scholarly journals Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein: Striking Differences between Different Virus Subtypes

2007 ◽  
Vol 81 (11) ◽  
pp. 5995-6006 ◽  
Author(s):  
Krister Melén ◽  
Leena Kinnunen ◽  
Riku Fagerlund ◽  
Niina Ikonen ◽  
Karen Y. Twu ◽  
...  
Keyword(s):  
Host Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Ns1 Protein ◽  
Nonstructural Protein 1 ◽  
Importin Α ◽  
Localization Signal ◽  
Ns1a Protein

ABSTRACT Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin α isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin α/β pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.

scholarly journals Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses

2007 ◽  
Vol 81 (7) ◽  
pp. 3058-3067 ◽  
Author(s):  
Christina Ehrhardt ◽  
Thorsten Wolff ◽  
Stephan Pleschka ◽  
Oliver Planz ◽  
Wiebke Beermann ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Glycogen Synthase ◽  
Nonstructural Protein ◽  
Akt Pathway ◽  
Ns1 Protein ◽  
Nonstructural Protein 1 ◽  
Cell Death Program ◽  
Pi3k Activation

ABSTRACT Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3β and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.

scholarly journals Epigenetic Modification Is Regulated by the Interaction of Influenza A Virus Nonstructural Protein 1 with the De Novo DNA Methyltransferase DNMT3B and Subsequent Transport to the Cytoplasm for K48-Linked Polyubiquitination

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Shi Liu ◽  
Li Liu ◽  
Gang Xu ◽  
Zhongying Cao ◽  
Qing Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Influenza A Virus ◽  
Influenza A ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Epigenetic Modification ◽  
Nonstructural Protein ◽  
Ns1 Protein ◽  
Nonstructural Protein 1 ◽  
Stat Signaling

ABSTRACT The influenza virus nonstructural protein 1 (NS1) is a nonstructural protein that plays a major role in antagonizing host interferon responses during infection. However, a clear role for the NS1 protein in epigenetic modification has not been established. In this study, NS1 was found to regulate the expression of some key regulators of JAK-STAT signaling by inhibiting the DNA methylation of their promoters. Furthermore, DNA methyltransferase 3B (DNMT3B) is responsible for this process. Upon investigating the mechanisms underlying this event, NS1 was found to interact with DNMT3B but not DNMT3A, leading to the dissociation of DNMT3B from the promoters of the corresponding genes. In addition, the interaction between NS1 and DNMT3B changed the localization of DNMT3B from the nucleus to the cytosol, resulting in K48-linked ubiquitination and degradation of DNMT3B in the cytosol. We conclude that NS1 interacts with DNMT3B and changes its localization to mediate K48-linked polyubiquitination, subsequently contributing to the modulation of the expression of JAK-STAT signaling suppressors. IMPORTANCE The nonstructural protein 1 (NS1) of the influenza A virus (IAV) is a multifunctional protein that counters cellular antiviral activities and is a virulence factor. However, the involvement of NS1 in DNA methylation during IAV infection has not been established. Here, we reveal that the NS1 protein binds the cellular DNMT3B DNA methyltransferase, thereby inhibiting the methylation of the promoters of genes encoding suppressors of JAK-STAT signaling. As a result, these suppressor genes are induced, and JAK-STAT signaling is inhibited. Furthermore, we demonstrate that the NS1 protein transports DNMT3B to the cytoplasm for ubiquitination and degradation. Thus, we identify the NS1 protein as a potential trigger of the epigenetic deregulation of JAK-STAT signaling suppressors and illustrate a novel mechanism underlying the regulation of host immunity during IAV infection.

scholarly journals The RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1β Secretion

