Analysis of rotavirus non-structural protein NSP5 by mass spectrometry reveals a complex phosphorylation pattern

Sotelo, Pablo H.; Schümann, Michael; Krause, Eberhard; Chnaiderman, Jonás

doi:10.1016/j.virusres.2009.12.006

Cited by 14 publications

(13 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3. finding suggests that RVB NSP5 may also be a phosphoprotein as is typical for RVA NSP5, nevertheless an important serine site of phosphorylation has not been identified. Furthermore, three regions (aa 1-7, 48-77, 121 to C terminus) of the NSP5 amino acid sequences are conserved between porcine and other RVBs, which were different from the regions that were conserved between RVAs and RVCs (Sotelo et al, 2010). However, this fact suggests the possibility that the N-and C-terminal regions of RVB NSP5, despite low level amino acid identity, are also critical for formation of viroplasm-like structures as with RVA NSP5 (Fabbretti et al, 1999;Mohan et al, 2003).…”

Section: Discussionmentioning

confidence: 83%

“…2). Although the positions and mechanisms of phosphorylational modification of the NSP5 protein are still unclear, NSP5 is known to be activated by complex hyperphospholyration of serine residues among RVAs (Poncet et al, 1997;Afrikanova et al, 1998;Eichwald et al, 2002Eichwald et al, , 2004bSen et al, 2006;Bar-magen et al, 2007;Sotelo et al, 2010). While this Table 2 NSP5 sequence identities (%) in nucleotide (upper right) and amino acids (lower left) among genotypes grouped by phylogenetic analysis.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains

Suzuki

Soma

Miyazaki

et al. 2012

Infection, Genetics and Evolution

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains

Suzuki

Soma

Miyazaki

et al. 2012

Infection, Genetics and Evolution

View full text Add to dashboard Cite

“…NSP5 is an O-linked glycoprotein (21) with observed molecular masses of 26, 28, and 32 to 35 kDa that represent its many phosphorylated isoforms (22)(23)(24)(25). NSP5 interacts with NSP2, VP1, and VP2 and is found in complexes with NSP2, NSP6, VP1, VP2, VP3, and VP6 (26)(27)(28).…”

mentioning

confidence: 99%

A Novel Form of Rotavirus NSP2 and Phosphorylation-Dependent NSP2-NSP5 Interactions Are Associated with Viroplasm Assembly

Criglar

Crawford

et al. 2014

J Virol

108

View full text Add to dashboard Cite

Rotavirus (RV) replication occurs in cytoplasmic inclusions called viroplasms whose formation requires the interactions of RV proteins NSP2 and NSP5; however, the specific role(s) of NSP2 in viroplasm assembly remains largely unknown. To study viroplasm formation in the context of infection, we characterized two new monoclonal antibodies (MAbs) specific for NSP2. These MAbs show high-affinity binding to NSP2 and differentially recognize distinct pools of NSP2 in RV-infected cells; a previously unrecognized cytoplasmically dispersed NSP2 (dNSP2) is detected by an N-terminal binding MAb, and previously known viroplasmic NSP2 (vNSP2) is detected by a C-terminal binding MAb. Kinetic experiments in RV-infected cells demonstrate that dNSP2 is associated with NSP5 in nascent viroplasms that lack vNSP2. As viroplasms mature, dNSP2 remains in viroplasms, and the amount of diffuse cytoplasmic dNSP2 increases. vNSP2 is detected in increasing amounts later in infection in the maturing viroplasm, suggesting a conversion of dNSP2 into vNSP2. Immunoprecipitation experiments and reciprocal Western blot analysis confirm that there are two different forms of NSP2 that assemble in complexes with NSP5, VP1, VP2, and tubulin. dNSP2 associates with hypophosphorylated NSP5 and acetylated tubulin, which is correlated with stabilized microtubules, while vNSP2 associates with hyperphosphorylated NSP5. Mass spectroscopy analysis of NSP2 complexes immunoprecipitated from RV-infected cell lysates show both forms of NSP2 are phosphorylated, with a greater proportion of vNSP2 being phosphorylated compared to dNSP2. Together, these data suggest that dNSP2 interacts with viral proteins, including hypophosphorylated NSP5, to initiate viroplasm formation, while viroplasm maturation includes phosphorylation of NSP5 and vNSP2.

show abstract

“…Since most of the high-tuned regulation of the cell cycle progression is dependent on phosphorylation cascades [61] and one of the identified proteins (NSP5) shows a hyperphosphorylated pattern [62, 63], mediated by essential kinases involved in the cell progression (such are CK1α (casein kinase 1 alpha) [24], casein kinase 2 [64] or protein kinase C [65]). We hypothesized that the hyperphosphorylated form of NSP5 could deregulate the activity of the kinases mentioned above.…”

Section: Resultsmentioning

confidence: 99%

Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle

et al. 2017

View full text Add to dashboard Cite

In infected cells rotavirus (RV) replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT) network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2) involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.

show abstract

Analysis of rotavirus non-structural protein NSP5 by mass spectrometry reveals a complex phosphorylation pattern

Cited by 14 publications

References 30 publications

Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains

Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains

A Novel Form of Rotavirus NSP2 and Phosphorylation-Dependent NSP2-NSP5 Interactions Are Associated with Viroplasm Assembly

Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle

Contact Info

Product

Resources

About