Minimum molecular architectures for transcription and replication of the influenza virus

Abstract

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA-PB1-PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA-PB1 and PB1-PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1-PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA-PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA. T he genome of influenza virus is composed of eight negativestrand viral RNA (vRNA) segments, which altogether encode 10 different viral proteins (1). The vRNA polymerase is involved in both transcription and replication (for reviews see refs. 2-4). In transcription, the RNA polymerase catalyzes not only RNA polymerization but also the cleavage of host cell mRNA to generate capped RNA primers (5-8) and polyadenylation of mRNA (9-11). The RNA polymerase also carries an apparent proofreading (12). The RNA polymerase is composed of one molecule each of three viral proteins, PB1, PB2 and PA (13). The PB1 protein plays central roles in both RNA polymerase assembly (14, 15), and RNA polymerization (16,17). The PB1 gene also encodes a short immunogenic peptide by the ϩ1 reading frame with killing activity of host immune cells (18). The PB2 subunit plays a role in recognition and binding of capped RNA for generation of primers for RNA synthesis (19-21). The consensus sequence for RNA cap-binding exists in this subunit (22), but the catalytic site for capped RNA cleavage was suggested to locate in PB1 (23). The function of PA remains unidentified, but mutations in the PA gene affect replication (reviewed in ref. 24).Because the content of RNA polymerase in virions is very low (1, 3, 4) and moreover it is tightly associated with vRNA (13), it is difficult to purify large amounts of the RNA polymerase. The efficiency of reconstitution in vitro is too low to get the RNA polymerase in active form (25,26). For the large-scale production, attempts have been made to develop expression systems of the recombinant RNA polymerase. The initial success was achieved by using a vaccinia virus and was used for in vivo studies (27,28). However, because of the low level of expression and the cytopathic effect of vaccinia virus vector, this system is not suitable for large-scal...

Detection and Characterization of Influenza A Virus PA-PB2 Interaction through a Bimolecular Fluorescence Complementation Assay

“…The observation that PA interacts with PB2 in the absence of PB1 raises an interesting question of whether the PA-PB2 dimer could perform any polymerase activity independent of the intact heterotrimeric complex. Several studies have shown that the PB1-PA binary complex can efficiently synthesize cRNA in the absence of PB2 protein (17,31). Future studies are needed to address the role the PA-PB2 dimer can play in virus polymerase activity.…”

Internal Initiation of Influenza Virus Replication of Viral RNA and Complementary RNA in Vitro

“…De Novo Initiation via the Internal Initiation of TemplatesWe found that the replication initiation site of the vRNA was located at the second nucleotide of the 3Ј-end, which disagrees with the previous reports (7,22). Therefore, we examined first the initiation nucleotide of v84 using [␥- 32 (Fig.…”

“…The replication of the influenza virus is believed to be initiated without primers because the RNA genome (vRNA) purified from the virus was found to possess a terminal triphosphate at the 5Ј-end (30). The purified RdRp heterotrimer was found to initiate transcription from the 3Ј-end of vRNA without primers (7,22). Recently, the internal initiation and primer realignment model of influenza virus replication was proposed by Brownlee and co-workers (22).…”

“…2F). This was the model RNA template identical to that used by Honda et al (7). Deng et al (22) used a two-RNA system.…”

“…The RdRp heterotrimer purified from an influenza virion consists of three subunits, PB1, PB2, and PA (6). The purified influenza virus RdRp from insect and mammalian cells catalyzes AG and cap-1 primed transcription and polyadenylation (model of viral transcription) and de novo initiation (model of viral genome replication) in vitro (7)(8)(9).…”