The T = 4 tetravirus and T = 3 nodavirus capsid proteins undergo closely similar autoproteolysis to produce the N-terminal beta and C-terminal, lipophilic gamma polypeptides. The gamma peptides and the N termini of beta also act as molecular switches that determine their quasi equivalent capsid structures. The crystal structure of Providence virus (PrV), only the second of a tetravirus (the first was N omega V), reveals conserved folds and cleavage sites, but the protein termini have completely different structures and the opposite functions of those in N omega V. N termini of beta form the molecular switch in PrV, whereas gamma peptides play this role in N omega V. PrV gamma peptides instead interact with packaged RNA at the particle two-folds by using a repeating sequence pattern found in only four other RNA- or membrane-binding proteins. The disposition of peptide termini in PrV is closely related to those in nodaviruses, suggesting that PrV may be closer to the primordial T = 4 particle than N omega V.

Providence virus (PrV) is a member of the family Tetraviridae, a family of small, positive-sense, ssRNA viruses that exclusively infect lepidopteran insects. PrV is the only known tetravirus that replicates in tissue culture. We have analysed the genome and characterized the viral translation products, showing that PrV has a monopartite genome encoding three ORFs: (i) p130, unique to PrV and of unknown function; (ii) p104, which contains a read-through stop signal, producing an N-terminal product of 40 kDa (p40) and (iii) the capsid protein precursor (p81). There are three 2A-like processing sequences: one at the N terminus of p130 (PrV-2A₁) and two more (PrV-2A₂ and PrV-2A₃) at the N terminus of p81. Metabolic radiolabelling identified viral translation products corresponding to all three ORFs in persistently infected cells and showed that the read-through stop in p104 and PrV-2A₃ in p81 are functional in vivo and these results were confirmed by in vitro translation experiments. The RNA-dependent RNA polymerase domain of the PrV replicase is phylogenetically most closely related to members of the families Tombusviridae and Umbraviridae rather than to members of the family Tetraviridae. The unique genome organization, translational control systems and phylogenetic relationship with the replicases of (+ve) plant viruses lead us to propose that PrV represents a novel family of small insect RNA viruses, distinct from current members of the family Tetraviridae.

Providence Virus (PrV) is a non-envoloped, T = 4 icosahedral beta-tetravirus that undergoes autocatalytic cleavage of its coat protein precursor after capsid assembly. This is also a well characterized function of Nudaurelia capensis ω virus (NωV), a member of the related omegatetraviruses, whose x-ray structure has been determined. Virus-like particle (VLP) production of PrV in a recombinant baculovirus expression system was attempted to obtain high VLP yields for comparison of structural and autocatalytic active site properties between these virus groups. This resulted in insoluble aggregates of PrV coat protein even though NωV VLPs have been successfully produced in the same system. Betatetraviruses may be more dependent on compartmentalization and availability of their full-length genome for proper folding and assembly. However, crystals were grown of limited quantities of authentic PrV produced in cell culture and a partial X-ray data set collected to 3.8 Å resolution. The virus particle position and orientation in the unit cell was determined by space group consideration and rotation function analysis. A phasing model, based on NωV, was developed to initiate the structure solution of PrV.

At the International Congress of Virology (ICV) in San Francisco in July 2005, the International Committee on Taxonomy of Viruses (ICTV) held a Plenary Session. This note summarizes the reports made to the meeting by the President and the chairs of the ICTV subcommittees acknowledged at the end of this note. It also reports the results of elections to positions on the ICTV Executive Committee (EC) and changes made to the statutes that govern how ICTV operates.

ABSTRACT Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 104-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.

ABSTRACT Gene expression of the nonsegmented negative-strand RNA viruses is determined by the position of each gene relative to that the single 3′ promoter. The general order of genes among all of the viruses of the order Mononegavirales is highly conserved. In previous work we generated recombinant viruses in which the order of the three central genes of the prototypical rhabdovirus, vesicular stomatitis virus, was rearranged to all six possible permutations. While some of these viruses replicated less well than the wild type when assayed by single-step growth analyses in BSC-1 cells, others replicated as well or slightly better. In the work reported here, we used competition assays to compare the fitness of the viruses with alternative gene orders to that of the wild-type (wt) virus. We found that the relative fitness of these recombinant viruses depended on the multiplicity of infection (MOI) but not on the population size. However, during competitions at low MOI, when complementation cannot compensate for the defects of the populations with rearranged genomes, the virus with the wt gene order was always the most fit.

