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...

PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.

The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA.

A collection of influenza A virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the Cterminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) compete with the wild-type PB2 protein in this reconstituted system. These experiments showed that many of the mutants behaved as dominant negative; i.e., they were unable to induce cat gene expression but interfered with wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or ␤-globin mRNA as a primer. One of the mutants, I299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher ␤-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.