In generating NS1 C-terminal truncation mutants of a human influenza A strain, we were able to ablate the functions of NS1 in that strain, demonstrating its potent inhibition potential. Interestingly, using these human viruses, we were also able to compare the NS1 functions in a laboratory strain of influenza virus, and we have characterized key differences in their manners of inhibition of antiviral responses. Importantly, we have demonstrated from our current study that the human influenza A NS1 protein has multiple capacities to inhibit the innate immune responses to influenza A virus infection, which gives it an advantage to human influenza virus. However, this strong virulence factor is rendered incompetent with C-terminal truncations as these deletions lead to a gross destabilization of NS1 expression. This suggests that the C terminus of human influenza A virus NS1 is important for the stability of NS1 leading to viral pathogenesis, and mutants that contain this type of truncation would be virtual NS1 knockouts and thus potential options for an efficacious vaccine.
We have investigated, for the first time, an ex vivo model of human influenza A virus infection using a human isolate of influenza A virus and human primary cells to better understand the role of the NS1 protein of influenza virus in its pathogenesis and in its efficient replication and evasion of host immunity. Previous studies have demonstrated the importance of the C-terminal region of the influenza A virus NS1 protein during infection by showing that truncating the C-terminal domain of the NS1 results in a decreased viral growth rate and decreased viral NS1, HA, and M protein expression (12
). However, our model is unique in its ability to study human NS1 mutant viruses using primary human cells. Using human TX WT virus, we found that this virus efficiently expressed viral proteins in a human lung epithelial cell line (A549), in primary HTBE cells, and in DCs. As DCs support influenza virus infection but do not release infectious viral particles (for mice, see reference 35
; data not shown for humans), viral replication was demonstrated in these cells by analyzing Western blots for viral proteins in the present study. Our comparison of TX WT viral replication with the NS1-truncated TX 1-99 and TX 1-126 viruses supports a role for the NS1 protein in enhancing the expression of other viral proteins in relevant human cells. Although TX 1-99 and TX 1-126 appeared to replicate well in the low-IFN environment of 7-day-old eggs, they exhibited diminished viral replication in A549 cells and DCs, as demonstrated by the poor expression of the HA, M1, and NS1 proteins. In HTBE cell infections, most of the infected cells were nonciliated, consistent with the known tropism of human influenza viruses (40
). There were no significant differences in the numbers of infected cells during early TX WT or TX 1-126 infections (9.5 h postinfection). However, the number of infected cells was decreased at later times in HTBE infections with TX 1-126 virus (25 h postinfection). These results are consistent with the lack of differences in viral titers between these two viruses at 9 h postinfection. However, at 25 h, more than 50% of the cells on the culture were infected with TX WT, and only approximately 30% were infected with TX 1-126. Poor viral replication can be explained by a possible impairment in the ability of the mutant NS1 protein to dimerize (70
), resulting in lost augmentation of viral replication and aberrant IFN antagonistic functions. Nevertheless, the comparable virus titers at early time points of HTBE cell infections and limited viral replication in HTBE cells, A549 cells, and DCs suggest that NS1 C-terminal truncation mutants will replicate enough to provide antigen for immunity but not replicate efficiently to become pathogenic, both of which traits are prerequisites for vaccine design.
We previously showed that the NS1 protein of a mouse-adapted laboratory strain of influenza virus (PR8) has the ability to inhibit the production of IFN and the activation of human DCs, and therefore an influenza PR8 virus lacking the NS1 protein (deltaNS1) is an efficient stimulant of DC activation (15
). We now analyzed human DC infections with PR8 influenza virus and human influenza virus strain TX WT. These two viruses displayed very different abilities to replicate and stimulate innate and adaptive immune responses. TX WT virus infections demonstrated a marked increased in production of NP and NS1 mRNA and protein in human DCs compared with PR8, suggesting its enhanced ability of replication in human DCs. There was some activation of the type I IFN induction pathway, secretion of type I IFN, and stimulation of IFN signaling with minimal increases in IFN-responsive gene expression, all evidence for the initiation of an antiviral state in human DCs. However, during later stages of TX WT infections, the expression of IFN-responsive genes was diminished, whereas PR8 infections of human DCs resulted in a gradual increase in IFN-responsive gene expression, which surpassed that in TX WT infections. And this respective inability of the PR8 virus to completely block human DC activation resulted in increased naïve CD4 stimulation. A likely explanation is reflected in a recent study (32
) that showed that the NS1 protein of TX WT is more potent at inducing a general host shutoff of gene expression than the NS1 of PR8 virus. The amino acid residues F103 and M106 of NS1 mediated interaction with the cellular factor CPSF, resulting in inhibition of cellular mRNA processing. Since most human influenza strains, like TX WT, bear F103 and M106 residues in their NS1 proteins (Influenza Virus Resource, NCBI), it is possible that NS1-CPSF interactions potentiate the ability of human influenza viruses to overcome the type I IFN system in humans, as demonstrated in this study with reduced IP10, MxA, STAT1, and IFI56K expression in human DCs infected with human influenza A virus compared to human DCs infected with PR8. Nevertheless, it remains to be determined how influenza viruses expressing NS1 proteins that do not interact with CPSF, such as the PR8 virus, are able to replicate to high levels and cause disease in their hosts. Nevertheless, our results underscore the need to study human influenza viruses in human systems to understand the impact during natural infection.
