No abstract

The PA subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. The aminoterminal third of the protein is responsible for the induction of proteolysis. We mutated five potential casein kinase II phosphorylation sites located in the amino-terminal third of the protein. The influenza virus RNA polymerase is a heterotrimer formed by the PB1, PB2, and PA subunits. It associates with nucleoprotein (NP)-complexed viral RNA (vRNA) to form virion ribonucleoproteins (vRNPs). In influenza virus-infected cells, the vRNPs direct two types of RNA synthesis: mRNA synthesis (transcription) and vRNA amplification (replication). For mRNA synthesis, 5Ј-capped oligonucleotides derived from cellular mRNAs by cap-snatching are used as primers (21). These primers are elongated until polyadenylation occurs at a signal of five to seven U residues close to the 5Ј end of the template (24,(32)(33)(34). Replication, in contrast, occurs without primer. The vRNA template is copied to form full-length positive-stranded RNA (cRNA), which serves as a template for vRNA synthesis (18,21). Free NP is required as an antitermination factor to ignore the polyadenylation signal during the synthesis of cRNA (39). However, a detailed picture of the mechanism of the transcription-replication switch is still lacking.The PB1 subunit contains several sequence motifs characteristic of the vRNA-dependent RNA polymerases (31). These motifs have been shown to be essential for vRNA synthesis (6), suggesting that PB1 is the polymerase itself. PB2 protein binds CAP1 structures (7, 41) and might contain the endonucleolytic activity responsible of the cleavage of host mRNA precursors (8, 23). The phenotype of viral temperature-sensitive (ts) mutants indicates that the PA subunit is involved in vRNA replication (reviewed in reference 25), but its precise role in this process is unknown. The PA subunit induces a generalized proteolytic process when expressed individually from cloned cDNA (36), and the amino-terminal third of the molecule (positions 1 to 247) is sufficient to activate this proteolysis (38).We recently showed that the PA protein is phosphorylated in vivo and that it is a substrate of casein kinase II in vitro (37). PA protein contains 11 potential phosphorylation sites for casein kinase II in its molecule, 8 of them located in a cluster inside the first 247 N-terminal amino acids. Therefore we produced point mutations of several putative casein kinase II phosphorylation sites located at the amino-terminal third of the protein and studied the consequences of these genetic changes in the activity of the mutated PA proteins. Some of these PA mutants presented decreased ability to induce proteolysis. Interestingly, the capacity of these mutants to support replication of model vRNA in a polymerase reconstituted in vivo from cloned cDNAs strongly correlated with their proteolysis induction, but all mutants were as active as wil...

We describe a method for African swine fever (ASF) virus purification based on equilibrium centrifugation in Percoll density gradients of extracellular virions produced in infected VERO cells that yielded about 15 +/- 9% recovery of the starting infectious virus particles. The purified virus preparations were essentially free of a host membrane fraction (vesicles) that could not be separated from the virus by previously described purification methods. The purified virus sedimented as a single component in sucrose velocity gradients with a sedimentation coefficient of 3,500 +/- 300S, showed a DNA-protein ratio of 0.18 +/- 0.02 and a specific infectivity of 2.7 X 10(7) PFU/micrograms of protein, and remained fully infectious after storage at -70 degrees C for at least 7 months. The relative molecular weights of the 34 polypeptides detected in purified virus particles ranged from 10,000 to 150,000. Some of these proteins were probably cellular components that might account for the reactivity of purified virus with antiserum against VERO cells.