Mind the Gap: How Some Viruses Infect Their Hosts

Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (Cryo-ET) provide structural insights into complex biological processes. The podoviridae are dsDNA containing phage with short, non-contractile tails which nevertheless translocate their DNA into the cytoplasm of their host cells. Liu et al. [1] used a combination of cryo-EM and cryo-ET to study the structural changes accompanying infection of P. marinus by the phage P-SSP7 and thereby provide unique molecular insight into the process by which the DNA transits from phage to host during infection

Whole Blood in Trauma

Abstract: Hemorrhage is a leading cause of death in trauma patients. Whole blood administration in trauma has gained a renewed interest in recent years, however the concept is not new at all, with its origins spanning over 100 years. Field experience in United States’ military conflicts has provided important data on the efficacy of whole blood in reducing mortality and amount of blood product administration. Civilian trials however have not necessarily shown similar results. The safety of whole blood however does not seem to be in question given the available data, and is an acceptable option for blood replacement in the hemorrhaging trauma patient

Detection of intermediates and kinetic control during assembly of bacteriophage P22 procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈50 nm). In this work we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly

DNA Packaging: A New Class of Molecular Motors

DNA is packaged into preformed bacteriophage capsids to liquid crystalline density by the action of a portal protein complex. Single molecule packaging studies indicate that this is a new and extremely powerful class of molecular motors

Role of reversibility in viral capsid growth: A paradigm for self-assembly

Self-assembly at submicroscopic scales is an important but little understood phenomenon. A prominent example is virus capsid growth, whose underlying behavior can be modeled using simple particles that assemble into polyhedral shells. Molecular dynamics simulation of shell formation in the presence of an atomistic solvent provides new insight into the self-assembly mechanism, notably that growth proceeds via a cascade of strongly reversible steps and, despite the large variety of possible intermediates, only a small fraction of highly bonded forms appear on the pathway.Comment: 4 pages, 4 figures (slightly shorter version, new Fig.2); further minor change

Understanding the Concentration Dependence of Viral Capsid Assembly Kinetics - the Origin of the Lag Time and Identifying the Critical Nucleus Size

The kinetics for the assembly of viral proteins into a population of capsids can be measured in vitro with size exclusion chromatography or dynamic light scattering, but extracting mechanistic information from these studies is challenging. For example, it is not straightforward to determine the critical nucleus size or the elongation time (the time required for a nucleated partial capsid to grow completion). We show that, for two theoretical models of capsid assembly, the critical nucleus size can be determined from the concentration dependence of the assembly reaction half-life and the elongation time is revealed by the length of the lag phase. Furthermore, we find that the system becomes kinetically trapped when nucleation becomes fast compared to elongation. Implications of this constraint for determining elongation mechanisms from experimental assembly data are discussed.Comment: Submitted to Biophysical Journa

Rewriting Nature’s Assembly Manual for a ssRNA Virus

Satellite Tobacco Necrosis Virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit relying on the polymerase of its helper virus TNV for replication. The genome contains a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP binding motif -A.X.X.A- in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such Packaging Signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP binding motif, the relative placement of PS stem-loops, their number and their folding propensity. CP binding has an electrostatic contribution but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all –A.X.X.A- motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs assembly is partially restored although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV, and identify an efficient approach for production of stable viruslike particles encapsidating non-native RNAs or other cargoes

Investigation of N-terminal domain charged residues on the assembly and stability of HIV-1CA

ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion as well as viral infectivity. Previous in vivo experiments generated two N-terminal domain charge change mutants (E45A and E128A/R132A) that showed an increase in stability of the viral core. This increase in core stability resulted in decreased infectivity, suggesting the need for a delicate balance of favorable and unfavorable interactions to both allow assembly and facilitate uncoating following infection. Purified CA protein can be triggered to assemble into tubelike structures through the use of a high salt buffer system. The requirement for high salt suggests the need to overcome charge/charge repulsion between subunits. The mutations mentioned above lie within a highly charged region of the N-terminal domain of CA, away from any of the proposed protein/protein interaction sites. We constructed a number of charge mutants in this region (E45A, E45K, E128A, R132A, E128A/ R132A, K131A, and K131E) and evaluated their effect on protein stability in addition to their effect on the rate of CA assembly. We find that the mutations alter the rate of assembly of CA without significantly changing the stability of the CA monomer. The changes in rate for the mutants studied are found to be due to varying degrees of electrostatic repulsion between the subunits of each mutant

Hamiltonian path analysis of viral genomes

Cryo-electron microscopy (EM) is undergoing a revolution, enabling the study of viral pathogens in unprecedented detail. The asymmetric EM reconstruction of bacteriophage MS2 at medium resolution (8.7 Å) by Koning et al.1, and the subsequent reconstruction at even higher resolution (3.6 Å) by Dai et al.2 revealed the structures of both the protein shell and the asym- metric genomic RNA and the unique maturation protein (A). It is the start of a wave of such structural data for viruses, and calls for the development of new analytical tools to describe the results. One approach is Hamiltonian path analysis (HPA) that we introduced to describe repeated, sequence-specific contacts between the MS2 genome and its protein shell3. Here, we describe how HPA is consistent with the new structures and, in turn, how it extends our understanding beyond the structural data alone