Virally induced inhibition of cellular protein synthesis, or shutoff, is a process in which the virus hijacks cellular machinery for its own benefit. Viruses rely on host cell machinery for replication and production of progeny virions. Therefore, viral control of cellular transcription/translation machinery for preferential production of viral proteins is beneficial for the virus. Inhibition of host protein synthesis also prevents the cellular antiviral response, which can contribute to efficient virus production and spread. In the present study, we identified the domain within the PA subunit of the influenza virus polymerase complex that critically affects expression of exogenous protein, as confirmed by cotransfection experiments. We also showed that PA suppresses cellular protein synthesis and has an effect on the induction of apoptosis in virus-infected cells. Our results indicate an additional role for PA gene products in the control of host cell protein synthesis machinery, which is likely to affect viral production and cytopathogenicity of infected cells.
Influenza virus is known to shut off host cell protein synthesis following infection (3
). It is highly likely that multiple viral proteins and their functions contribute to the overall magnitude of host protein synthesis shutoff. In fact, some viral proteins have been identified to have functions that contribute to shutoff. NS1 blocks nuclear export of mRNA and inhibits mRNA splicing (7
). The viral polymerase complex removes 5′ methyl caps from host cell pre-mRNA for the synthesis of viral mRNA (32
) and also degrades cellular RNA polymerase II (14
). These viral protein functions likely alter the steady-state levels of cellular mRNAs. However, analysis of mRNA levels using a cDNA microarray assay indicated that influenza virus infection only partially affected mRNA levels, whereas a significant downregulation of cellular mRNAs, which would explain the profound inhibition of host protein synthesis, was not observed in infected cells (33
). The data suggest that downregulation of cellular transcription or transcripts may not be the major factor that mediates host protein synthesis shutoff but that there may be an additional mechanism(s) involved in the control of host cell protein synthesis in infected cells.
Our data presented here clearly indicate that PA gene products have a significant impact on host protein synthesis shutoff. Comparison of protein synthesis shutoff by each polymerase component, as well as by NS1, clearly implicates PA as the major factor for host protein synthesis shutoff (). A role for PA in shutoff of host protein synthesis is also supported by our failure and the inability of others to generate cell lines that stably express PA (34
). The molecular mechanism by which PA induces shutoff is unclear at this stage. However, previous reports implicate the proteolytic activity of PA in shutoff of both cellular and viral proteins (16
). In these studies, the impact of PA expression on the steady-state levels of coexpressed proteins was evaluated by pulse-chase experiments, which suggest that the expression of PA affects the half-lives of coexpressed proteins. However, the rate of degradation of coexpressed proteins in this study was not high enough to explain the level of reduction we observed in cells labeled for 30 min without chase (). Furthermore, a study of the crystal structure of the PA N-terminal domain found no obvious protease active site, and an additional in vitro
protease assay supported no detectable proteolytic activity in the N-terminal domain of PA (29
). Taking this together with our data, we anticipate that PA contributes to shutoff not through the proteolytic degradation of existing proteins but rather by inhibiting the synthesis of new protein. Coexpression of eGFP and Cal PA, both tagged with the same Flag tag, resulted in suppression of eGFP synthesis (). PA expression was also suppressed, although this was not as significant as eGFP suppression. This result may indicate that, even in the absence of the 5′ untranslated region, there might be a mechanism that allows for viral proteins to escape the suppressive effect. Also, in infected cells, viral protein synthesis increases while cellular protein production is strongly blocked (). It is unclear how viral proteins are selectively produced; however, it is possible that PA is involved in selective shutoff of nonviral protein synthesis.
Interestingly, there was a difference in the activity of host protein synthesis shutoff between the avian and human virus PA proteins examined. PAs from an avian virus (Nan) or the avian-origin pH1N1 (Cal) demonstrated much more efficient shutoff of host protein synthesis than those from a human virus (NC) or a mouse-adapted human virus (WSN) (). By characterizing the shutoff effect of Cal/WSN chimeric PA N-terminal fragments, we identified regions responsible for the difference in the activity, which include a flexible loop and the helix α4 (). To determine if these residues were conserved among avian or human isolates, we analyzed the PA sequences of 5,643 avian and 4,782 human influenza viruses. The results indicate that most of the avian, but not human, viruses contain residues 57R, 62I, 65S, and 100V (), which enhanced the reduction in protein expression from cotransfected cDNAs (). Therefore, the sequence data support the idea that avian influenza virus contains a PA gene that is more effective in shutoff activity. It is not clear if this difference in PA activity has significance for host-specific virus growth, although it is conceivable that strong host protein shutoff by avian virus PA gene products could be an important factor in preventing the antiviral response in avian hosts. In mammalian hosts, a critical function of NS1 in suppressing the innate immune response is well established (35
). The in vivo
role of avian virus NS1 in chickens has not yet been studied in detail. However, a recent study using NS1 mutant viruses suggests that NS1 does not suppress IFN gene expression efficiently in vivo
. It was suggested that, in chickens, other functions of NS1, such as its ability to inhibit apoptosis, might be more critical for maintaining the virus in an avian host (38
). It is unknown how much of an effect PA has in antagonizing the innate immune response in avian hosts. However, it is possible that influenza viruses develop different mechanisms to escape the antiviral response to achieve the best transmission efficiency in specific hosts.
Amino acid differences between avian and human PA proteins
Expression of an N-terminal domain of PA comprised of 257 residues was sufficient to inhibit protein synthesis (). This is consistent with a previous study, using deletion mutants, which showed that the N-terminal 247 residues are sufficient for reduced accumulation of coexpressed proteins (28
). However, our data with chimeric PA N-terminal fragments uncovered the presence of a particular domain that determines the activity, located at helix α4 and the flexible loop of amino acids 51 to 57 (). The fact that these regions are proximally located in the crystal structure suggests that a possible interaction with a cellular protein(s) through the helix/loop domain is required for the suppressive activity of PA.
Recently, an additional PA gene product, termed PA-X, has been reported (18
). PA-X contains the region which reflects the difference in reducing protein synthesis between WSN and Cal. In fact, Cal PA-X repressed expression of cotransfected gene products more efficiently than WSN PA-X (), supporting our findings that the flexible loop (residues 51 to 74) and helix α4 determine the difference in the activity between WSN and Cal. In addition, both Cal and WSN PA-X showed stronger suppressive activity than PA N-terminal fragments, indicating that unique sequences in the C-terminal region of PA-X also play an important role in reducing protein expression (). Although the mechanism is unclear at this stage, it is highly likely that mRNA degradation is a key process in reducing protein expression, as mutations at the endonuclease active sites (D108A and K134A) completely abolished the repressive activity () (18
). Further studies into the mechanism of how PA-X efficiently suppresses protein synthesis are required to unveil its role in virus replication and pathogenesis.