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, simultaneouly, 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.
Rotavirus genome replication and the first steps of virus morphogenesis take place in cytoplasmic viral factories, called viroplasms, containing four structural (VP1, VP2, VP3 and VP6) and two nonstructural (NSP2 and NSP5) proteins. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm-like structures (VLS). In the present work, VLS formation was shown upon co-expression of NSP5 with the core protein VP2 despite the absence of NSP2, indicating a central role for NSP5 in VLS assembly. Since VP2 and NSP2 also induce NSP5 hyperphosphorylation, the possible correlation between VLS formation and the NSP5 phosphorylation status was investigated without evidence of a direct link. In VLS induced by NSP2, the polymerase VP1 was recruited, while the middle layer protein VP6 was not, forming instead tubular structures. On the other hand, VLS induced by VP2 were able to recruit both VP1 and VP6. More importantly, in VLS formed when NSP5 was expressed with both inducers, all viroplasmic proteins were found co-localized, resembling their distribution in viroplasms. Our results suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins. A new role for VP2 as an inducer of viroplasms and of NSP5 hyperphosphorylation is also described. These data may contribute to the understanding of rotavirus morphogenesis.
Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.Rotavirus is a major etiologic agent of severe gastroenteritis in infants and young children worldwide (20,21,33). The virion (defined as a triple-layered particle [TLP]) contains a genome consisting of 11 segments of double-stranded RNA (dsRNA) and is made up of three concentric layers of proteins: the outer layer consists of the two proteins VP7 and VP4, the intermediate layer of VP6, and the internal (core) layer of VP2, with VP1 and VP3 attached at its inside as minor components (29, 41). After entry into the host cell, the virion loses the outer layer to become a double-layered particle (DLP), which is active in transcription of viral mRNAs from the dsRNA genome. The viral RNA-dependent RNA polymerase (RdRp) acts as both the transcriptase and the replicase. Several lines of evidence indicate that VP1 is the viral RdRp: (i) VP1 contains sequence motifs that are shared by RdRps of other RNA viruses (31); (ii) VP1 has NTP-binding activity and, when cross-linked with the nucleotide analog 8-azido-ATP, inhibits RNA transcription (50); (iii) VP1 specifically recognizes the 3Ј end of viral mRNAs (35); and (iv) recombinant VP1 can direct template-dependent minus-strand synthesis in vitro in the presence of VP2 (37, 54).Despite partial characterization of rotavirus replication intermediates (3,19,36,37,54), molecular details of viral genome replication and of the different steps of viral morphogenesis still remain to be elucidated. It has been shown that VP1 and VP2, the scaffolding protein of viral cores, are...
Background: Due to its extremely high strength, the interaction between biotin and (strept)avidin has been exploited for a large number of biotechnological applications. Site-specific biotinylation of proteins in vivo can be achieved by co-expressing in mammalian cells the protein of interest fused to a 15 amino acid long Biotin Acceptor Peptide (BAP) and the bacterial biotin-protein ligase BirA, which specifically recognizes and attaches a biotin to the single lysine residue of the BAP sequence. However, this system is mainly based on the contemporaneous use of two different plasmids or on induction of expression of two proteins through an IRES-driven mechanism.
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