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Rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules.

ABSTRACT: Rotavirus viroplasms are cytosolic, electron-dense inclusions corresponding to the viral machinery of replication responsible for viral template transcription, dsRNA genome segments replication and assembly of new viral cores. We have previously observed that, over time, those viroplasms increase in size and decrease in number. Therefore, we hypothesized that this process was dependent on the cellular microtubular network and its associated dynamic components. Here, we present evidence demonstrating that viroplasms are dynamic structures, which, in the course of an ongoing infection, move towards the perinuclear region of the cell, where they fuse among each other, thereby gaining considerably in size and, simultaneously, explaining the decrease in numbers. On the viral side, this process seems to depend on VP2 for movement and on NSP2 for fusion. On the cellular side, both the temporal transition and the maintenance of the viroplasms are dependent on the microtubular network, its stabilization by acetylation, and, surprisingly, on a kinesin motor of the kinesin-5 family, Eg5. Thus, we provide for the first time deeper insights into the dynamics of rotavirus replication, which can explain the behavior of viroplasms in the infected cell.

SUBMITTER: Eichwald C

PROVIDER: S-EPMC3479128 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules.

Eichwald Catherine C Arnoldi Francesca F Laimbacher Andrea S AS Schraner Elisabeth M EM Fraefel Cornel C Wild Peter P Burrone Oscar R OR Ackermann Mathias M

PloS one 20121023 10

Rotavirus viroplasms are cytosolic, electron-dense inclusions corresponding to the viral machinery of replication responsible for viral template transcription, dsRNA genome segments replication and assembly of new viral cores. We have previously observed that, over time, those viroplasms increase in size and decrease in number. Therefore, we hypothesized that this process was dependent on the cellular microtubular network and its associated dynamic components. Here, we present evidence demonstra ...[more]

PMID: 23110139

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Project description:One step of the life cycle common to all rotaviruses (RV) studied so far is the formation of viroplasms, membrane-less cytosolic inclusions providing a microenvironment for early morphogenesis and RNA replication. Viroplasm-like structures (VLS) are simplified viroplasm models consisting of complexes of nonstructural protein 5 (NSP5) with the RV core shell VP2 or NSP2. We identified and characterized the domains required for NSP5-VP2 interaction and VLS formation. VP2 mutations L124A, V865A, and I878A impaired both NSP5 hyperphosphorylation and NSP5/VP2 VLS formation. Moreover, NSP5-VP2 interaction does not depend on NSP5 hyperphosphorylation. The NSP5 tail region is required for VP2 interaction. Notably, VP2 L124A expression acts as a dominant-negative element by disrupting the formation of either VLS or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV species A to H. Detailed knowledge of the protein interaction interface required for viroplasm formation may facilitate the design of broad-spectrum antivirals to block RV replication.IMPORTANCE Alternative treatments to combat rotavirus infection are a requirement for susceptible communities where vaccines cannot be applied. This demand is urgent for newborn infants, immunocompromised patients, adults traveling to high-risk regions, and even for the livestock industry. Aside from structural and physiological divergences among RV species studied before now, all replicate within cytosolic inclusions termed viroplasms. These inclusions are composed of viral and cellular proteins and viral RNA. Viroplasm-like structures (VLS), composed of RV protein NSP5 with either NSP2 or VP2, are models for investigating viroplasms. In this study, we identified a conserved amino acid in the VP2 protein, L124, necessary for its interaction with NSP5 and the formation of both VLSs and viroplasms. As RV vaccines cover a narrow range of viral strains, the identification of VP2 L124 residue lays the foundations for the design of drugs that specifically block NSP5-VP2 interaction as a broad-spectrum RV antiviral.

Project description:Rotavirus (RV) viroplasms are cytosolic inclusions where both virus genome replication and primary steps of virus progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, that contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wild-type (wt) VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in coexpression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCE The spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens.

Project description:The precise characteristics that distinguish normal and oncogenic RAS signaling remain obscure. Here, we show that oncogenic RAS and BRAF induce perinuclear relocalization of several RAS pathway proteins, including the kinases CK2 and p-ERK1/2 and the signaling scaffold KSR1. This spatial reorganization requires endocytosis, the kinase activities of MEK-ERK and CK2, and the presence of KSR1. CK2? colocalizes with KSR1 and Rab11, a marker of recycling endosomes, whereas p-ERK associates predominantly with a distinct KSR1-positive endosomal population. Notably, these perinuclear signaling complexes (PSC) are present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. PSCs are also transiently induced by growth factors (GF) in nontransformed cells with delayed kinetics (4-6 hours), establishing a novel late phase of GF signaling that appears to be constitutively activated in tumor cells. PSCs provide an essential platform for RAS-induced phosphorylation and activation of the prosenescence transcription factor C/EBP? in primary MEFs undergoing senescence. Conversely, in tumor cells, C/EBP? activation is suppressed by 3'UTR-mediated localization of Cebpb transcripts to a peripheral cytoplasmic domain distinct from the PSC region. Collectively, our findings indicate that sustained PSC formation is a critical feature of oncogenic RAS/BRAF signaling in cancer cells that controls signal transmission to downstream targets by regulating selective access of effector kinases to substrates such as C/EBP?.Significance: In addressing the long-standing question of the difference between normal and oncogenic RAS pathway signaling, this study shows that oncogenic RAS specifically triggers constitutive endocytosis-dependent movement of effector kinases to a perinuclear region, thereby creating connections to unique downstream targets such as the core prosenescence and the inflammatory regulatory transcription factor C/EBP?. Cancer Res; 78(4); 891-908. ©2017 AACR.

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Rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules.

Publications

Rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules.

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