2016 ◽  
Vol 90 (8) ◽  
pp. 4105-4114 ◽  
Author(s):  
Miyu Moriyama ◽  
I-Yin Chen ◽  
Atsushi Kawaguchi ◽  
Takumi Koshiba ◽  
Kyosuke Nagata ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Nlrp3 Inflammasome ◽  
Influenza A ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Domain ◽  
Interleukin 1Β ◽  
Ns1 Protein ◽  
Caspase 1 ◽  
Nonstructural Protein 1

ABSTRACTInflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1β (IL-1β) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1β secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1β secretion. However, the precise mechanism by which NS1 inhibits IL-1β secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1β secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1β secretion.IMPORTANCEInnate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1β secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results will facilitate the development of new anti-inflammatory drugs.

scholarly journals ISG15 conjugation system targets the viral NS1 protein in influenza A virus–infected cells

2010 ◽  
Vol 107 (5) ◽  
pp. 2253-2258 ◽  
Author(s):  
Chen Zhao ◽  
Tien-Ying Hsiang ◽  
Rei-Lin Kuo ◽  
Robert M. Krug
Keyword(s):  
Antiviral Activity ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Rna Binding ◽  
Acceptor Site ◽  
Ns1 Protein ◽  
Conjugation System ◽  
Infected Cells ◽  
Ns1a Protein

ISG15 is an IFN-α/β–induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a “loss of function” mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.

scholarly journals Glycine 184 in Nonstructural Protein NS1 Determines the Virulence of Influenza A Virus Strain PR8 without Affecting the Host Interferon Response

2010 ◽  
Vol 84 (24) ◽  
pp. 12761-12770 ◽  
Author(s):  
Sabine Steidle ◽  
Luis Martínez-Sobrido ◽  
Markus Mordstein ◽  
Stefan Lienenklaus ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Interferon Response ◽  
Binding Motif ◽  
Ns1 Protein ◽  
Viral Virulence ◽  
Functional Rna

ABSTRACT The nonstructural protein NS1 of influenza A virus counteracts the interferon (IFN) system and thereby promotes viral replication. NS1 has acquired different mechanisms to limit induction of IFN. It prevents double-stranded RNA (dsRNA) and RIG-I-mediated activation of interferon regulatory factor 3 (IRF3), and it blocks posttranscriptional processing of cellular mRNAs by binding to the cleavage and polyadenylation specificity factor (CPSF). Using a mouse-adapted A/PR/8/34 virus and reverse genetics to introduce specific mutations in NS1 which eliminate one or both functions, we determined the relative contributions of these two activities of NS1 to viral virulence in mice. We found that a functional RNA-binding motif was required for IFN suppression and virulence. Restoration of CPSF binding in the NS1 protein of wild-type A/PR/8/34 virus, which cannot bind CPSF due to mutations in the central binding motif at positions 103 and 106, resulted in enhanced virulence. Surprisingly, if CPSF binding was abolished by substituting glycine for arginine at position 184 in the classical NS1-CPSF binding motif, the mutant virus replicated much more slowly in mice, although the mutated NS1 protein continued to repress the IFN response very efficiently. Our results show that a functional RNA-binding motif is decisive for NS1 of A/PR/8/34 virus to suppress IFN induction. They further demonstrate that in addition to its contribution to CPSF binding, glycine 184 strongly influences viral virulence by an unknown mechanism which does not involve the IFN system.

scholarly journals The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

2019 ◽  
Vol 295 (4) ◽  
pp. 1153-1164 ◽  
Author(s):  
Alexander S. Jureka ◽  
Alex B. Kleinpeter ◽  
Jennifer L. Tipper ◽  
Kevin S. Harrod ◽  
Chad M. Petit
Keyword(s):  
Influenza A ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Direct Interaction ◽  
Ns1 Protein ◽  
Single Strain ◽  
Nonstructural Protein 1 ◽  
Marked Deficit

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

scholarly journals Inhibition of Retinoic Acid-Inducible Gene I-Mediated Induction of Beta Interferon by the NS1 Protein of Influenza A Virus

