Mapping the Phosphoproteome of Influenza A and B Viruses by Mass Spectrometry

Self Cite

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.T he segmented single-strand negative-sense RNA genome of influenza A virus is contained in viral ribonucleoprotein (vRNP) complexes. Within the vRNP, the genomic RNA termini associate with the viral RNA-dependent RNA polymerase while the rest of the RNA is bound to oligomeric nucleoprotein (NP). The oligomeric NP is arranged in a double-helical conformation within the vRNP (1, 2). The RNA polymerase carries out transcription of the genomic viral RNA (vRNA) into mRNA and replication through a cRNA intermediate that serves as a template for the synthesis of vRNA (reviewed in references 3 and 4). NP is essential for both transcription and replication of full-length RNA genome segments by the viral RNA polymerase. However, it is dispensable for the transcription and replication of short model RNA templates (5).NP is a crescent-shaped protein with a head and a body domain which are separated by a highly basic region that is involved in RNA binding (6, 7). NP can homo-oligomerize by the insertion of the tail-loop of one NP molecule into a groove in the body domain of a neighboring NP molecule (8). NP homo-oligomerization is essential for RNP complex activity on full-length templates (5,8,9). Recent work suggested that NP needs to be maintained in a monomeric conformation prior to assembly into vRNPs (10, 11). In particular, it was found that NP readily forms trimers and tetramers in solution and that, although both the monomeric and trimeric/tetrameric forms can bind RNA, only monomers of NP can form oligomers on RNA that resemble oligomeric NP in vRNPs. However, how NP is maintained in a monomeric form prior to assembly into vRNPs remains unclear.Mapping of the phosphoproteome of influenza A and B viruses identified multiple phosphorylation sites within NP (12). Three sites, S165, S457, and S402/403, were present within the homooligomerization domain of NP, and it was hypothesized that phosphorylation at these sites might prevent NP oligomerization by inhibiting the interaction between the tail-loop and groove of neighboring NP monomers. Ribonucleoprotein reconstitution assays suggested that reversible phosphorylation at positions S402/403 and S457 is not essential for RNP complex activity, whereas reversible phosphorylation at position S165 was required (12). A separate study also detected phosphorylation at NP S165 and showed that mutation of S165 to aspartic acid, which mimics constitutive phosphorylation, resulted in predominantly monomeric NP in vitro (10). However, the biological significance of t...

mentioning

confidence: 99%

Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

Turrell

Hutchinson

Vreede

et al. 2015

Self Cite

“…For instance, a mass spectrometry study of the phosphoproteome of influenza A and B viruses revealed that almost all viral proteins, namely, PB2, PB1, PA, HA, NA, NP, M1, M2, NS1, and NEP, are phosphorylated (6). In addition, NP undergoes ubiquitination and deubiquitination, which may play an important role in regulating viral transcription and replication (7).…”

mentioning

confidence: 99%

Sumoylation of Influenza A Virus Nucleoprotein Is Essential for Intracellular Trafficking and Virus Growth

Han

Chang

et al. 2014

Viruses take advantage of host posttranslational modifications for their own benefit. It was recently reported that influenza A virus proteins interact extensively with the host sumoylation system. Thereby, several viral proteins, including NS1 and M1, are sumoylated to facilitate viral replication. However, to what extent sumoylation is exploited by influenza A virus is not fully understood. In this study, we found that influenza A virus nucleoprotein (NP) is a bona fide target of sumoylation in both NPtransfected cells and virus-infected cells. We further found that NP is sumoylated at the two most N-terminal residues, lysines 4 and 7, and that sumoylation at lysine 7 of NP is highly conserved across different influenza A virus subtypes and strains, including the recently emerged human H7N9 virus. While NP stability and polymerase activity are little affected by sumoylation, the NP sumoylation-defective WSN-NP K4,7R virus exhibited early cytoplasmic localization of NP. The growth of the WSN-NP K4,7R virus was highly attenuated compared to that of the wild-type WSN virus, and the lysine residue at position 7 is indispensable for the virus's survival, as illustrated by the rapid emergence of revertant viruses. Thus, sumoylation of influenza A virus NP is essential for intracellular trafficking of NP and for virus growth, illustrating sumoylation as a crucial strategy extensively exploited by influenza A virus for survival in its host. IMPORTANCEHost posttranslational modifications are heavily targeted by viruses for their own benefit. We and others previously reported that influenza A virus interacts extensively with the host sumoylation system. However, the functional outcomes of viral sumoylation are not fully understood. Here we found that influenza A virus nucleoprotein (NP), an essential component for virus replication, is a new target of SUMO. This is the first study to find that NP from different influenza A viruses, including recently emerged H7N9, is sumoylated at conserved lysine 7. Our data further illustrated that sumoylation of influenza A virus NP is essential for intracellular trafficking of NP and virus growth, indicating that influenza A virus relies deeply on sumoylation to survive in host cells. Strategies to downregulate viral sumoylation could thus be a potential antiviral treatment.

“…It should also be noted that the peptide of 101 to 105 aa in BM1 satisfied the pattern of a monopartite NLS but was found to lack nuclear import activity. Among the five phosphorylation sites identified in BM1 in the present study, 236S and 237S were reported in a previous study (28), but the roles of the phosphorylation of the these residues in virus life cycle were not investigated. M1 and BM1 were both phosphorylated proteins (29).…”

Section: Discussionmentioning

confidence: 56%

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

Cao

Jiang

et al. 2014

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...