A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

Cao, Shuai; Liu, Xiaoling; Yu, Maorong; Li, Jing; Jia, Xiaojuan; Bi, Yuhai; Sun, Lei; Gao, George F.; Liu, Wenjun

doi:10.1128/jvi.06586-11

Cited by 72 publications

(62 citation statements)

References 54 publications

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“…The conclusion in the Berkhoff et al abstract that "alanine replacements for each of the nine amino acids of the M1 58-66 were tolerated to various extents, except for the anchor residue at the second position" (3) is reasonable and reflected in our report (2). Cao et al (4) demonstrate that the nuclear export signal overlaps the M1 58-66 epitope and reveals how the nuclear export signal motif tolerates substantial sequence variability (3). Most interestingly, all mutant viral epitopes tested by chromium release cytolysis assay were no longer targeted by M1 58-66 cytotoxic T lymphocyte (CTLs) (figure 4M in ref.…”

Section: Reply To Van De Sandt and Rimmelzwaan: Matching Epitope Dispsupporting

confidence: 72%

Reply to van de Sandt and Rimmelzwaan: Matching epitope display with functional avidity

Keskin¹,

Reinhold²,

Zhang³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Section: Reply To Van De Sandt and Rimmelzwaan: Matching Epitope Dispsupporting

confidence: 72%

Reply to van de Sandt and Rimmelzwaan: Matching epitope display with functional avidity

Keskin¹,

Reinhold²,

Zhang³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The transfected wild-type Myc-M1 protein was localized predominantly in the cytoplasm, as described previously, as it bears both nuclear export signal (NES) and NLS peptides (39). Nevertheless, a nuclear localization was obvious when the protein was coexpressed with importin-␣1; in this case, the overexpressed importin-␣1 would break the balance of nuclear export and import, thus greatly enhancing the nuclear import ability of M1 (Fig.…”

Section: Resultsmentioning

confidence: 61%

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

Wang

Zhao

et al. 2013

J Virol

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bPhosphorylation of viral proteins plays important roles in the influenza A virus (IAV) life cycle. By using mass spectrometry, we identified tyrosine 132 (Y132) as a phosphorylation site of the matrix protein (M1) of the influenza virus A/WSN/1933(H1N1). Phosphorylation at this site is essential to the process of virus replication by controlling the nuclear import of M1. We further demonstrated that the phosphorylated tyrosine is crucial for the binding of M1 to the nuclear import factor importin-␣1, since any substitutions at this site severely reduce this protein-protein interaction and damage the importin-␣1-mediated nuclear import of M1. Additionally, the tyrosine phosphorylation which leads to the nuclear import of M1 is blocked by a Janus kinase inhibitor. The present study reveals a pivotal role of this tyrosine phosphorylation in the intracellular transportation of M1, which controls the process of viral replication. The genome of influenza A virus (IAV) consists of eight segments of negative-sense RNA coding for 14 viral proteins (1-4). With the exception of the newly found PB1 N40, PA-X, and M42 proteins, the remaining 11 viral proteins have been described as phosphorylated proteins either in virions or in infected cells. These remaining proteins include the RNA polymerase PA, PB1, and PB2 proteins, the nucleoprotein (NP), the two envelope proteins, i.e., hemagglutinin (HA) and neuraminidase (NA), nonstructural protein 1 (NS1), the nuclear export protein (NEP), the PB1-F2 protein, and the two matrix proteins, M1 and M2 (5-14). Phosphorylation of viral proteins plays an important role in the virus life cycle. Remarkably, phosphorylation of NP controls its intracellular distribution and then affects the transportation of viral RNPs (vRNPs) (15, 16). The phosphorylation of NS1 affects its double-stranded RNA (dsRNA)-binding capacity and has been shown to have critical roles in virus replication (13,17). M1, the most abundant protein in virions, also has multiple functions in the influenza A virus life cycle, including uncoating, transcription, nuclear export of vRNPs, assembly, and budding. Early studies indicated that M1 contains phosphoserine and phosphothreonine residues (5, 18) and has the potential to be phosphorylated by protein kinase C (PKC) and extracellular signal-regulated protein kinases (ERKs) (19). Recently, several phosphorylation sites on M1 (including a phosphotyrosine) were reported (14). However, the phosphorylated tyrosine of M1 has not yet been identified, and phosphorylated residues on M1 have not been confirmed functionally (20,21). There is evidence that abnormal modification of M1 by hyperphosphorylation causes its retention in the nucleus (22), which further suggests that this important posttranslational modification of M1 is finely regulated for productive virus growth. In order to further understand the effect of phosphorylation on the functions of M1, we determined the status of M1 phosphorylation and identified tyrosine residue 132 as a phosphorylation site which is critical...

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“…This could be due to altered levels of expression of M1 and M2, which are known to affect viral replication both in cell cultures and in vivo (36); to the M1 protein being fused to the 2A autoproteolytic cleavage site at the C-terminal end (Fig. 1), which could affect M1 functions; or to the presence of alternative proteins encoded by the influenza virus M segment being disrupted during Ms virus generation (18,(37)(38)(39). Surprisingly, the defect in replication of the Ms and Ms/NSs viruses was more evident at high temperatures (37°C and 39°C) than at a low temperature (33°C) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

Nogales

DeDiego

Topham

et al. 2016

J Virol

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Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease which is most effectively prevented through vaccination. Segments 7 (M) and 8 (NS) of the influenza virus genome encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing M or NS viral segments individually or modified M or NS viral segments combined in which the overlapping open reading frames of matrix 1 (M1)/M2 for the modified M segment and the open reading frames of nonstructural protein 1 (NS1)/nuclear export protein (NEP) for the modified NS segment were split by using the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at nonpermissive high temperatures, whereas high viral titers could be obtained at permissive low temperatures (33°C). Furthermore, viruses containing the M split segment were highly attenuated in vivo, while they retained their immunogenicity and provided protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and that such attenuated viruses represent an excellent option as safe, immunogenic, and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of the M segment to encode M1 and M2 from two separate, nonoverlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product. Influenza viruses are enveloped pathogens that belong to the Orthomyxoviridae family and contain a segmented genome of eight single-stranded RNA molecules with negative polarity (1). Influenza virus infections cause both seasonal epidemics and occasional pandemics when novel viruses are introduced into humans (2). Despite comprehensive vaccination programs, the World Health Organization (WHO) estimates that the global disease burden from influenza results in 1 billion infections, 3 million to 5 million cases of severe disease, and between 300,000 and 500,000 deaths annually (3). Therefore, infection with influenza virus poses a threat to human health and results in significant negative economic impacts on society every year (4). The public health concerns posed by influenza viruses are aggravated by their efficient transmission and the limited antiviral therapeutic options (5). Hence, vaccination remains our best medical intervention to protect humans against influenza virus (6), even though the effectiveness of current vaccines is suboptimal (7). To date, the U.S. Food and Drug Administration (FDA) approves three types of influenza virus vaccines for human use: inactivated vir...

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A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

Cited by 72 publications

References 54 publications

Reply to van de Sandt and Rimmelzwaan: Matching epitope display with functional avidity

Reply to van de Sandt and Rimmelzwaan: Matching epitope display with functional avidity

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

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

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