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  1. Article ; Online: Rotavirus reverse genetics: A tool for understanding virus biology.

    Papa, Guido / Burrone, Oscar R

    Virus research

    2021  Volume 305, Page(s) 198576

    Abstract: Rotaviruses (RVs) are considered to be one of the most common causes of viral gastroenteritis in young children and infants worldwide. Before recent developments, studies on rotavirus biology have suffered from the lack of an effective reverse genetics ( ... ...

    Abstract Rotaviruses (RVs) are considered to be one of the most common causes of viral gastroenteritis in young children and infants worldwide. Before recent developments, studies on rotavirus biology have suffered from the lack of an effective reverse genetics (RG) system to generate recombinant rotaviruses and study the precise roles of the viral proteins in the context of RV infection. Lately a fully-tractable plasmid-only based RG system for rescuing recombinant rotaviruses has been developed leading to a breakthrough in the RV field. Since then, the reproducibility and improvements of this technology have led to the generation of several recombinant rotaviruses with modifications on different gene segments, which has allowed the manipulation of viral genes to characterise the precise roles of viral proteins during RV replication cycle or to encode exogenous proteins for different purposes. This review will recapitulate the different RG approaches developed so far, highlighting any similarities, differences and limitations of the systems as well as the gene segments involved. The review will further summarise the latest recombinant rotaviruses generated using the plasmid-only based RG system showing the enormous potentials of this technique to shed light on the still unanswered questions in rotavirus biology.
    MeSH term(s) Biology ; Child ; Child, Preschool ; DNA Viruses/genetics ; Humans ; Infant ; Reproducibility of Results ; Reverse Genetics/methods ; Rotavirus/genetics ; Rotavirus/metabolism ; Rotavirus Infections ; Viral Proteins/genetics ; Virus Replication/genetics ; Viruses, Unclassified/genetics
    Chemical Substances Viral Proteins
    Language English
    Publishing date 2021-09-21
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 605780-9
    ISSN 1872-7492 ; 0168-1702
    ISSN (online) 1872-7492
    ISSN 0168-1702
    DOI 10.1016/j.virusres.2021.198576
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Rotavirus reverse genetics: A tool for understanding virus biology

    Papa, Guido / Burrone, Oscar R.

    Virus research. 2021 Nov., v. 305

    2021  

    Abstract: Rotaviruses (RVs) are considered to be one of the most common causes of viral gastroenteritis in young children and infants worldwide. Before recent developments, studies on rotavirus biology have suffered from the lack of an effective reverse genetics ( ... ...

    Abstract Rotaviruses (RVs) are considered to be one of the most common causes of viral gastroenteritis in young children and infants worldwide. Before recent developments, studies on rotavirus biology have suffered from the lack of an effective reverse genetics (RG) system to generate recombinant rotaviruses and study the precise roles of the viral proteins in the context of RV infection. Lately a fully-tractable plasmid-only based RG system for rescuing recombinant rotaviruses has been developed leading to a breakthrough in the RV field. Since then, the reproducibility and improvements of this technology have led to the generation of several recombinant rotaviruses with modifications on different gene segments, which has allowed the manipulation of viral genes to characterise the precise roles of viral proteins during RV replication cycle or to encode exogenous proteins for different purposes.This review will recapitulate the different RG approaches developed so far, highlighting any similarities, differences and limitations of the systems as well as the gene segments involved. The review will further summarise the latest recombinant rotaviruses generated using the plasmid-only based RG system showing the enormous potentials of this technique to shed light on the still unanswered questions in rotavirus biology.
    Keywords Rotavirus ; genes ; research ; reverse genetics ; viral gastroenteritis ; viruses
    Language English
    Dates of publication 2021-11
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 605780-9
    ISSN 1872-7492 ; 0168-1702
    ISSN (online) 1872-7492
    ISSN 0168-1702
    DOI 10.1016/j.virusres.2021.198576
    Database NAL-Catalogue (AGRICOLA)

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  3. Article ; Online: IP6-stabilised HIV capsids evade cGAS/STING-mediated host immune sensing.

