Protein crystallization in vivo

Protein crystallization in vivo provides some fascinating examples of biological self-assembly. Here, we provide a selective survey to show the diversity of functions for which protein crystals are used, and the physical properties of the crystals thatare exploited. Where known, we emphasize how the nature of the protein-protein interactions leads to control of the crystallization behaviour.Comment: 17 pages, 1 figur

Super-Resolution Microscopy: A Virus’ Eye View of the Cell

It is difficult to observe the molecular choreography between viruses and host cell components, as they exist on a spatial scale beyond the reach of conventional microscopy. However, novel super-resolution microscopy techniques have cast aside technical limitations to reveal a nanoscale view of virus replication and cell biology. This article provides an introduction to super-resolution imaging; in particular, localisation microscopy, and explores the application of such technologies to the study of viruses and tetraspanins, the topic of this special issue

Mechanisms of viral capsid assembly around a polymer

Capsids of many viruses assemble around nucleic acids or other polymers. Understanding how the properties of the packaged polymer affect the assembly process could promote biomedical efforts to prevent viral assembly or nanomaterials applications that exploit assembly. To this end, we simulate on a lattice the dynamical assembly of closed, hollow shells composed of several hundred to 1000 subunits, around a flexible polymer. We find that assembly is most efficient at an optimum polymer length that scales with the surface area of the capsid; significantly longer than optimal polymers often lead to partial-capsids with unpackaged polymer `tails' or a competition between multiple partial-capsids attached to a single polymer. These predictions can be tested with bulk experiments in which capsid proteins assemble around homopolymeric RNA or synthetic polyelectrolytes. We also find that the polymer can increase the net rate of subunit accretion to a growing capsid both by stabilizing the addition of new subunits and by enhancing the incoming flux of subunits; the effects of these processes may be distinguishable with experiments that monitor the assembly of individual capsids.Comment: 7 figure

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

Stochastic kinetics of viral capsid assembly based on detailed protein structures

We present a generic computational framework for the simulation of viral capsid assembly which is quantitative and specific. Starting from PDB files containing atomic coordinates, the algorithm builds a coarse grained description of protein oligomers based on graph rigidity. These reduced protein descriptions are used in an extended Gillespie algorithm to investigate the stochastic kinetics of the assembly process. The association rates are obtained from a diffusive Smoluchowski equation for rapid coagulation, modified to account for water shielding and protein structure. The dissociation rates are derived by interpreting the splitting of oligomers as a process of graph partitioning akin to the escape from a multidimensional well. This modular framework is quantitative yet computationally tractable, with a small number of physically motivated parameters. The methodology is illustrated using two different viruses which are shown to follow quantitatively different assembly pathways. We also show how in this model the quasi-stationary kinetics of assembly can be described as a Markovian cascading process in which only a few intermediates and a small proportion of pathways are present. The observed pathways and intermediates can be related a posteriori to structural and energetic properties of the capsid oligomers

Molecular studies on the sweet potato virus disease and its two causal agents

The studies presented in this thesis contribute to an increased understanding of the molecular aspects, variability and interaction of the two most important viral pathogens of sweet potato (Ipomoea batatas L): Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), which cause the severe sweet potato virus disease (SPVD) when co-infecting sweet potato plants. SPVD is the most important disease affecting sweet potato in Africa, and may be the most important virus disease of sweet potato globally. The coat protein gene sequences of several African SPFMV isolates were determined and compared by phylogenetic analyses. Results showed that East African SPFMV isolates were genetically distinct. They could furthermore be divided into two serotypes which differed in their ability to systemically infect the sweet potato cultivar Tanzania. The aetiology of SPVD was studied in sweet potato plants co-infected with SPFMV and SPCSV using nucleic acid hybridisation, bioassays, tissue printing and thin section immunohistochemistry. Resistance to SPFMV in East African sweet potato cultivars was found to be due to inhibition of virus replication rather than movement and resistance was suppressed by infection with SPCSV, resulting in a ca. 600-fold increase in titres of SPFMV. Furthermore, in SPVD affected plants SPFMV is detected outside of the phloem, whereas SPCSV is detected only inside the phloem, which suggests novel as yet unknown mechanisms how SPCSV synergises SPFMV. The genomic sequence of SPCSV was determined. It was composed of two RNA molecules (9407 and 8223 nucleotides), representing the second largest (+)ssRNA genome of plant viruses. The genomic organization of SPCSV revealed novel features for the genus Crinivirus, such as i) the presence of a gene putatively encoding an ribonuclease III-like protein, ii) near-identical, 208 nucleotides long 3’-sequences on both viral RNAs, and iii) the placement of the SHP gene at a new position on the genome of SPCSV relative to other closteroviridae. Northern analyses showed the presence of several sub-genomic RNAs, of which the accumulation was temporally regulated in infected tissues. The 5’-ends of seven sub-genomic RNAs were determined using a PCR based method, which indicated that the sgRNAs were capped