RIG-I is the major sensor for detection of influenza virus infection by DCs and the resultant IFN and cytokine expression in mouse systems (30
). Furthermore, NS1 is known to bind to RIG-I-containing complexes, decreasing RIG-I-mediated NF-κB and IRF3 activation (48
), which leads to decreased expression of type I IFN and IFN-related genes (22
). These lines of evidence taken together suggest that infections with TX WT would show greater inhibition of RIG-I-mediated activation of downstream transcription factors such as NF-κB, IRF3, and c-Jun than infections with attenuated TX 1-99 and TX 1-126 viruses (60
). Activation of these proteins was determined by phosphorylation at specific serine residues. Indeed, an increase was observed in phosphorylation of NF-κB/RelA, c-Jun, and IRF3 at S396 in DCs infected with TX 1-99 and TX 1-126 (data not shown) versus those infected with TX WT. There was also inhibition of RelA, IRF3, and c-Jun activation in PR8-infected human DCs. Nevertheless, there was a gradual increase in the production of antiviral genes in those cells. Here, we further demonstrate and support the hypothesis that the NS1 of TX WT inhibits the establishment of an antiviral state not only by expression of diminishing IFN-responsive genes but also by blocking RIG-I signaling in human DC infections, making TX WT more efficient at the evasion of innate and adaptive immunity. These results conflict with those obtained from experiments studying infections with the A/Udorn/72 laboratory influenza virus strain (43
), underscoring again the need to conduct studies with human viruses in human cells to gain insights into the mechanisms of regulation of host gene expression by influenza virus in humans.
The augmented DC activation by NS1 mutant viruses was corroborated by increased proinflammatory cytokine gene transcription and protein secretion including type I IFN following TX 1-99 and TX 1-126 infections, which led to the activation of the IFN-responsive genes IP10, IFI56K, and ISG54 as well as RIG-I and STAT1 (data not shown). Additionally, the NS1 protein of a human influenza virus isolate can efficiently block IFN-related genes (antiviral state) in human DCs, contributing to the establishment of infection in humans. Moreover, although the NS1 of TX WT uses multiple mechanisms to inhibit establishment of an antiviral state (32
), truncating the C terminus of NS1 efficiently enhances the ability of the virus to activate DCs and allows increased upregulation and secretion of IFN, IFN-related genes, and proinflammatory cytokines (15
The efficient activation of DCs by the TX 1-99 and TX 1-126 NS1 C-terminal deletion mutants leads to potent stimulation of T cells. This may explain the strong induction of B- and T-cell responses of TX 1-126 virus in nonhuman primates (5
). For an efficient induction of adaptive immune responses, DCs must upregulate MHC-I and MHC-II, as well as costimulatory molecules, to the membrane surface (2
) for proper antigen presentation and stimulation of NK, T, and B cells. We observed increased MHC-II (data not shown) and CD86 expression in DCs infected with TX 1-99 and TX 1-126 over that of TX WT infections. And this increased CD86 surface expression was primarily in Flu NP+
cells, suggesting that CD86 upregulation is mostly dependent on infection. Furthermore TX 1-99- and TX 1-126-infected DCs stimulated increased IFN-γ secretion from T cells compared to conventional DCs infected with TX WT. The increased IFN-γ suggests a Th1 polarity, which is believed to be beneficial for clearance of influenza virus infections (11
). These observations correlate well with the efficacy in protection achieved using NS1 C-terminal truncation mutants from other influenza virus strains in mice and pigs (55
In this study, we show that NS1 C-terminal deletions that disrupt NS1 functions in a human strain of influenza decrease the rate and efficiency of viral replication, which is concomitant with an impaired ability to prevent molecular and cellular antiviral mechanisms. This strongly supports the idea that such mutant viruses might be used as live attenuated vaccine viruses for human influenza. Live attenuated vaccine viruses must replicate sufficiently to produce viral antigen and stimulate immune response and memory but not inflict serious pathology and disease (6
). Strong supporting evidence for demonstrations from our system that NS1 C-terminal deletion mutants are possible live attenuated human influenza vaccines comes from a recent study using macaques (5
). In this study, macaques were mock infected, vaccinated with inactivated TX WT virus, or infected with live TX 1-126 virus. Macaques infected with live TX 1-126 acquired humoral and cellular immunity to influenza A virus that was more robust than that acquired with inactivated virus, as demonstrated by higher levels of specific HA antibody production and an increase in the percentages of influenza virus-specific CD4 and CD8 T cells. An added benefit to the use of these mutants as human influenza virus vaccines is that they are generated by reverse genetics techniques, which is predicted to curtail the length of production from the current time of months to weeks (28
). Availability of efficient live attenuated influenza virus vaccines would be advantageous in efforts to protect the public against the increasing resistance of influenza A virus to current antiviral treatments, as well as potential influenza A virus pandemics (26