Architecture of the chikungunya virus replication organelle

Laurent, Timothée; Kumar, Pravin; Liese, Susanne; Zare, Farnaz; Pernebrink, Mattias; Carlson, Andreas; Carlson, Lars A.

doi:10.1101/2022.04.05.487153

Cited by 3 publications

(14 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The simplest model explaining this observation is that the[-] RNA varies in the extent to which it is double stranded in different viral RCs and it is this that correlates with both cleavage and the suppression of replication. Certainly, recent electron cryotomography studies on CHIKV replication spherules would support the notion that this viruses [-] RNA is sequestered away in a double stranded state, consistent with the lack of cleavage we observed in our studies(Laurent et al 2022;Tan et al 2022). Interestingly, these same studies suggest that the viral helicase nsp3 is either not associated with the replicase proteins found at the neck of the spherule, or if it is then this association in on the cytoplasmic rather than the lumen face.…”

supporting

confidence: 91%

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand

Herod

Ward

Tuplin

et al. 2022

RNA

View full text Add to dashboard Cite

Genome replication of positive strand RNA viruses requires the production of a complementary negative strand RNA that serves as a template for synthesis of more positive strand progeny. Structural RNA elements are important for genome replication, but while they are readily observed in the positive strand, evidence of their existence in the negative strand is more limited. We hypothesised that this was due to viruses differing in their capacity to allow this latter RNA to adopt structural folds. To investigate this, ribozymes were introduced into the negative strand of different viral constructs; the expectation being that if RNA folding occurred, negative strand cleavage and suppression of replication would be seen. Indeed this was what happened with hepatitis C virus (HCV) and feline calicivirus (FCV) constructs. However, little or no impact was observed for chikungunya virus (CHIKV), human rhinovirus (HRV), hepatitis E virus (HEV) and yellow fever virus (YFV) constructs. Reduced cleavage in the negative strand proved to be due to duplex formation with the positive strand. Interestingly, ribozyme-containing RNAs also remained intact when produced in vitro by the HCV polymerase, again due to duplex formation. Overall, our results show that there are important differences in the conformational constraints imposed on the folding of the negative strand between different positive strand RNA viruses.

show abstract

supporting

confidence: 91%

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand

Herod

Ward

Tuplin

et al. 2022

RNA

View full text Add to dashboard Cite

show abstract

“…So far, we investigated the conformation of sections of the dsRNA within spherules, but did not yet study its overall packaging. Using the volume measurements we previously performed for individual spherules (14), and assuming dsRNA to have a diameter of 24 Å (excluding its hydration shell), we found that the traced amount of dsRNA occupied 7.6 ± 0.9 % of the spherule lumen (Fig. 2A).…”

Section: The Rna Occupies the Spherule Lumen Uniformlymentioning

confidence: 90%

“…The total resolved amount of dsRNA in the five classes reflects 52% of the genome length. The remaining 48% of genome might be present in more variable dsRNA conformations that were averaged out, or as single-stranded RNA (14).…”

Section: Discussionmentioning

confidence: 99%

“…The acquisition of the cryo-electron tomograms analysed in this study was detailed in our previous study (14). In addition, the two tomograms are available at the electron microscopy databank (EMDB) with accession numbers EMD-15582 and EMD-15583.…”

Section: Cryo-electron Tomography Datamentioning

confidence: 99%

“…We previously showed that cryo-electron tomography can resolve structural details of the CHIKV spherule (14). A first characterization of the filamentous material in the spherule lumen indicated that each spherule contains a single copy of the viral genome, ~80% of which is present in dsRNA form.…”

Section: Subtomogram Averaging Unveils Different Conformation Of the ...mentioning

confidence: 99%

See 2 more Smart Citations

The organization of double-stranded RNA in the chikungunya virus replication organelle

Laurent

Carlson

2022

Preprint

Self Cite

View full text Add to dashboard Cite

Alphaviruses are mosquito-borne, positive-sense single-stranded RNA viruses. Amongst the alphaviruses, chikungunya virus is notable as a large source of human illness, especially in tropical and subtropical regions. When they invade a cell, alphaviruses generate dedicated organelles for viral genome replication, so-called spherules. Spherules form as outward-facing buds at the plasma membrane, and it has recently been shown that the thin membrane neck that connects this membrane bud with the cytoplasm is guarded by a two-megadalton protein complex that contains all the enzymatic functions necessary for RNA replication. The lumen of the spherules contains a single copy of the negative-strand template RNA, present in a duplex with newly synthesized positive-sense RNA. Less is known about the organization of this double-stranded RNA as compared to the protein components of the spherule. Here, we analyzed cryo-electron tomograms of chikungunya virus spherules in terms of the organization of the double-stranded RNA replication intermediate. We find that the double-stranded RNA has a shortened apparent persistence length as compared to unconstrained double-stranded RNA. Around half of the genome is present in either of five conformations identified by subtomogram classification, each representing a relatively straight segment of ~25-32 nm. Finally, the RNA occupies the spherule lumen at a homogeneous density, but has a preferred orientation to be perpendicular to a vector pointing from the membrane neck towards the spherule center. Taken together, this analysis lays another piece of the puzzle of the highly coordinated alphavirus genome replication.

show abstract

In situ characterization of late Chikungunya virus replication organelle

Girard

Bihan

Lai-Kee-Him

et al. 2023

Preprint

View full text Add to dashboard Cite

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculo-skeletal disease in humans. The viral RNA genome replication occurs in membrane spherules named replication organelles (ROs). In this work, we investigate the native structural organization of CHIKV ROs in their cellular context using in situ cryogenic-electron microscopy approaches at late replication stage. We observed previously unreported diameter heterogeneity of ROs at the plasma membrane of infected human cells. CHIKV ROs were only marginally detected in cytopathic vacuoles where they are homogeneous in size, suggesting a finely regulated internalization process. Our data show that ROs maintained at the plasma membrane beyond the first viral cycle are dynamically active both in viral RNA replication, in its export to the cell cytosol, but also in the production of viral proteins. We suggest that late CHIKV ROs have an amplifying role or represent an alternative pathway in the production of infectious viral particles. All these observations bring new insight into the CHIKV life cycle in human cells.

show abstract

Architecture of the chikungunya virus replication organelle

Cited by 3 publications

References 50 publications

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand

The organization of double-stranded RNA in the chikungunya virus replication organelle

In situ characterization of late Chikungunya virus replication organelle

Contact Info

Product

Resources

About