    Papa, Guido / Albecka, Anna / Mallery, Donna / Vaysburd, Marina / Renner, Nadine / James, Leo C

    EMBO reports

    2023  Volume 24, Issue 5, Page(s) e56275

    Abstract: HIV-1 uses inositol hexakisphosphate (IP6) to build a metastable capsid capable of delivering its genome into the host nucleus. Here, we show that viruses that are unable to package IP6 lack capsid protection and are detected by innate immunity, ... ...

    Abstract HIV-1 uses inositol hexakisphosphate (IP6) to build a metastable capsid capable of delivering its genome into the host nucleus. Here, we show that viruses that are unable to package IP6 lack capsid protection and are detected by innate immunity, resulting in the activation of an antiviral state that inhibits infection. Disrupting IP6 enrichment results in defective capsids that trigger cytokine and chemokine responses during infection of both primary macrophages and T-cell lines. Restoring IP6 enrichment with a single mutation rescues the ability of HIV-1 to infect cells without being detected. Using a combination of capsid mutants and CRISPR-derived knockout cell lines for RNA and DNA sensors, we show that immune sensing is dependent upon the cGAS-STING axis and independent of capsid detection. Sensing requires the synthesis of viral DNA and is prevented by reverse transcriptase inhibitors or reverse transcriptase active-site mutation. These results demonstrate that IP6 is required to build capsids that can successfully transit the cell and avoid host innate immune sensing.
    MeSH term(s) Humans ; Capsid/metabolism ; HIV Infections ; Host-Pathogen Interactions ; Immunity, Innate ; Nucleotidyltransferases/genetics ; Nucleotidyltransferases/metabolism ; Membrane Proteins/metabolism
    Chemical Substances Nucleotidyltransferases (EC 2.7.7.-) ; Membrane Proteins
    Language English
    Publishing date 2023-03-27
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2020896-0
    ISSN 1469-3178 ; 1469-221X
    ISSN (online) 1469-3178
    ISSN 1469-221X
    DOI 10.15252/embr.202256275
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus.

    Strauss, Sebastian / Acker, Julia / Papa, Guido / Desirò, Daniel / Schueder, Florian / Borodavka, Alexander / Jungmann, Ralf

    eLife

    2023  Volume 12

    Abstract: Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that ... ...

    Abstract Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.
    MeSH term(s) Virus Replication ; Ribonucleoproteins/genetics ; Ribonucleoproteins/metabolism ; Viral Nonstructural Proteins/genetics ; Cell Line ; Rotavirus/genetics ; RNA/metabolism ; RNA, Viral/genetics ; RNA, Viral/metabolism
    Chemical Substances Ribonucleoproteins ; Viral Nonstructural Proteins ; RNA (63231-63-0) ; RNA, Viral
    Language English
    Publishing date 2023-01-26
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.68670
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Rotavirus research: 2014-2020.

    Caddy, Sarah / Papa, Guido / Borodavka, Alexander / Desselberger, Ulrich

    Virus research

    2021  Volume 304, Page(s) 198499

    Abstract: Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way ... ...

    Abstract Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. This review follows different stages of the viral replication cycle and summarizes highlights of structure-function studies of rotavirus-encoded proteins (both structural and non-structural), molecular mechanisms of viral replication including involvement of cellular proteins and lipids, the spectrum of viral genomic and antigenic diversity, progress in understanding of innate and acquired immune responses, and further developments of prevention of rotavirus-associated disease.
    MeSH term(s) Animals ; Child ; Child, Preschool ; Cryoelectron Microscopy ; Gastroenteritis ; Humans ; Infant ; Mammals ; Rotavirus/physiology ; Rotavirus Infections ; Virus Replication/genetics
    Language English
    Publishing date 2021-07-02
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 605780-9
    ISSN 1872-7492 ; 0168-1702
    ISSN (online) 1872-7492
    ISSN 0168-1702
    DOI 10.1016/j.virusres.2021.198499
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Viroplasms: Assembly and Functions of Rotavirus Replication Factories.

    Papa, Guido / Borodavka, Alexander / Desselberger, Ulrich

    Viruses

    2021  Volume 13, Issue 7

    Abstract: Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. ... ...

