Discrete spatio-temporal regulation of tyrosine phosphorylation directs influenza A virus M1 protein towards its function in virion assembly
Fig 1
Phosphorylation of M1 Y132 late in the infection cycle is needed for efficient virus replication.
A) M1 was immunoprecipitated with M1-specific antibodies from WSN-infected A549 cells (5 MOI; 2, 4, 6, 8 hpi) followed by analysis of M1 tyrosine phosphorylation in western blot by using pY antibodies. IP efficiency was analyzed by detection of M1. Blots are representative of two independent experiments. N-fold increase in pY M1 was analyzed by densitometric analysis of respective band intensities of pY M1 and total M1. Phosphorylation intensities were normalized to respective M1 amounts and pY detected 6 hpi was arbitrarily set to 1. B) MS/MS spectrum of the tryptic peptide EIALSYSAGALACCMGLIYNR of M1 isolated from WSN virus particles showing phosphorylation at Y132. LC-MS/MS analyses were performed on a Proxeon Easy-nLC coupled to a QExactice mass spectrometer. The MS data were processed using MaxQuant software (version 1.5.2.8. with integrated Andromeda search engine). C) Plaque phenotypes of WSN WT (left), M1 Y10F (middle) and M1 Y132A (right). Dilutions from standard plaque assays were stained with neutral red. D) A549 cells were infected with 0.01 MOI WSN WT, M1 Y10F or M1 Y132A. Virus-containing supernatants were harvested 8, 24, 32 and 48 hpi. Titers were determined by standard plaque assays and are depicted as mean (±SD) of three independent experiments. Statistical significance was analyzed by two-way Anova followed by Dunett’s multiple comparison test (*p≤0.05, ****p≤0.0001). E) A549 cells were infected with 1 MOI WSN WT, M1 Y10F or M1 Y132A and expression of viral proteins PB1, NP, HA, M1, NS1 and M2 was analyzed 4, 6, 8 and 9 hpi. ERK2 expression served as loading control. Blots are representative of three independent experiments. F) A549 cells were infected with 5 MOI WSN WT and activation of JAK2 and STAT3 was analyzed by detection of phosphorylation of Y1007/Y1008 (JAK2) and Y705 (STAT3), respectively. Expression of viral PA was analyzed to confirm efficient infection. JAK2 and tubulin expression served as loading controls. Blots are representative of three independent experiments. G) A549 cells were transfected with JAK1-, JAK2- or a combination of specific siRNAs for 48 h and were subsequently infected with 0.1 MOI WSN WT or M1 Y132A. Virus-containing supernatants were harvested 8 hpi and viral titers were determined by standard plaque assays and are depicted as mean (±SD) of three independent experiments. Statistical significance was analyzed by two-way Anova followed by Dunett’s multiple comparisons test (***p≤0.001).