The mechanism of translation initiation on Type 1 picornavirus IRESs.

Picornavirus Type 1 IRESs comprise five principal domains (dII-dVI). Whereas dV binds eIF4G, a conserved AUG in dVI was suggested to stimulate attachment of 43S ribosomal preinitiation complexes, which then scan to the initiation codon. Initiation on Type 1 IRESs also requires IRES trans-acting factors (ITAFs), and several candidates have been proposed. Here, we report the in vitro reconstitution of initiation on three Type 1 IRESs: poliovirus (PV), enterovirus 71 (EV71), and bovine enterovirus (BEV). All of them require eIF2, eIF3, eIF4A, eIF4G, eIF4B, eIF1A, and a single ITAF, poly(C) binding protein 2 (PCBP2). In each instance, initiation starts with binding of eIF4G/eIF4A. Subsequent recruitment of 43S complexes strictly requires direct interaction of their eIF3 constituent with eIF4G. The following events can differ between IRESs, depending on the stability of dVI. If it is unstructured (BEV), all ribosomes scan through dVI to the initiation codon, requiring eIF1 to bypass its AUG. If it is structured (PV, EV71), most initiation events occur without inspection of dVI, implying that its AUG does not determine ribosomal attachment.

VP1 B-C and D-E loops of bovine enterovirus cluster B can effectively display foot-and-mouth disease virus type O-conserved neutralizing epitope.

On the basis of generation of an infectious cDNA clone for the BHM26 strain of bovine enterovirus cluster B (BEV-B), 22 sites on different loops of the BHM26 capsid were selected according to an alignment of its sequence with the structural motifs of BEV-A strain VG-5-27 for insertion of the foot-and-mouth disease virus (FMDV) type O-conserved neutralizing epitope 8E8. Two recombinant viruses, rBEV-A1 and rBEV-DE, in which the FMDV epitope was inserted into the VP1 B-C or D-E loops, were rescued by transfection of BHK-21 cells with the in vitro-transcribed RNA of the recombinant BHM26 genome-length cDNA constructs. The two epitope-inserted viruses were genetically stable and exhibited growth properties similar to those of their parental virus in BHK-21 and IBRS-2 cells, which are susceptible to both BEV and FMDV. However, the two recombinant BEVs (rBEVs) had a significantly lower growth titre than those of the parental virus BHM26 in MDBK and Marc145 cells, which are susceptible to BEV but not to FMDV. These results indicated that insertion of the FMDV epitope into the VP1 B-C or D-E loops of the BEV particle altered the replication properties of BEV. In addition, the two rBEVs were sensitive to neutralization by the FMDV type O-specific mAb 8E8, and anti-FMDV IgG antibodies were induced in mice by intramuscular inoculation with the rBEV-A1 and rBEV-DE viruses. Our results demonstrate that the VP1 B-C and D-E loops of the BEV-B particle can effectively display a foreign epitope, making this an attractive approach for the design of BEV-vectored and epitope-based vaccines.

In vitro assembly of an empty picornavirus capsid follows a dodecahedral path.

The Picornaviridae are a large family of small, spherical RNA viruses that includes numerous pathogens. The picornavirus structural proteins VP0, VP1, and VP3 are believed to first form protomers, which then form 14S particles and subsequently assemble to form empty and RNA-filled particles. 14S particles have long been presumed to be pentamers. However, the structure of the 14S particles, their mechanism of assembly, and the role of empty particles during infection are all unknown. We established an in vitro assembly system for bovine enterovirus (BEV) by using purified baculovirus-expressed proteins. By Rayleigh scattering, we determined that 14S particles are 488 kDa, confirming they are pentamers. Image reconstructions based on negative-stain electron microscopy showed that 14S particles have 5-fold symmetry, and their structures correlate extremely well with the corresponding pentamer from crystal structures of mature BEV. Purified 14S particles readily assemble in response to increasing ionic strength or temperature to form 5.8-MDa 12-pentamer particles, indistinguishable from native empty particles. Surprisingly, empty particles were sufficiently stable that, under physiological conditions, dissociation is unlikely to be a biologically relevant reaction. This suggests that empty particles are not a storage form of 14S particles, at least for bovine enterovirus, but are either a dead-end product or direct precursor into which viral RNA is packaged by as-yet-unidentified machinery.

