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  1. Article ; Online: Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein.

    Lei, Jian / Kusov, Yuri / Hilgenfeld, Rolf

    Antiviral research

    2017  Volume 149, Page(s) 58–74

    Abstract: The multi-domain non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus ... genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known ... CoVs: the ubiquitin-like domain 1 (Ubl1), the Glu-rich acidic domain (also called "hypervariable region ...

    Abstract The multi-domain non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome, with an average molecular mass of about 200 kD. Nsp3 is an essential component of the replication/transcription complex. It comprises various domains, the organization of which differs between CoV genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known CoVs: the ubiquitin-like domain 1 (Ubl1), the Glu-rich acidic domain (also called "hypervariable region"), a macrodomain (also named "X domain"), the ubiquitin-like domain 2 (Ubl2), the papain-like protease 2 (PL2
    MeSH term(s) Amino Acid Sequence ; Coronaviridae/genetics ; Coronaviridae/metabolism ; Glutamine/chemistry ; Humans ; Models, Molecular ; Papain/chemistry ; Papain/metabolism ; Protein Binding ; Protein Conformation ; Protein Domains ; Protein Interaction Domains and Motifs ; Structure-Activity Relationship ; Ubiquitin/chemistry ; Ubiquitin/metabolism ; Viral Nonstructural Proteins/chemistry ; Viral Nonstructural Proteins/metabolism
    Chemical Substances Ubiquitin ; Viral Nonstructural Proteins ; Glutamine (0RH81L854J) ; Papain (EC 3.4.22.2)
    Keywords covid19
    Language English
    Publishing date 2017-11-08
    Publishing country Netherlands
    Document type Journal Article ; Review
    ZDB-ID 306628-9
    ISSN 1872-9096 ; 0166-3542
    ISSN (online) 1872-9096
    ISSN 0166-3542
    DOI 10.1016/j.antiviral.2017.11.001
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    In stock of ZB MED Cologne/Königswinter

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  2. Article ; Online: Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

    Babot, Marion / Boulard, Yves / Agouda, Samira / Pieri, Laura / Fieulaine, Sonia / Bressanelli, Stéphane / Gervais, Virginie

    Journal of virology

    2024  Volume 98, Issue 4, Page(s) e0157523

    Abstract: ... transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays ... structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and ... understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and ...

    Abstract As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA
    Importance: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.
    MeSH term(s) Humans ; Coronavirus Papain-Like Proteases/chemistry ; Coronavirus Papain-Like Proteases/metabolism ; COVID-19/virology ; Endoplasmic Reticulum/metabolism ; Phospholipids ; RNA, Viral/metabolism ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; Viral Nonstructural Proteins/genetics ; Viral Nonstructural Proteins/metabolism ; Virus Replication
    Chemical Substances Coronavirus Papain-Like Proteases (EC 3.4.22.2) ; papain-like protease, SARS-CoV-2 (EC 3.4.22.2) ; Phospholipids ; RNA, Viral ; Viral Nonstructural Proteins
    Language English
    Publishing date 2024-03-14
    Publishing country United States
    Document type Journal Article
    ZDB-ID 80174-4
    ISSN 1098-5514 ; 0022-538X
    ISSN (online) 1098-5514
    ISSN 0022-538X
    DOI 10.1128/jvi.01575-23
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  3. Article ; Online: Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5′ UTR of the viral genome

    Lemak, Sofia / Skarina, Tatiana / Flick, Robert / Patel, Deepak T / Stogios, Peter J / Savchenko, Alexei

    bioRxiv

    Abstract: ... an extensive structure-function analysis of Nsp3 and determined the crystal structures of the Ubiquitin-like 1 ... suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5′ UTR of viral genome during ... translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein. ...

    Abstract Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the Ubiquitin-like 1 (Ubl1), Nucleic Acid Binding (NAB), β-coronavirus-Specific Marker (βSM) domains and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5′ UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5′ UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5′ UTR of viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein.
    Keywords covid19
    Language English
    Publishing date 2024-05-09
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2024.05.09.593331
    Database COVID19

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  4. Article ; Online: X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions.

    Chen, Yafang / Savinov, Sergey N / Mielech, Anna M / Cao, Thu / Baker, Susan C / Mesecar, Andrew D

    The Journal of biological chemistry

    2015  Volume 290, Issue 42, Page(s) 25293–25306

    Abstract: ... structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains ... that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 ... to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating ...

    Abstract Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication.
    MeSH term(s) Amino Acid Sequence ; Crystallography, X-Ray ; Molecular Sequence Data ; Murine hepatitis virus/chemistry ; Protein Conformation ; Sequence Homology, Amino Acid ; Substrate Specificity ; Viral Nonstructural Proteins/chemistry ; Viral Nonstructural Proteins/physiology
    Chemical Substances Viral Nonstructural Proteins
    Keywords covid19
    Language English
    Publishing date 2015-08-19
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1074/jbc.M115.662130
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  5. Article ; Online: Topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning.

    Oostra, Monique / Hagemeijer, Marne C / van Gent, Michiel / Bekker, Cornelis P J / te Lintelo, Eddie G / Rottier, Peter J M / de Haan, Cornelis A M

    Journal of virology

    2008  Volume 82, Issue 24, Page(s) 12392–12405

    Abstract: ... hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus ... the replication complexes to these membranes. For 3 of the 16 coronavirus nsps-nsp3, nsp4, and nsp6-multiple ... of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed ...

    Abstract Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps-nsp3, nsp4, and nsp6-multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.
    MeSH term(s) Cell Line ; Cell Membrane/metabolism ; Coronavirus/genetics ; Coronavirus/metabolism ; Gene Deletion ; Glycoproteins/genetics ; Glycoproteins/metabolism ; Hydrophobic and Hydrophilic Interactions ; Mutation/genetics ; Protein Binding ; Viral Nonstructural Proteins/genetics ; Viral Nonstructural Proteins/metabolism ; Virus Internalization ; Virus Replication
    Chemical Substances Glycoproteins ; Viral Nonstructural Proteins
    Keywords covid19
    Language English
    Publishing date 2008-12
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80174-4
    ISSN 1098-5514 ; 0022-538X
    ISSN (online) 1098-5514
    ISSN 0022-538X
    DOI 10.1128/JVI.01219-08
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