Tyrosine 132 Phosphorylation of Influenza A Virus M1 Protein Is Crucial for Virus Replication by Controlling the Nuclear Import of M1

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The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-␣. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication. IMPORTANCEIt is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.

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confidence: 99%

Phosphorylation Controls the Nuclear-Cytoplasmic Shuttling of Influenza A Virus Nucleoprotein

Zheng

Wang

et al. 2015

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“…M1 and BM1 were both phosphorylated proteins (29). Our group showed previously that a phosphorylation site in M1 (132Y) is required for the nuclear import of M1 (24). In the present study, we showed that phosphorylation of 80T and 84S, which are located 293T cells were transfected with constructs encoding EGFP-tagged WT or mutant NLS1.…”

Section: Discussionmentioning

confidence: 62%

“…After centrifugation at 4°C with 12,000 rpm for 10 min, the supernatant of the cell lysates were incubated at 4°C for 4 h with either monoclonal anti-BM1 antibody or an antibody isotype preconjugated to protein G-agarose beads. After three washes, the precipitated proteins conjugated on beads were treated with or without alkaline phosphatase (ALP) at 37°C for 1 h, centrifuged at 4°C with 5,000 rpm for 5 min, and separated by Phos tag SDS-PAGE as described previously (24). The gel was silver stained, and bands of interest were collected from the gel and subjected to nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) analysis conducted by the Technological Platform of the Institute of Zoology, Chinese Academy of Sciences (LCQ Deca XP Plus; Thermo).…”

Section: Cells and Reagentsmentioning

confidence: 99%

“…The matrix protein of influenza A virus (M1) is a multifunctional protein playing important roles in several steps of the influenza virus life cycle. Crucially, the M1 protein facilitates the nuclear export of vRNP by shuttling between the cytoplasm and nuclei of infected cells under the regulation of nuclear localization signal (NLS), nuclear export signal (NES), and phosphorylation (22)(23)(24)(25)(26). However, BM1 shares only ca.…”

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confidence: 99%

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Characterization of the Nucleocytoplasmic Shuttle of the Matrix Protein of Influenza B Virus

Cao

Jiang

et al. 2014

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Influenza B virus is an enveloped negative-strand RNA virus that contributes considerably to annual influenza illnesses in human. The matrix protein of influenza B virus (BM1) acts as a cytoplasmic-nuclear shuttling protein during the early and late stages of infection. The mechanism of this intracellular transport of BM1 was revealed through the identification of two leucinerich CRM1-dependent nuclear export signals (NESs) (3 to 14 amino acids [aa] and 124 to 133 aa), one bipartite nuclear localization signal (NLS) (76 to 94 aa), and two phosphorylation sites (80T and 84S) in BM1. The biological function of the NLS and NES regions were determined through the observation of the intracellular distribution of enhanced green fluorescent protein (EGFP)-tagged signal peptides, and wild-type, NES-mutant, and NLS-mutant EGFP-BM1. Furthermore, the NLS phosphorylation sites 80T and 84S, were found to be required for the nuclear accumulation of EGFP-NLS and for the efficient binding of EGFP-BM1 to human importin-␣1. Moreover, all of these regions/sites were required for the generation of viable influenza B virus in a 12-plasmid virus rescue system. IMPORTANCEThis study expands our understanding of the life cycle of influenza B virus by defining the dynamic mechanism of the nucleocytoplasmic shuttle of BM1 and could provide a scientific basis for the development of antiviral medication. Influenza B virus, one of the major causative agents of flu, is an enveloped negative-strand RNA virus containing eight segmented strands of genome RNA, which encodes 12 viral proteins, consisting of four membrane proteins (HA, NA, BM2, and NB) (1-3), three subunits of viral RNA polymerase (PA, PB1, and PB2) (4), nucleoprotein (NP) (5), matrix protein BM1 (6), nuclear export protein (NEP), and nonstructural protein NS1 (7).The seventh RNA segment of the viral genome, encodes the viral proteins BM1 and BM2 which are expressed separately through the use of a stop-start pentanucleotide mechanism of regulation (1, 8). BM2 is an oligomeric membrane protein with ion channel activity (9-12), transported through trans-Golgi network (13) which participates in process of viral ribonucleoprotein complex (vRNP) incorporation into virions (14-17). In contrast to the detailed studies of BM2, our understanding of the function of BM1 in the virus life cycle has been limited, with previous studies showing that BM1 is related to mouse adaptation (18) and cold adaptation (19,20).Influenza A and B viruses are closely related and composed of proteins with similar functions (21). The matrix protein of influenza A virus (M1) is a multifunctional protein playing important roles in several steps of the influenza virus life cycle. Crucially, the M1 protein facilitates the nuclear export of vRNP by shuttling between the cytoplasm and nuclei of infected cells under the regulation of nuclear localization signal (NLS), nuclear export signal (NES), and phosphorylation (22-26). However, BM1 shares only ca. 30% sequence identity of amino acids with M1, showing no c...

“…Posttranslational modifications of M1 play important roles in the regulation of its cellular localization and function. Phosphorylation of M1 at Y132 mediates the nuclear import of M1 (23), whereas SUMOylation of M1 regulates the nuclear export of vRNPs and promotes virion assembly and budding (21). Ubiquitination of M1 has been implicated in IAV release from the endosomes (24).…”

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confidence: 99%

Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells

Gao

Liu

et al. 2015

Influenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication. IMPORTANCEAlthough the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication. Influenza A virus (IAV) is a significant cause of morbidity and mortality in both humans and animal species owing to its ability to cause yearly epidemics and occasional pandemics (1-3). Like other viruses, IAV hijacks the host cellular machinery to support its life cycle. Thus, identification and characterization of the interactions between viral components and specific host factors would help provide an understanding of the mechanisms by which the viruses acquire human pandemic potential.IAV belongs to the Orthomyxoviridae family, and its genome consists of eight negative-sense RNA segments encoding up to 17 viral proteins (4-9). The viral RNA (vRNA) exists as a form of viral ribonucleoprotein complexes (vRNPs) containing vRNA, nucleoprotein (NP), and three viral polymerase proteins (PB1, PB2, and PA). Unlike most other RNA viruses, influenza virus transcribes and replicates its genome in the nucleus. Thus, after it enters a host cell, vRNPs enter the nucleus to complete transcription and replication (10). The newly synthesized vRNPs are exported from the nucleus for packaging into progeny virions (11). In this regard, efficient nuclear export of vRNPs is essential for productive infection.NS2, also kn...