The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex

The RNA genome of influenza virus is transcribed and replicated by the viral RNA polymerase complex in the cell nucleus. We have generated green fluorescent protein (GFP)-tagged polymerase subunits to study the assembly of the polymerase complex. Our results show that individually expressed polymerase basic protein 1 (PB1) and polymerase acidic protein (PA) subunits were distributed in both the cytoplasm and the nucleus, while the polymerase basic protein 2 (PB2) subunit accumulated in the nucleus. Although it has been reported that PB1 alone accumulates in the nucleus, we demonstrate that PB1 requires the coexpression of PA for efficient nuclear accumulation. Our results support a model which proposes that PB1 and PA are transported into the nucleus as a complex.     

Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1.

Influenza virus polymerase complex is a heterotrimer consisting of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Of these, only PB1, which has been implicated in RNA chain elongation, possesses the four conserved motifs (motifs I, II, III, and IV) and the four invariant amino acids (one in each motif) found among all viral RNA-dependent RNA or RNA-dependent DNA polymerases. We have modified an assay system developed by Huang et al. (T.-J. Huang, P. Palese, and M. Krystal, J. Virol. 64:5669-5673, 1990) to reconstitute the functional polymerase activity in vivo. Using this assay, we have examined the requirement of each of these motifs of PB1 in polymerase activity. We find that each of these invariant amino acids is critical for PB1 activity and that mutation in any one of these residues renders the protein nonfunctional. We also find that in motif III, which contains the SSDD sequence, the signature sequence of influenza virus RNA polymerase, SDD is essentially invariant and cannot accommodate sequences found in other RNA viral polymerases. However, conserved changes in the flanking sequences of SDD can be partially tolerated. These results provide the experimental evidence that influenza virus PB1 possesses a similar polymerase module as has been proposed for other RNA viruses and that the core SDD sequence of influenza virus PB1 represents a sequence variant of the GDN in negative-stranded nonsegmented RNA viruses, GDD in positive-stranded RNA virus and double-stranded RNA viruses, or MDD in retroviruses.     

A Rab11- and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNA

The viral RNA (vRNA) genome of influenza A virus is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins (RNPs), and trafficked to the plasma membrane through uncertain means. Using fluorescent in situ hybridization to detect vRNA as well as the live cell imaging of fluorescently labeled RNPs, we show that an early event in vRNA cytoplasmic trafficking involves accumulation near the microtubule organizing center in multiple cell types and viral strains. Here, RNPs colocalized with Rab11, a pericentriolar recycling endosome marker. Cytoplasmic RNP localization was perturbed by inhibitors of vesicular trafficking, microtubules, or the short interfering RNA-mediated depletion of Rab11. Green fluorescent protein (GFP)-tagged RNPs in living cells demonstrated rapid, bidirectional, and saltatory movement, which is characteristic of microtubule-based transport, and also cotrafficked with fluorescent Rab11. Coprecipitation experiments showed an interaction between RNPs and the GTP-bound form of Rab11, potentially mediated via the PB2 subunit of the polymerase. We propose that influenza virus RNPs are routed from the nucleus to the pericentriolar recycling endosome (RE), where they access a Rab11-dependent vesicular transport pathway to the cell periphery.     

RNA聚合酶——抗流感病毒的新靶点

RNA聚合酶是由PA、PB1和PB2三个亚基构成的蛋白质复合物,在流感病毒基因组的转录复制过程中发挥着重要作用。随着研究的不断深入,RNA聚合酶已经成为抗流感病毒药物重要的靶点。本文介绍了RNA聚合酶各个亚基结构、功能以及RNA聚合酶抑制剂的研究进展。     

The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes

The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.