    Abstract Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein-RNA condensates that may be formed via liquid-liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.
    MeSH term(s) Animals ; Capsid Proteins/genetics ; Cytoplasm/virology ; Cytosol/metabolism ; Humans ; Phosphorylation ; RNA-Binding Proteins/metabolism ; Rotavirus/genetics ; Rotavirus/metabolism ; Rotavirus Infections/virology ; Viral Nonstructural Proteins/metabolism ; Viral Replication Compartments/metabolism ; Viral Replication Compartments/physiology ; Virus Assembly ; Virus Replication/genetics
    Chemical Substances Capsid Proteins ; NSP5 protein, rotavirus group A ; RNA-Binding Proteins ; Viral Nonstructural Proteins ; NS35 protein, rotavirus (138414-65-0)
    Language English
    Publishing date 2021-07-12
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 2516098-9
    ISSN 1999-4915 ; 1999-4915
    ISSN (online) 1999-4915
    ISSN 1999-4915
    DOI 10.3390/v13071349
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus

    Sebastian Strauss / Julia Acker / Guido Papa / Daniel Desirò / Florian Schueder / Alexander Borodavka / Ralf Jungmann

    eLife, Vol

    2023  Volume 12

    Abstract: Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that ... ...

    Abstract Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3’ untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.
    Keywords RNA viruses ; biomolecular condensates ; ribonucleoproteins ; RNA imaging ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 612
    Language English
    Publishing date 2023-01-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article: Viroplasms: Assembly and Functions of Rotavirus Replication Factories

    Papa, Guido / Borodavka, Alexander / Desselberger, Ulrich

    Viruses. 2021 July 12, v. 13, no. 7

    2021  

    Abstract: Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. ... ...

    Abstract Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein–RNA condensates that may be formed via liquid–liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.
    Keywords Rotavirus ; droplets ; genome ; reverse genetics ; separation ; viral inclusion bodies ; virion ; virus replication
    Language English
    Dates of publication 2021-0712
    Publishing place Multidisciplinary Digital Publishing Institute
    Document type Article
    ZDB-ID 2516098-9
    ISSN 1999-4915
    ISSN 1999-4915
    DOI 10.3390/v13071349
    Database NAL-Catalogue (AGRICOLA)

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  9. Article ; Online: The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

    Vetter, Janine / Papa, Guido / Tobler, Kurt / Rodriguez, Javier M / Kley, Manuel / Myers, Michael / Wiesendanger, Mahesa / Schraner, Elisabeth M / Luque, Daniel / Burrone, Oscar R / Fraefel, Cornel / Eichwald, Catherine

    mBio

    2024  Volume 15, Issue 4, Page(s) e0049924

    Abstract: Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are ... ...

    Abstract Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle.
    Importance: The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.
    MeSH term(s) Rotavirus/genetics ; Viral Replication Compartments/metabolism ; Viral Nonstructural Proteins/metabolism ; Cryoelectron Microscopy ; Virus Replication/physiology ; RNA ; Peptides
    Chemical Substances Viral Nonstructural Proteins ; RNA (63231-63-0) ; Peptides
    Language English
    Publishing date 2024-03-12
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2557172-2
    ISSN 2150-7511 ; 2161-2129
    ISSN (online) 2150-7511
    ISSN 2161-2129
    DOI 10.1128/mbio.00499-24
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article: Rotavirus research: 2014–2020

    Caddy, Sarah / Papa, Guido / Borodavka, Alexander / Desselberger, Ulrich

    Virus research. 2021 Oct. 15, v. 304

    2021  

    Abstract: Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5–6 years rotavirus research has benefitted in a major way ... ...

    Abstract Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5–6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. This review follows different stages of the viral replication cycle and summarizes highlights of structure-function studies of rotavirus-encoded proteins (both structural and non-structural), molecular mechanisms of viral replication including involvement of cellular proteins and lipids, the spectrum of viral genomic and antigenic diversity, progress in understanding of innate and acquired immune responses, and further developments of prevention of rotavirus-associated disease.
    Keywords Rotavirus ; antigenic variation ; birds ; cryo-electron microscopy ; gastroenteritis ; genomics ; humans ; intestines ; plasmids ; research ; reverse genetics ; structural biology ; tomography ; virus replication ; viruses
    Language English
    Dates of publication 2021-1015
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 605780-9
    ISSN 1872-7492 ; 0168-1702
    ISSN (online) 1872-7492
    ISSN 0168-1702
    DOI 10.1016/j.virusres.2021.198499
    Database NAL-Catalogue (AGRICOLA)

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