Modification of nonstructural protein 1 of influenza A virus by SUMO1.

Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.

Influenza A virus replication induces cell cycle arrest in G0/G1 phase.

Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G(0)/G(1)-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G(0)/G(1) phase into S phase. Consistent with the G(0)/G(1)-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G(1) into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G(0)/G(1)-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G(0)/G(1) phase were observed compared to those in either unsynchronized cells or cells synchronized in the G(2)/M phase. G(0)/G(1)-phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G(0)/G(1)-phase cell cycle arrest in infected cells.

Intracellular distribution of NS1 correlates with the infectivity and interferon antagonism of an avian influenza virus (H7N1).

Highly pathogenic avian influenza viruses of subtype H7N1 that emerged during an outbreak in 1999 and 2000 in Italy differ from their low-pathogenicity precursor viruses by changes in several genes, including three mutations in the NS1 protein. Two of them involve amino acid exchanges located within or closely adjacent to the nuclear export signal of NS1. The third mutation resulted in a new stop codon and thereby a C-terminal truncation of the NS1 protein of the highly pathogenic viruses. To find out whether these mutations contribute to the phenotypic differences between the highly pathogenic and low pathogenic viruses, we generated recombinants of the highly pathogenic A/ostrich/Italy/984/00 strain that contained the nuclear export signal and/or the extended C terminus of NS1 of a low pathogenic virus (A/chicken/Italy/1082/99). Using these recombinants we could demonstrate that replication rate and spread of infection in chicken fibroblast cultures, as well as infectivity for chicken embryos is reduced, whereas the mean death time for chicken embryos is increased, when the highly pathogenic virus acquires the NS1 motifs of the low pathogenic virus. Analysis of beta interferon transcription in chicken fibroblasts infected with the recombinants revealed that the mutations observed in the nuclear export signal of the highly pathogenic viruses were responsible for the enhanced interferon antagonism of these viruses. Cell fractionation and immunofluorescence studies in chicken fibroblasts showed that the nuclear export signal of the highly pathogenic viruses is responsible for cytoplasmic accumulation of NS1, whereas the C-terminal truncation promotes transport into the nucleoli. Comparative analysis in human A549 cells indicated that intracellular distribution of NS1 is host specific. Taken together, these observations support the concept that compartmentalization of NS1 within the cell contributes to the pathogenicity of avian influenza viruses.

Mutations at positions 186 and 194 in the HA gene of the 2009 H1N1 pandemic influenza virus improve replication in cell culture and eggs.

Obtaining suitable seed viruses for influenza vaccines poses a challenge for public health authorities and manufacturers. We used reverse genetics to generate vaccine seed-compatible viruses from the 2009 pandemic swine-origin influenza virus. Comparison of viruses recovered with variations in residues 186 and 194 (based on the H3 numbering system) of the viral hemagglutinin showed that these viruses differed with respect to their ability to grow in eggs and cultured cells. Thus, we have demonstrated that molecular cloning of members of a quasispecies can help in selection of seed viruses for vaccine manufacture.

Human RNA polymerase I-driven reverse genetics for influenza a virus in canine cells.

We have established a human RNA polymerase I (pol I)-driven influenza virus reverse genetics (RG) system in the Madin-Darby canine kidney 33016-PF cell line, which is approved for influenza vaccine manufacture. RNA pol I polymerases are generally active only in cells of species closely related to the species of origin of the polymerases. Nevertheless, we show that a nonendogenous RNA pol I promoter drives efficient rescue of influenza A viruses in a canine cell line. Application of this system allows efficient generation of virus strains and presents an alternative approach for influenza vaccine production.