Use of filter carrier technique to measure the persistence of avian influenza viruses in wet environmental conditions.

A germ carrier technique was adapted for the determination of the persistence of influenza viruses in moist environments. The technique was employed with 3 low pathogenic avian influenza viruses (H4N6, H5N1, and H6N8), one human influenza virus (H1N1), and two model viruses (NDV and ECBO) in lake water at five different temperatures (30, 20, 10, 0, and -10°C). Viral quantitation was carried out at regular intervals on cell culture for a maximum duration of 16 weeks. Serial data were analyzed by linear regression model to calculate T-90 values (time required for one log reduction in the virus titer). Persistence of all of the viruses was highest at -10°C followed by 0, 10, 20, and 30°C. At -10°C, the single freeze-thaw cycle resulted in an abrupt decline in the virus titer, followed by long term persistence. Generally, influenza viruses persisted shorter than model viruses while ECBO has the highest survival time in lake water. Individual influenza viruses differed in their persistence at all temperatures. The findings of the present study suggest that AIV can remain infectious in lake water for extended periods of time at low temperatures.

Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton via the nonstructural protein 3Cpro.

Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofed animals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cell morphology and rearrangement of cytoplasmic membranes, which are a consequence of virus replication. We show here, by confocal immunofluorescence and electron microscopy, that the changes in morphology of FMDV-infected cells involve changes in the distribution of microtubule and intermediate filament components during infection. Despite the continued presence of centrosomes in infected cells, there is a loss of tethering of microtubules to the microtubule organizing center (MTOC) region. Loss of labeling for gamma-tubulin, but not pericentrin, from the MTOC suggests a targeting of gamma-tubulin (or associated proteins) rather than a total breakdown in MTOC structure. The identity of the FMDV protein(s) responsible was determined by the expression of individual viral nonstructural proteins and their precursors in uninfected cells. We report that the only viral nonstructural protein able to reproduce the loss of gamma-tubulin from the MTOC and the loss of integrity of the microtubule system is FMDV 3C(pro). In contrast, infection of cells with another picornavirus, bovine enterovirus, did not affect gamma-tubulin distribution, and the microtubule network remained relatively unaffected.

The ultrastructure of the developing replication site in foot-and-mouth disease virus-infected BHK-38 cells.

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus of the Picornaviridae: Infection by picornaviruses results in a major rearrangement of the host cell membranes to create vesicular structures where virus genome replication takes place. In this report, using fluorescence and electron microscopy, membrane rearrangements in the cytoplasm of FMDV-infected BHK-38 cells are documented. At 1.5-2.0 h post-infection, free ribosomes, fragmented rough endoplasmic reticulum, Golgi and smooth membrane-bound vesicles accumulated on one side of the nucleus. Newly synthesized viral RNA was localized to this region of the cell. The changes seen in FMDV-infected cells distinguish this virus from other members of the Picornaviridae, such as poliovirus. Firstly, the collapse of cellular organelles to one side of the cell has not previously been observed for other picornaviruses. Secondly, the membrane vesicles, induced by FMDV, appear distinct from those induced by other picornaviruses such as poliovirus and echovirus 11 since they are relatively few in number and do not aggregate into densely packed clusters. Additionally, the proportion of vesicles with double membranes is considerably lower in FMDV-infected cells. These differences did not result from the use of BHK-38 cells in this study, as infection of these cells by another picornavirus, bovine enterovirus (a close relative of poliovirus), resulted in morphological changes similar to those reported for poliovirus-infected cells. With conventional fixation, FMDV particles were not seen; however, following high-pressure freezing and freeze-substitution, many clusters of virus-like particles were seen.