No abstract

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.

Viruses are a paradigm of the economy of genome resources, reflected in their multiplication strategy and for their own structure. Although there is enormous structural diversity, the viral genome is always enclosed within a proteinaceous coat, and most virus species are haploid; the only exception to this rule are the highly pleomorphic enveloped viruses. We performed an in-depth characterization of infectious bursal disease virus (IBDV), a nonenveloped icosahedral dsRNA virus with a bisegmented genome. Up to 6 natural populations can be purified, which share a similar protein composition but show higher sedimentation coefficients as particle density increases. Stoichiometry analysis of their genome indicated that these biophysical differences correlate with the copy number of dsRNA segments inside the viral capsid. This is a demonstration of a functional polyploid icosahedral dsRNA virus. We show that IBDV particles with greater genome copy number have higher infectivity rates. Our results show an unprecedented replicative strategy for dsRNA viruses and suggest that birnaviruses are living viral entities encompassing numerous functional and structural characteristics of positive and negative ssRNA viruses.cargo volume ͉ IBDV ͉ icosahedral dsRNA viruses ͉ life cycle ͉ viral polyploidy

During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNAprotein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp 38 of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.genome uncoating | X-ray analysis | 3D cryo-EM | picornavirus H uman rhinoviruses (HRVs) cause the common cold. Although seldom severe, this disease is widespread and frequent in man; HRVs thus have considerable economic impact due to expenditure on medication and lost working days. More than 150 serotypes belong to the genus Enteroviruses (EVs) of the Picornaviridae family, which includes serious human and animal pathogens. In addition to phylogenetic classification into species A, -B, and -C, HRVs are divided into a minor receptor group (12 HRV-A) that bind low-density lipoprotein receptors (LDLRs), and a major receptor group (more than 89 HRV-A and -B serotypes) that use intercellular adhesion molecule 1 (ICAM-1) for cell entry (1). HRV-C binds an unknown receptor (2).The EV icosahedral shell is built from four viral proteins (VP1-4) that encase a single-stranded (+)-sense RNA genome. Sixty copies each of these four polypeptides assemble on a T = 1 (pseudo T = 3) lattice, ∼30 nm in diameter. VP1, VP2, and VP3 are surface-exposed; the small myristoylated VP4 is internal. In the mature virion, the N-terminal extensions of VP1, VP2, and VP3, together with the entire VP4, interact in an intricate network beneath the shell (Fig. S1) (3, 4).In the cytosol, the viral RNA is translated into a ∼250 kDa precursor polyprotein that is processed by viral proteinases. Assembly of the viral shell involves immature pentamers built from VP0, VP1, and VP3. VP2 and VP4 arise late in infection through VP0 cl...

The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.

The transcription and translation signals of the S-layer gene (slpA) from Thermus thermophilus HB8 have been used to express a thermostable kanamycin adenyl transferase gene in this organism. The chimaeric resistance gene was inserted in vitro into slpA to produce different inactive forms of the gene, which were used to transform T. thermophilus HB8. After 48 hours of incubation at 70 degrees C, only two constructions that contained the kat gene flanked by Thermus sequences from both sides of slpA were able to produce protein layer (P100)-defective mutants. The mutants obtained with both constructions showed identical protein patterns, in which a major 50 kDa protein and two other minor proteins were tentatively identified as P100 fragments, expressed from the extreme 5' end of slpA. They also exhibited important phenotypic defects, such as slow growth in liquid broth, a tendency to aggregate as 'rotund bodies', a twisted filamentous shape, and an extreme sensitivity to lysozyme, suggesting protective and shaping roles for the S-layer in T. thermophilus HB8. These results also demonstrate for the first time the feasibility of using selective antibiotic-resistance markers in extreme thermophiles.

Recent years have witnessed the emergence of bacterial semiorganelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo, resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM). Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality of the encapsulin for an in vitro surface-immobilized catalysis in a cascade pathway with an additional enzyme, glucose oxidase. We also demonstrate the in vivo functionality of the encapsulin for cellular uptake using mammalian macrophages. Unraveling both the structure and functionality of the encapsulins allows transforming biological nanocompartments into functional systems.