We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a GC content of 40.3%, and is predicted to contain 2,079 coding sequences.

Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IEl49laa and IE2579aa, produced from differentially spliced transcripts under control of the iel/ie2 promoter-enhancer. A deletion mutation removing all 406 IEl49laa-specific amino acids was engineered into the viral genome and this mutant (RC303AAcc) was propagated on an IE149.aa-expressing human fibroblast cell line (ihfiel.3). RC303AAcc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois >3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303AAcc correlated with the ability to express IE2579aa, consistent with a role for IEl49laa in positive autoregulation of the iel/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IEl491aa under high moi conditions. iel-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.

Positive-strand RNA viruses such as poliovirus replicate their genomes on intracellular membranes of their eukaryotic hosts. Electron microscopy has revealed that purified poliovirus RNA-dependent RNA polymerase forms planar and tubular oligomeric arrays. The structural integrity of these arrays correlates with cooperative RNA binding and RNA elongation and is sensitive to mutations that disrupt intermolecular contacts predicted by the polymerase structure. Membranous vesicles isolated from poliovirus-infected cells contain structures consistent with the presence of two-dimensional polymerase arrays on their surfaces during infection. Therefore, host cytoplasmic membranes may function as physical foundations for two-dimensional polymerase arrays, conferring the advantages of surface catalysis to viral RNA replication.

Protein primers are used to initiate genomic synthesis of several RNA and DNA viruses, although the structural details of the primer-polymerase interactions are not yet known. Poliovirus polymerase binds with high affinity to the membrane-bound viral protein 3AB but uridylylates only the smaller peptide 3B in vitro. Mutational analysis of the polymerase identified four surface residues on the three-dimensional structure of poliovirus polymerase whose wild-type identity is required for 3AB binding. These mutants also decreased 3B uridylylation, arguing that the binding sites for the membrane tether and the protein primer overlap. Mutation of flanking residues between the 3AB binding site and the polymerase active site specifically decreased 3B uridylylation, likely affecting steps subsequent to binding. The physical overlap of sites for protein priming and membrane association should facilitate replication initiation in the membrane-associated complex.The replication of linear nucleic acids without loss of coding information from the ends is a problem that has been solved in several ways by cellular and viral genomes. Solutions include those used by vaccinia virus, which primes DNA replication from hairpins that can be refolded to regenerate the terminal sequences (1, 2), by Drosophila, which repair damaged chromosomal termini by recombination (3), by many eukaryotic cells, which add end-specific telomeric sequences postreplicatively, and by viruses such as adenovirus and ⌽29, which use protein primers that are covalently linked to the initiating nucleotides (reviewed in Ref. 4). For mammalian positive-strand RNA viruses such as poliovirus and hepatitis C, two mechanisms are suspected: de novo initiation (5-8) and protein priming (9) from the genomic ends.The protein primer for the synthesis of poliovirus RNA includes, at a minimum, the 22-amino acid viral peptide 3B (also called VPg), which is found covalently linked to the 5Ј ends of all newly synthesized positive and negative strands. Poliovirus translates its proteins as a single, large polyprotein that is cleaved into the proteins required for virion formation, host modification, and RNA replication. In many cases, proteolytic precursors have functions distinct from those of the limit digestion products. Evidence for the use of 3B. As a protein primer comes from in vitro experiments in which it was demonstrated that 3B is uridylylated in the presence of UTP, the poliovirus RNA-dependent RNA polymerase (3D), and an RNA template (9). The RNA used to template the uridylylation of 3B can either be poly(A), poliovirus RNA, or the small cis replication enhancer RNA (10, 11), an internal sequence required for RNA replication in infected cells (12). Whether 3B itself serves as the primer within infected cells and how it is brought into the RNA replication complex are not yet known.Evidence for direct binding between polymerase and 3B was observed in the two-hybrid system (13), although a stronger signal was observed between the polymerase and a larger polypeptide th...

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.Picornaviruses have a single copy, positive-strand RNA genome with a small peptide, VPg (3B), covalently attached to the 5Ј-terminal nucleotide and a poly(A) segment at the 3Ј end. RNA replication occurs in the cytoplasm of infected cells, proceeding through negative-strand intermediates which, in turn, serve as templates for production of progeny positive strands. A long-standing question has been how both positiveand negative-strand syntheses are initiated. Characterization of a reaction catalyzed by poliovirus 3D polymerase, in which the tyrosine of the VPg peptide was trans-esterified (uridylylated) in the presence of a poly(A) template to form VPg-pU (pU), provided insight into the mechanism of strand initiation (34): given the ability of poliovirus 3D polymerase to uridylylate VPg, the peptide-nucleotide conjugate could serve as the protein primer for progeny RNA strand synthesis. VPg must bind directly to poliovirus polymerase, because it can serve as an enzymatic substrate; furthermore, VPg-polymerase interactions have been observed in two-hybrid experiments (42). While the binding site on the poliovirus 3D polymerase for the VPg substrate of the uridylylation reaction has not yet been characterized, the binding site for its likely proteolytic precursor, 3AB, has been identified as a distinct site on the back of the palm of the polymerase molecule via extensive alaninescanning mutagenesis (22).Although VPg uridylylation could provide a protein primer for use in either positive-or negative-strand synthesis, this reaction is not sufficient to describe the mechanism of initiation for viral RNA synthesis in infected cells. For example, the use of a poly(A) template for VPg uridylylation does not provide specificity for a particular virus. This specificity could be provided if an RNA sequence or structure within the picornavirus genome were the authentic RNA template for VPg uridylylation...