Phosphorylation of the influenza A virus protein PB1-F2 by PKC is crucial for apoptosis promoting functions in monocytes

Mitzner, David; Dudek, Sabine Eva; Studtrucker, Nicole; Anhlan, Darisuren; Mazur, Igor; Wissing, Josef; Jänsch, Lothar; Wixler, Ludmilla; Bruns, Karsten; Sharma, Alok; Wray, Victor; Henklein, Peter; Ludwig, Stephan; Schubert, Ulrich S.

doi:10.1111/j.1462-5822.2009.01343.x

Cited by 64 publications

(47 citation statements)

References 69 publications

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“…Furthermore, besides the well-established role of PKCs in activating the downstream RAF/MEK/ERK kinases, PKCs have been found to directly phosphorylate several viral proteins (36,37). Our study provides further evidence that PKCβ may be an important cellular component that interacts with viral proteins including apoptin.…”

Section: Discussionsupporting

confidence: 48%

Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

et al. 2010

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The chicken anemia virus-derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein-apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34 + cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma.

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Section: Discussionsupporting

confidence: 48%

Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

et al. 2010

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“…This kinase also phosphorylates two amino acids in PB1-F2 (9). PKC␣, as well as other PKCs, is rapidly activated upon influenza virus infection (6,14).…”

Section: Discussionmentioning

confidence: 99%

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

Hsiang

Zhou

Krug

2012

J Virol

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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...

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“…The PB1-F2 protein contributes to viral polymerase activity (13,31,34,58), induces apoptosis by interacting with host cell mitochondria (24,25,40,61), and affects host immune responses directed toward the virus (29,54). Full-length PB1-F2 is frequently maintained at 87 to 90 amino acids (24,33), with specific amino acid residues that are associated with virulence (1), including a critical one at position 66 (14).…”

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confidence: 99%

Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens

Weeks-Gorospe

Hurtig

Iverson

et al. 2012

J Virol

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A combination of viral, bacterial, and host factors contributes to the severity and overall mortality associated with influenza virus-bacterium superinfections. To date, the virulence associated with the recently identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae superinfection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a nonhuman host species (swine). Using this rationale, we developed the hypothesis that naturally occurring viruses expressing variants of genes, like the PB1-F2 gene, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from superinfection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial superinfection, are broadly associated with enhanced pathogenicity in swine in a bacterium-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with Streptococcus pyogenes superinfection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of superinfections with specific Gram-positive respiratory pathogens.

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Phosphorylation of the influenza A virus protein PB1-F2 by PKC is crucial for apoptosis promoting functions in monocytes

Cited by 64 publications

References 69 publications

Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

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

Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens

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