ABSTRACT The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-Å resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.

ABSTRACT During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.

Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila , which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila . The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells.

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia β virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.

The nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3′ 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3′ termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3.

Nodamura virus (NoV) was the first isolated member of the Nodaviridae, and is the type species of the alphanodavirus genus. The alphanodaviruses infect insects; NoV is unique in that it can also lethally infect mammals. Nodaviruses have bipartite positive-sense RNA genomes in which RNA1 encodes the RNA-dependent RNA polymerase and the smaller genome segment, RNA2, encodes the capsid protein precursor. To facilitate the study of NoV, we generated infectious cDNA clones of its two genomic RNAs. Transcription of these NoV1 and NoV2 cDNAs in mammalian cells led to viral RNA replication, protein synthesis, and production of infectious virus. Subgenomic RNA3 was produced during RNA replication and encodes nonstructural proteins B1 and B2 in overlapping ORFs. Site-directed mutagenesis of these ORFs, followed by SDS–PAGE and MALDI–TOF mass spectrometry analyses, showed synthesis of B1 and two forms of B2 (B2-134 and B2-137) during viral replication. We also characterized a point mutation in RNA1 far upstream of the RNA3 region that resulted in decreased RNA3 synthesis and RNA2 replication, and a reduced yield of infectious particles. The ability to reproduce the entire life cycle of this unusual nodavirus from cDNA clones will facilitate further analysis of NoV RNA replication and pathogenesis.

Virions of the alphanodavirus Pariacoto virus (PaV) have T=3 icosahedral symmetry and are assembled from multiple copies of a precursor protein that is cleaved into two mature capsid proteins after assembly. The crystal structure of PaV shows that the N-terminal approximately 30 amino acid residues of the subunits surrounding the 5-fold axes interact extensively with icosahedrally ordered regions of the encapsidated positive-sense genomic RNAs. We found that wild-type PaV particles also contain a minor capsid protein that is truncated by 24 residues at its N terminus. Reverse genetic experiments showed that translation of this protein initiated at the second AUG of the capsid protein open reading frame. When either the longer or shorter version of the capsid protein was expressed independently of the other, it assembled into virus particles and underwent maturational cleavage. Virions that lacked the shorter capsid protein retained infectivity for cultured insect cells and Galleria mellonella larvae.

To date, the insect nodavirus flock house virus (FHV) is the only virus of a higher eukaryote that has been shown to undergo a full replicative cycle and produce infectious progeny in the yeast Saccharomyces cerevisiae. The genome of FHV is composed of two positive-sense RNA segments: RNA1, encoding the RNA replicase, and RNA2, encoding the capsid protein precursor. When yeast cells expressing FHV RNA replicase were transfected with a chimeric RNA composed of a selectable gene flanked by the termini of RNA2, the chimeric RNA was replicated and transmitted to daughter cells indefinitely. In the work reported here, we developed a system in which a selectable chimeric RNA replicon was transcribed from an inducible RNA polymerase II (polII) promoter in vivo in yeast. To render marker gene expression absolutely dependent on RNA replication, the primary polII transcript was made negative in sense and contained an intron that blocked the translation of cryptic transcripts from the opposite DNA strand. The RNA products of DNA-templated transcription, processing, and RNA replication were characterized by Northern blot hybridization and primer extension analysis. Marker gene expression and colony growth under selective conditions depended strictly on FHV RNA replication, with background colonies arising at a frequency of fewer than 1 in 10(8) plated cells. The utility of the system was demonstrated by introducing a second chimeric replicon and showing that at least two different selectable markers could be simultaneously expressed by means of RNA replication. This is the first example of FHV RNA1-dependent selectable marker expression initiated in vivo and will greatly facilitate the identification and characterization of the requirements and inhibitors of RNA replication.

ABSTRACT Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.

The insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis. Here we report that RNA1 mutants deficient in RNA3 synthesis failed to support RNA2 replication. This effect was not caused by alterations in the RdRp catalytic subunit nor by a lack of the proteins encoded by RNA3. Furthermore, RNA3 supplied in trans from an exogenous source restored RNA2 replication. These data indicate that RNA3 transactivates the replication of RNA2, a novel property for a viral RNA. We propose that the RNA3 dependence of RNA2 replication serves to coordinate replication of the FHV genome segments.