We report an extensive characterization of two H5N1 influenza viruses, SW/FJ/01 and SW/FJ/03, that were isolated from pigs in Fujian Province, southern China. The two swine viruses share high homology with a duck virus, DK/ZJ/00, that has been characterized previously. The virus SW/FJ/03 has a unique five-amino-acid deletion in the NS gene. The two swine influenza viruses replicated in the lungs of mice without prior adaptation, but they displayed low pathogenicity in mice. Interestingly, the viruses displayed differing levels of pathogenicity in chickens. The SW/FJ/03 virus was less pathogenic than SW/FJ/01, even though the HA protein of SW/FJ/03 bears multiple basic amino acids in its cleavage site, a known marker for high pathogenicity. Using reverse genetics, we generated a series of single-gene recombinants by switching the genes of the SW/FJ/01 and SW/FJ/03 viruses, and we then tested their replication and lethality in chickens. We demonstrated that the five-amino-acid deletion at positions 191 to 195 in the NS1 protein contributes to the attenuation of the SW/FJ/03 virus in chickens and that the attenuation phenotype of this virus in chickens may be associated with its decreased ability to antagonize IFN-α/β production in host cells.
Our data also support the hypothesis that pigs can serve as an intermediate host for influenza viruses to evolve in a direction toward being able to replicate more efficiently in animals other than their natural hosts. We found that there are only 23 amino acid differences between the genomes of the SW/FJ/01 and DK/ZJ/00 viruses, but the replication phenotypes of these two viruses in mice are quite different. DK/ZJ/00 does not replicate in mice (4
), whereas SW/FJ/01 replicates well in the mouse lung and kills mice at high doses (105
). SW/FJ/01 does not, however, contain the markers 627K and 701N in the PB2 protein, which are associated with the replication and virulence of H5N1 influenza viruses in mice (9
). This suggests the existence of additional mutations that contribute to the replication of H5N1 viruses in mammals. The SW/FJ/01 and SW/FJ/03 viruses were isolated from the same region of Fujian Province, and besides the deletion in the NS gene of SW/FJ/03, there are only five amino acid differences between the two isolates. Two of these five amino acids (HA 444L and M2 26L) in SW/FJ/03 are the same as those in DK/ZJ/00, implying that the virus may have circulated in this region and been transmitted back and forth between waterfowl and pigs.
NS1 has multiple regulatory functions during influenza virus infection (11
). Here we confirmed that the deletion of the amino acids at positions 191 to 195 in the NS1 protein plays an important role in the attenuation of the SW/FJ/03 or R-SW/FJ/01-03 virus in chickens and in its ability to antagonize host cell IFN-α/β production. There are several possible explanations for how this deletion could contribute to the low-pathogenicity phenotype and the inability to antagonize IFN. First, the deletion is located within the effector domain, whose main function is the stabilization and/or facilitation of NS1 dimerization (41
). Interestingly, it was shown that truncation of the C-terminal domain significantly decreased the stability of an NS1 protein from a swine influenza virus (34
) but not that of NS1 from the influenza virus WSN (41
). We found that the amount of NS1 protein in cells infected with R-SW/FJ/03 or SW/FJ/01-03NS was significantly lower than that in cells infected with R-SW/FJ/01 or SW/FJ/03-01NS (Fig. ) and that the SW/FJ/03 NS1 protein exhibited decreased stability compared to the SW/FJ/01 NS1 protein (Fig. ). It is thus possible that the deletion in the effector domain affects NS1 protein stabilization, resulting in reduced amounts of the NS1 protein in infected cells. The NS1 protein is responsible for IFN antagonist activity in mammalian (6
) and chicken (23
) cells. Decreased NS1 protein levels could therefore lead to a decreased ability to antagonize IFN production. Second, the deletion in the effector domain could lead to impaired multimerization of the NS1 protein, which would weaken NS1 binding to RNA (40
). The NS1 protein of influenza A virus binds to double-stranded RNA and prevents double-stranded RNA-mediated activation of protein kinase R (1
). It is this kinase that induces IFN-α/β synthesis (6
). Therefore, the NS1 deletion could lead to the attenuated phenotype through a decreased NS1 protein level and/or a reduction in NS1's RNA binding ability.
The deletion of amino acids 191 to 195 of the NS1 protein of SW/FJ/03 negatively affects the ability of NS1 to bind the cellular 30-kDa subunit CPSF. CPSF is an essential component of the 3′-end processing machinery of cellular pre-mRNAs, and the influenza virus NS1 protein is known to interact with the 30-kDa subunit and to inhibit 3′-end formation of cellular pre-mRNAs (21
). The inhibition of cellular pre-mRNA processing and subsequent protein translation are among the antiviral mechanisms employed by influenza virus (17
). Two distinct domains in the C-terminal effector domain, namely, the well-characterized region around amino acid 186 (39
) and the recently identified middle region around positions 103 and 106 (16
), have been proved to be required for binding to the host factor CPSF, resulting in an inhibition of cellular mRNA processing. In the present study, we proved that the amino acid deletion at positions 191 to 195 of NS1 of the SW/FJ/03 virus impairs NS1 binding to the 30-kDa CPSF subunit and may lead to more cellular pre-mRNA, including antiviral IFN-α/β pre-mRNA and proteins.
The nucleotide deletion in the SW/FJ/03 NS gene also results in a five-amino-acid deletion at positions 39 to 43 of the NS2 protein. The NS2 protein of influenza A virus mediates the nuclear export of viral ribonucleoproteins (28
), and mutations within the nuclear export signal motif (amino acids 12 to 21) of NS2 affect the export of viral ribonucleoproteins and limit viral growth in cell culture and in mice (15
). Although the function, if any, of the NS2 region affected by the deletion at positions 39 to 43 is unknown, we believe that it is unlikely that this deletion in NS2 affects the attenuation of the SW/FJ/03 virus in chickens.
Several reports suggest that mutations or deletions in the NS1 protein contribute to the virulence of influenza viruses in different hosts (3
). In this study, we found that the amino acid deletion at positions 191 to 195 of the NS1 protein attenuated the SW/FJ/03 virus in chickens and was critical for the virus to antagonize the host cell IFN response. Our results further strengthen the view that the NS1 protein is an important virulence factor for influenza viruses and that multiple domains within this protein may be suitable targets for the development of antiviral drugs and attenuated vaccines.