Roles of the Phosphorylation of Specific Serines and Threonines in the NS1 Protein of Human Influenza A Viruses

Abstract

We demonstrate that phosphorylation of the NS1 protein of a human influenza A virus occurs not only at the threonine (T) at position 215 but also at serines (Ss), specifically at positions 42 and 48. By generating recombinant influenza A/Udorn/72 (Ud) viruses that encode mutant NS1 proteins, we determined the roles of these phosphorylations in virus replication. At position 215 only a T-to-A substitution attenuated replication, whereas other substitutions (T to E to mimic constitutive phosphorylation, T to N, and T to P, the amino acid in avian influenza A virus NS1 proteins) had no effect. We conclude that attenuation resulting from the T-to-A substitution at position 215 is attributable to a deleterious structural change in the NS1 protein that is not caused by other amino acid substitutions and that phosphorylation of T215 does not affect virus replication. At position 48 neither an S-to-A substitution nor an S-to-D substitution that mimics constitutive phosphorylation affected virus replication. In contrast, at position 42, an S-to-D, but not an S-to-A, substitution caused attenuation. The S-to-D substitution eliminates detectable double-stranded RNA binding by the NS1 protein, accounting for attenuation of virus replication. We show that protein kinase C ␣ (PKC␣) catalyzes S42 phosphorylation. Consequently, the only phosphorylation of the NS1 protein of this human influenza A virus that regulates its replication is S42 phosphorylation catalyzed by PKC␣. In contrast, phosphorylation of Ts or Ss in the NS1 protein of the 2009 H1N1 pandemic virus was not detected, indicating that NS1 phosphorylation probably does not play any role in the replication of this virus. Influenza A viruses cause a highly contagious respiratory disease in humans, resulting in annual epidemics and periodic worldwide pandemics. The smallest of the eight negative-sense viral gene segments encodes the NS1 protein, a nonstructural protein that plays multiple crucial roles in virus replication (5). Most NS1 proteins are 230 to 237 amino acids long, although shorter forms have also been found. The NS1 protein comprises two functional domains: the N-terminal (amino acids 1 to 73) RNA-binding domain (RBD) and the C-terminal (amino acids 74 to 230/237) effector domain (ED), which binds several cellular proteins.The NS1 protein undergoes three posttranslational modifications: phosphorylation by cellular kinases (4, 12, 13), ISG15 modification by the interferon (IFN)-induced ISG15 conjugation system (17, 21), and modification by SUMO (11,20). It has been reported that these modifications affect the NS1 protein and the phenotype of the virus (4, 17, 21). Phosphorylation of threonines (Ts) of the NS1 protein was observed many years ago (12, 13). It was reported that T at position 215 (T215) of the NS1 protein of the human H3N2 influenza A/Udorn/72 virus (Ud) is phosphorylated and that this phosphorylation is important for virus replication (4). The basis for the latter conclusion was that a recombinant Ud virrus encoding an NS1 protein with a T-to...

The Influenza A Virus Protein NS1 Displays Structural Polymorphism

“…This in turn would permit NS1 to participate in specific protein-protein or RNA-protein interactions that allow for the known multifunctionality of NS1 (7). Such conformational plasticity could provide a mechanism for auto-regulating NS1 functions, depending on its temporal distribution, posttranslational modifications (phosphorylation [36,37] and sumoylation [38,39]), and nuclear or cellular localization, during the course of virus infection.…”

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

“…Phosphorylation was the first posttransla-tional modification described to affect NS1 (25), and although early reports established that NS1 phosphorylation is important for efficient viral replication (26,27), a recent study provided strong evidence that, at least for the A/Udorn/72 virus strain, only one out of three identified phosphorylation events affects NS1's functions (28). Two additional posttranslational modifications affecting NS1 were almost simultaneously described, both belonging to the group of UBL proteins (proteins exhibiting a tertiary structure similar to that of ubiquitin).…”

Interactome Analysis of the Influenza A Virus Transcription/Replication Machinery Identifies Protein Phosphatase 6 as a Cellular Factor Required for Efficient Virus Replication

“…Knockdown experiments in human cells revealed that physiological levels of PPP6C are required for efficient virus growth and for transcription and replication of the viral genome, suggesting an important role for PP6 and for the regulation of phosphorylation in the influenza A virus life cycle. Indeed, previous studies have identified numerous sites of phosphorylation in the proteomes of influenza A and B viruses, with roles for phosphorylation being implicated in virus entry and exit, nuclear localization, and protein oligomerization (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49).…”