis using diagnostic PCRs revealed that TSPV is highly prevalent in both anadromous and freshwater populations of threespine sticklebacks, infects almost all fish tissues, and is transmitted vertically to offspring obtained from in vitro fertilization in laboratory settings. Finally, TSPV was found in Sequence Reads Archives of transcriptome of Gasterosteus aculeatus, further demonstrating its wide distribution and unsought prevalence in samples. It is thus necessary to test the impact of TSPV on the biology of threespine sticklebacks, as this widespread virus could interfere with the behavioral, physiological, or immunological studies that employ this fish as a model system.
IMPORTANCE The threespine stickleback species complex is an important model system in ecological and evolutionary studies because of the large number of isolated divergent populations that are experimentally tractable. For similar reasons, its coevolution with the cestode parasite Schistocephalus solidus, its interaction with gut microbes, and the evolution of its immune system are of growing interest. Herein we describe the discovery of an RNA virus that infects both freshwater and anadromous populations of sticklebacks. We show that the virus is transmitted vertically in laboratory settings and found it in Sequence Reads Archives, suggesting that experiments using sticklebacks were conducted in the presence of the virus. This discovery can serve as a reminder that the presence of viruses in wild-caught animals is possible, even when animals appear healthy. Regarding threespine sticklebacks, the impact of Threespine Stickleback picornavirus (TSPV) on the fish biology should be investigated further to ensure that it does not interfere with experimental results.