Molecular Dynamics Studies of the Nucleoprotein of Influenza A Virus: Role of the Protein Flexibility in RNA Binding

The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73–90), loop 2 (200–214). In NP, loops 1 and 2 formed a 10–15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91–112) that interacted with the RNA grove (linker 360–373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein

DNA intercalator stimulates influenza transcription and virus replication

Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII). In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD), was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPIIa in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPIIa) to hyperphosphorylated RNAPII (RNAPIIo)

Influenza Polymerase Activity Correlates with the Strength of Interaction between Nucleoprotein and PB2 through the Host-Specific Residue K/E627

The ribonucleoprotein (RNP) complex is the essential transcription-replication machinery of the influenza virus. It is composed of the trimeric polymerase (PA, PB1 and PB2), nucleoprotein (NP) and RNA. Elucidating the molecular mechanisms of RNP assembly is central to our understanding of the control of viral transcription and replication and the dependence of these processes on the host cell. In this report, we show, by RNP reconstitution assays and co-immunoprecipitation, that the interaction between NP and polymerase is crucial for the function of the RNP. The functional association of NP and polymerase involves the C-terminal ‘627’ domain of PB2 and it requires NP arginine-150 and either lysine-627 or arginine-630 of PB2. Using surface plasmon resonance, we demonstrate that the interaction between NP and PB2 takes place without the involvement of RNA. At 33, 37 and 41°C in mammalian cells, more positive charges at aa. 627 and 630 of PB2 lead to stronger NP-polymerase interaction, which directly correlates with the higher RNP activity. In conclusion, our study provides new information on the NP-PB2 interaction and shows that the strength of NP-polymerase interaction and the resulting RNP activity are promoted by the positive charges at aa. 627 and 630 of PB2

The RNA Polymerase PB2 Subunit of Influenza A/HongKong/156/1997 (H5N1) Restrict the Replication of Reassortant Ribonucleoprotein Complexes

BACKGROUND: Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated. CONCLUSIONS/SIGNIFICANCE: Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes

Influenza Virus Ribonucleoprotein Complexes Gain Preferential Access to Cellular Export Machinery through Chromatin Targeting

In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration

Full Factorial Analysis of Mammalian and Avian Influenza Polymerase Subunits Suggests a Role of an Efficient Polymerase for Virus Adaptation

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern

Sequence analysis of the RNA polymerase gene of African horse sickness virus.

The gene encoding the inner core protein VP1 of African horse sickness virus (AHSV) serotype 9 has been cloned, expressed in vitro and entirely sequenced, completing molecular characterization of the AHSV genome. An analysis of the sequence supporting the identity of AHSV VP1 as the putative viral RNA polymerase is presented

Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3.

The gene encoding the outer capsid protein, VP2, of African horsesickness virus serotype 3 (AHSV-3) has been sequenced in its entirety from cDNA clones of the segment 2 RNA, and compared with the previously published VP2 gene sequence of AHSV-4. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50.5% identity to the amino acid sequence of AHSV-4 VP2. Two areas of high variability (approximately 35% identity) were identified. The N-proximal variable region (amino acids 128 to 309) exhibited significant hydrophilicity, suggesting a possible role in the determination of the serotype-specific immune response. VP2 of AHSV-3 has furthermore been expressed in a baculovirus expression system. The expressed protein was shown to react specifically with an anti-AHSV-3 serum in Western blots. Antibodies raised in rabbits and guinea-pigs against the recombinant VP2 neutralized the virus in a plaque reduction assay, confirming the identity of VP2 as the major neutralization-specific antigen of AHSV