Oligomerization paths of the nucleoprotein of influenza A virus

Tarus, Bogdan; Bakowiez, Olivier; Chenavas, Sylvie; Duchemin, Lucie; Estrozi, Leandro F.; Bourdieu, Christiane; Lejal, Nathalie; Bernard, Julie; Moudjou, Mohammed; Chevalier, Christophe; Delmas, Bernard; Ruigrok, Rob W.H.; Primo, Carmelo Di; Slama‐Schwok, Anny

doi:10.1016/j.biochi.2011.11.009

Cited by 42 publications

(95 citation statements)

References 26 publications

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“…However, RNP activity and viral growth were still affected, and we hypothesize that uncontrolled premature oligomerization of NP might hamper the efficient recruitment of NP into vRNPs. Indeed, this would be consistent with recent reports that monomeric NP can bind RNA and then form oligomers resembling those in vRNPs but that trimers of NP cannot oligomerize even though they readily bind RNA (10,11). Taking the data together, we propose that reversible phosphorylation of S165 of influenza A virus NP could be involved in the regulation of NP oligomerization prior to RNP assembly.…”

supporting

confidence: 91%

“…As phosphorylation at serine residues in baculovirus-infected cells is inefficient (14), we assume that the ratio of monomeric NP to oligomeric NP was largely independent of the effects of regulatory phosphorylation. Consistent with this, previous studies of bacterially expressed NP, which should not be phosphorylated, also found most NP present in oligomers but with a small monomeric fraction (7,11,15). We next used mutations at S165 to mimic the effects of constitutive phosphorylation or of a lack of phosphorylation.…”

supporting

confidence: 75%

“…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.…”

mentioning

confidence: 99%

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Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

Turrell

Hutchinson

Vreede

et al. 2015

J Virol

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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...

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supporting

confidence: 91%

supporting

confidence: 75%

mentioning

confidence: 99%

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Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

Turrell

Hutchinson

Vreede

et al. 2015

J Virol

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“…After the initial interaction, MxA could convert again into higher oligomeric structures exhibiting a low affinity for NP. In vitro NP is known to form oligomers even in the absence of RNA (46). Therefore, it may be possible that MxA reoligomerizes around newly synthesized NP oligomers, thereby inhibiting its function.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization and GTP-binding Requirements of MxA for Viral Target Recognition and Antiviral Activity against Influenza A Virus

Nigg

Pavlovic

2015

Journal of Biological Chemistry

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The IFN-induced human myxovirus resistance protein A (MxA) exhibits a broad antiviral activity against many viruses, including influenza A virus (IAV). MxA belongs to the family of dynamin-like GTPases and assembles in vitro into dimers, tetramers, and oligomeric ring-like structures. The molecular mechanism of action remains to be elucidated. Furthermore, it is not clear whether MxA exerts its antiviral activity in a monomeric and/or multimeric form. Using a set of MxA mutants that form complexes with defined stoichiometry, we observed that, in the presence of guanosine 5-O-(thiotriphosphate), purified MxA disassembled into tetramers and dimers. Dimeric forms did not further disassemble into monomers. Infection experiments revealed that besides wild-type MxA, dimeric and monomeric variants of MxA also efficiently restricted IAV at a replication step after primary transcription. Moreover, only dimeric MxA was able to form stable complexes with the nucleoprotein (NP) of IAV. MxA interacted with NP independently of other viral components. Interestingly, the dimeric form of MxA was able to efficiently bind to NP from several MxA-sensitive strains but interacted much more weakly with NP from the MxA-resistant PR8 strain derived from the H1N1 1918 lineage. Taken together, these data suggest that, during infection, a fraction of MxA disassembles into dimers that bind to NP synthesized following primary transcription in the cytoplasm, thereby preventing viral replication.The interferon system plays a pivotal role in the defense against viral infection. Interferons type I and III induce the expression of more than 300 proteins, many of them exhibiting intrinsic antiviral activity (1-3). The human myxovirus resistance protein A (MxA) 3 protein is induced exclusively by type I and type III interferons and restricts the replication of a large variety of RNA and DNA viruses (reviewed in Ref. 4). Mx proteins are members of the dynamin superfamily of large GTPases. They contain an amino-terminal globular GTPase (G) domain and a carboxy-terminal stalk domain (5, 6). The stalk domain harbors the GTPase effector domain and additional structural elements, including the L4 loop required for antiviral activity and target specificity (7-9). The G and stalk domains are connected via the bundle signaling element (BSE), which strongly resembles the BSE responsible for intramolecular signaling in dynamin (10). Mx proteins exhibit a high intrinsic rate of GTP hydrolysis, although the affinity to GTP is weak (11). Moreover, the human MxA protein forms dimers and tetramers and has the capacity to form higher oligomeric ringlike structures through a series of intra-and intermolecular interactions (5, 6, 12, 13). Introduction of mutations into the interfaces of the intermolecular interaction sites or hinge regions of MxA prevents assembly into higher-order multimeric forms. These mutations lead to the formation of MxA into tetramers, dimers, and monomers (5, 12).So far, little is known about the molecular mechanism of action of the human MxA...

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“…The influenza A NP is a component of the RNP complex and its oligomerization is required for the transcription and replication of full-length RNA genome segments, [98][99][100][101] but it needs to remain in a monomeric state before the assembly of the RNP complex. 102,103 The NP was phosphorylated at multiple sites, and reversible phosphorylation of S165, located in the "groove" of the NP which interacts with the "tail loop" of another NP, meditates NP oligomerization. 100,104 Substitution of S165 to phosphomimetics resulted in primarily monomeric NP, while a change to an alanine resulted in oligomers of the NP.…”

mentioning

confidence: 99%

<div>Phosphorylation of the viral coat protein regulates RNA virus infection</div>

Hoover

Kao

2016

VAAT

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Oligomerization paths of the nucleoprotein of influenza A virus

Cited by 42 publications

References 26 publications

Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

Oligomerization and GTP-binding Requirements of MxA for Viral Target Recognition and Antiviral Activity against Influenza A Virus

<div>Phosphorylation of the viral coat protein regulates RNA virus infection</div>

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