Unusual self-assembly properties of Norovirus Newbury2 virus-like particles.

In the Caliciviridae family of nonenveloped, positive-stranded RNA viruses, Noroviruses are major causes of human and animal gastroenteritis worldwide. The Norovirus T=3 icosahedral capsid is made of 180 copies of the VP1 protein, as exemplified in the crystal structure of the virus-like particle (VLP) of the human Norwalk virus (NV). It was previously shown that the ca 40-nm recombinant NV VLP can be disassembled and reassembled in vitro. Here we report on the disassembly and self-assembly properties for the related (VP1 sequence identity of 50%) bovine Newbury2 Norovirus (NB2) VLP. Using a panel of biophysical techniques, we show that while the NB2 VLP displays disassembly properties similar to the NV VLP, NB2-VP1 shows remarkable self-assembly properties heretofore unreported for NV-VP1 or any other calicivirus capsid protein. These properties include the capabilities of self-assembling not only into regular T=3 capsids but also into larger VLP (up to 76 nm in diameter) and of tolerating substitution of the spike domain for that of a distantly related Calicivirus. In conditions favoring the natural, T=3 capsid, NB2-VP1 reproducibly assembles by an apparent two-phase process. Our results establish a robust new system with which to probe the dynamics of viral capsid self-assembly.

Structure of the Triatoma virus capsid.

The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

Role of insert-1 of myosin VI in modulating nucleotide affinity.

Myosin VI is unique in its directionality among myosin superfamily members and also displays a slow and strain-dependent rate of ATP binding that allows for gating between its heads. In this study we demonstrate that leucine 310 is positioned by a class VI-specific insert, insert-1, so as to account for the selective hindrance of ATP versus ADP binding. Mutation of leucine 310 to glycine removes all influence of insert-1 on ATP binding. Furthermore, by analyzing myosin VI structures with either leucine 310 substituted to a glycine or complete removal of insert-1, we conclude that nucleotides may initially bind to myosin by their purine rings before docking their phosphate moieties. Otherwise, insert-1 could not exert a differential influence on ATP versus ADP binding.

The post-rigor structure of myosin VI and implications for the recovery stroke.

Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP.

Purification, crystallization and preliminary X-ray analysis of Triatoma virus (TrV) from Triatoma infestans.

Triatoma virus (TrV) is a viral pathogen of the blood-sucking reduviid bug Triatoma infestans, the most important vector of American human trypanosomiasis (Chagas' disease). TrV has been putatively classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work describes the purification of TrV particles from infected T. infestans and their crystallization and preliminary crystallographic analyses. Two different crystal forms, rhombohedral and orthorhombic, were obtained at room temperature by the hanging-drop vapour-diffusion technique using polyethylene glycol and polyethylene glycol monomethylether as precipitants. The rhombohedral crystals have unit-cell parameters a = b = 306.6, c = 788.4 A (hexagonal setting), diffract to 3.2 A resolution and contain one-third of the viral particle per asymmetric unit. The orthorhombic crystals have cell parameters a = 336, b = 351, c = 332 A, diffract to about 2.5 A resolution, and contain one-half of a virus particle in the asymmetric unit. A complete diffraction data set has been collected to 3.2 A resolution, using synchrotron radiation, from a single rhombohedral crystal under cryogenic conditions.