2006 ◽  
Vol 81 (2) ◽  
pp. 514-524 ◽  
Author(s):  
Masaki Mibayashi ◽  
Luis Martínez-Sobrido ◽  
Yueh-Ming Loo ◽  
Washington B. Cárdenas ◽  
Michael Gale ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Influenza A Virus ◽  
Transcriptional Activation ◽  
Influenza A ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Ns1 Protein ◽  
Beta Interferon ◽  
Nonstructural Protein 1 ◽  
Inducible Gene

ABSTRACT The retinoic acid-inducible gene I product (RIG-I) has been identified as a cellular sensor of RNA virus infection resulting in beta interferon (IFN-β) induction. However, many viruses are known to encode viral products that inhibit IFN-β production. In the case of influenza A virus, the viral nonstructural protein 1 (NS1) prevents the induction of the IFN-β promoter by inhibiting the activation of transcription factors, including IRF-3, involved in IFN-β transcriptional activation. The inhibitory properties of NS1 appear to be due at least in part to its binding to double-stranded RNA (dsRNA), resulting in the sequestration of this viral mediator of RIG-I activation. However, the precise effects of NS1 on the RIG-I-mediated induction of IFN-β have not been characterized. We now report that the NS1 of influenza A virus interacts with RIG-I and inhibits the RIG-I-mediated induction of IFN-β. This inhibition was apparent even when a mutant RIG-I that is constitutively activated (in the absence of dsRNA) was used to trigger IFN-β production. Coexpression of RIG-I, its downstream signaling partner, IPS-1, and NS1 resulted in increased levels of RIG-I and NS1 within an IPS-1-rich, solubilization-resistant fraction after cell lysis. These results suggest that RIG-I, IPS-1, and NS1 become part of the same complex. Consistent with this idea, NS1 was also found to inhibit IFN-β promoter activation by IPS-1 overexpression. Our results indicate that, in addition to sequestering dsRNA, the NS1 of influenza A virus binds to RIG-I and inhibits downstream activation of IRF-3, preventing the transcriptional induction of IFN-β.

scholarly journals Development of an HTS Assay for the Search of Anti-influenza Agents Targeting the Interaction of Viral RNA with the NS1 Protein

2008 ◽  
Vol 13 (7) ◽  
pp. 581-590 ◽  
Author(s):  
Marta Maroto ◽  
Yolanda Fernandez ◽  
Juan Ortin ◽  
Fernando Pelaez ◽  
M. Angerles Cabello
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Cytopathic Effect ◽  
Nonstructural Protein ◽  
Specific Interaction ◽  
Chemical Compounds ◽  
Viral Rna ◽  
Ns1 Protein ◽  
Rna Molecules ◽  
Novel Target

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. ( Journal of Biomolecular Screening 2008:581-590)

scholarly journals Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Hannah L. Turkington ◽  
Mindaugas Juozapaitis ◽  
Nikos Tsolakos ◽  
Eugenia Corrales-Aguilar ◽  
Martin Schwemmle ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virulence Factor ◽  
Influenza A ◽  
Nonstructural Protein ◽  
Functional Divergence ◽  
Protein A ◽  
Regulatory Subunit ◽  
Ns1 Protein ◽  
Influenza A Viruses ◽  
Hydrophobic Patch

ABSTRACT Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.

Influenza A viruses balance ER stress with host protein synthesis shutoff

2021 ◽  
Vol 118 (36) ◽  
pp. e2024681118
Author(s):  
Beryl Mazel-Sanchez ◽  
Justyna Iwaszkiewicz ◽  
Joao P. P. Bonifacio ◽  
Filo Silva ◽  
Chengyue Niu ◽  
...  
Keyword(s):  
Viral Replication ◽  
Er Stress ◽  
Host Cell ◽  
Messenger Rna ◽  
Influenza A ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Stress Induction ◽  
Nonstructural Protein 1

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

Sign in / Sign up

Export Citation Format

Share Document