The fruit fly Drosophila melanogaster is a model system for studying innate immunity, including antiviral host defense. Infection with drosophila C virus triggers a transcriptional response that is dependent in part on the Jak kinase Hopscotch. Here we show that successful infection and killing of drosophila with the insect nodavirus flock house virus was strictly dependent on expression of the viral protein B2, a potent inhibitor of processing of double-stranded RNA mediated by the essential RNA interference factor Dicer. Conversely, flies with a loss-of-function mutation in the gene encoding Dicer-2 (Dcr-2) showed enhanced susceptibility to infection by flock house virus, drosophila C virus and Sindbis virus, members of three different families of RNA viruses. These data demonstrate the importance of RNA interference for controlling virus replication in vivo and establish Dcr-2 as a host susceptibility locus for virus infections.

Borna disease virus is a neurotropic negativestrand RNA virus that infects a wide range of vertebrate hosts, causing disturbances in movement and behavior. We have cloned and sequenced the 8910-nucleotide viral genome by using RNA from Borna disease virus particles. The viral genome has complementary 3' and 5' termini and contains antisense information for five open reading frames. Homology to Filoviridae, Paramyxoviridae, and Rhabdoviridae is found in both cistronic and extracistronic regions. Northern analysis indicates that the virus transcribes mono-and polycistronic RNAs and uses terminatlon/polyadenylylation signals reminiscent ofthose observed in other negative-strand RNA viruses. Borna disease virus is likely to represent a previously unrecognized genus, bornaviruses, or family, Bornaviridae, within the order Mononegavirales.

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

Porcine circovirus 2 (PCV2) is a T‫1؍‬ nonenveloped icosahedral virus that has had severe impact on the swine industry. Here we report the crystal structure of an N-terminally truncated PCV2 virus-like particle at 2.3-Å resolution, and the cryo-electron microscopy (cryo-EM) image reconstruction of a full-length PCV2 virus-like particle at 9.6-Å resolution. This is the first atomic structure of a circovirus. The crystal structure revealed that the capsid protein fold is a canonical viral jelly roll. The loops connecting the strands of the jelly roll define the limited features of the surface. Sulfate ions interacting with the surface and electrostatic potential calculations strongly suggest a heparan sulfate binding site that allows PCV2 to gain entry into the cell. The crystal structure also allowed previously determined epitopes of the capsid to be visualized. The cryo-EM image reconstruction showed that the location of the N terminus, absent in the crystal structure, is inside the capsid. As the N terminus was previously shown to be antigenic, it may externalize through viral "breathing."

BDV uses a remarkably broad range of mechanisms to direct expression of its 8.9-kb genome. Although much remains to be elucidated, it is clear that BDV genome expression is modulated by the use of multiple strategies, including differential gene transcription, post-transcriptional modification, and translational efficiency. Further insights into the details of this multilevel system will be essential to understanding BDV biology, pathogenesis, and neurotropism.

Despite tremendous advances in high-resolution structure determination of virus particles, the organization of encapsidated genomes and their role during assembly are poorly understood. This article summarizes recent insights from structural, biochemical, and genetic analyses of icosahedral viruses that contain single-stranded, positive-sense RNA genomes. X-ray crystallography of several viruses in this category has provided tantalizing glimpses of portions of the packaged nucleic acid, contributing crucial information on how the genome might be folded within the virion. This information combined with theoretical considerations and data from molecular approaches suggests mechanisms by which coat proteins interact with genomic RNA to shape it into a conformation that is compatible with the geometry of the virion. It appears that RNA, in addition to its function as a repository for genetic information, plays an important structural role during assembly and can on occasion override the ability of the coat protein to form a particle with defined icosahedral symmetry.

The nodavirus Flock house virus (FHV) has a bipartite, positive-sense RNA genome that is packaged into an icosahedral particle displaying T‫3؍‬ symmetry. The high-resolution X-ray structure of FHV has shown that 10 bp of well-ordered, double-stranded RNA are located at each of the 30 twofold axes of the virion, but it is not known which portions of the genome form these duplex regions. The regular distribution of double-stranded RNA in the interior of the virus particle indicates that large regions of the encapsidated genome are engaged in secondary structure interactions. Moreover, the RNA is restricted to a topology that is unlikely to exist during translation or replication. We used electron cryomicroscopy and image reconstruction to determine the structure of four types of FHV particles that differed in RNA and protein content. RNA-capsid interactions were primarily mediated via the N and C termini, which are essential for RNA recognition and particle assembly. A substantial fraction of the packaged nucleic acid, either viral or heterologous, was organized as a dodecahedral cage of duplex RNA. The similarity in tertiary structure suggests that RNA folding is independent of sequence and length. Computational modeling indicated that RNA duplex formation involves both short-range and long-range interactions. We propose that the capsid protein is able to exploit the plasticity of the RNA secondary structures, capturing those that are compatible with the geometry of the dodecahedral cage.High-resolution X-ray analysis of icosahedral viruses has provided detailed insights into the organization of the viral capsid and the structure of the individual coat protein subunits. The encapsidated nucleic acid, on the other hand, is rarely visualized, in part because it does not conform to the icosahedral symmetry of the virus particle. There are exceptions to this, however, as exemplified by the plant viruses bean pod mottle virus (BPMV) (3) and satellite tobacco mosaic virus (STMV) (10) and the invertebrate nodaviruses Pariacoto virus (PaV) (20) and Flock house virus (FHV) (7). In these viruses, regions of the encapsidated single-stranded RNA genome interact with coat protein subunits at symmetrically equivalent positions, and an average structure of these ordered regions has been visualized at high resolution.BPMV, a Tϭ3 icosahedral virus, has a bipartite positivesense single-stranded RNA genome, with each RNA molecule packaged in a separate particle. In the crystal structure of particles containing RNA2 (ϳ3.6 kb), seven well-ordered ribonucleotides were visible near the icosahedral threefold axes of the virion (3). In addition, a total of 660 ribonucleotides corresponding to almost 20% of the packaged RNA could be modeled into the electron density. The overall structure of the ordered RNA is a single-stranded helix, which approximates that found for one strand of an A-type RNA duplex (3, 4). Because the RNA density in the X-ray map represents an average of the densities at symmetrically equivalent positions, the nucleot...

No abstract