The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3 termini ended with 5-UGAACC-3 instead of the 3 consensus sequence 5-UGACC-3, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3 consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3 consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3 end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3 consensus sequence.Rotavirus virions are icosahedral particles consisting of three layers of protein and containing 11 segments of doublestranded RNA (dsRNA) (8). The innermost protein layer has a Tϭ2 arrangement and is formed by the core lattice protein VP2 (reviewed in reference 29). Associated with the interior side of each of the 12 pentamers of the VP2 lattice is believed to be one copy each of the viral RNA-dependent RNA polymerase (RdRP) VP1 and the mRNA-capping enzyme VP3 (19,20). Based on structural studies of rotaviruses and other members of the family Reoviridae (29), it is thought that each genome segment exists as a tightly wound spiral around one of the 12 RdRP-capping complexes of the VP2 lattice (9). Collectively, the VP2 lattice, the RdRP-capping complexes, and the dsRNA genome make up the core of the virion. Doublelayered particles consisting of cores surrounded by the intermediate protein VP6 have transcriptase activity and are responsible for the synthesis of the 11 viral mRNAs (1, 37). The dsRNA genome probably exists as a liquid crystal within the core and, in this form, has the fluidity necessary for the dsRNA segments to slide through the anchored RdRP-capping enzyme complex during transcription (9). Nascent transcripts produced by the viral RdRP are extruded through channels located at the vert...

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.Lactococcus lactis strains are of considerable industrial and economic importance, as they are widely used in the production of a variety of fermented dairy products. A characteristic of Lactococcus strains is that they typically possess an abundance of plasmid DNA on which a number of significant technological traits are encoded. These include bacteriophage resistance, bacteriocin production, lactose assimilation, citrate utilization, and proteinase activity (11). Therefore, an extensive knowledge of lactococcal plasmid replication, partition, and stability functions is essential in order to ensure the stable maintenance of these traits. This information can also be applied for the generation of novel stable food-grade cloning and expression vectors for the manipulation of these hosts.There are two modes of bacterial plasmid replication, rolling circle (RC) and theta ( ), both of which have been identified in Lactococcus. The RC plasmids are classified on the basis of homologies within the region of the double-stranded origin and the gene encoding the replication initiation protein. Two classes of RC plasmid are evident in Lactococcus, pE194-like and pC194-like, of which pWV01 (26, 28) and pWC1 (33), respectively, are the prototypes. In general, replicons are classified according to their structural organization and the requirement for host-encoded proteins in the replication process. Originally, the best-characterized replicating plasmids were of gram-negative origin, and three classes designated A, B, and C were identified. Class A plasmids possess an origin of replication (ori), which consists of an AT-rich region, adjacent to a number of iterons which are essential in cis for replication initiation. This mode of replication requires a plasmid-encoded replication initiation protein (Rep) and is DNA polymerase I independent. Class B and C replicons do not harbor a typical ori sequence and require host-encoded DNA poly-

A novel picornavirus from commercial broiler chickens (Gallus gallus domesticus) has been identified and genetically characterized. The viral genome consists of a single-stranded, positive-sense RNA genome of >9243 nt excluding the poly(A) tail and as such represents one of the largest picornavirus genomes reported to date. The virus genome is GC-rich with a G+C content of 54.5 %. The genomic organization is similar to other picornaviruses: 5' UTR-L-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3' UTR. The partially characterized 5' UTR of >373 nt appears to possess a type II internal ribosomal entry site (IRES), which is also found in members of the genera Aphthovirus and Cardiovirus. This IRES exhibits significant sequence similarity to turkey 'gallivirus A'. The 3' UTR of 278 nt contains the conserved 48 nt 'barbell-like' structure identified in 'passerivirus', 'gallivirus', Avihepatovirus and some Kobuvirus genus members. A predicted large open reading frame (ORF) of 8592 nt encodes a potential polyprotein precursor of 2864 amino acids. In addition, the virus contains a predicted large L protein of 462 amino acids. Pairwise sequence comparisons, along with phylogenetic analysis revealed the highest percentage identity to 'Passerivirus A' (formerly called turdivirus 1), forming a monophyletic group across the P1, P2 and P3 regions, with <40, <40 and <50 % amino acid identity respectively. Reduced identity was observed against 'gallivirus A' and members of the Kobuvirus genus. Quantitative PCR analysis estimated a range of 4×10(5) to 5×10(8) viral genome copies g(-1) in 22 (73 %) of 30 PCR-positive faeces. Based on sequence and phylogenetic analysis, we propose that this virus is the first member of a potential novel genus within the family Picornaviridae. Further studies are required to investigate the pathogenic potential of this virus within the avian host.

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Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3 0 -consensus sequence (3 0 CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3 0 CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3 0 -sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN 0-5 CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3 0 CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines.