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

Genomic replication and partial assembly of Rotavirus takes place in cytoplasmic viral structures called viroplasms. NSP5 is a viral phosphoprotein localized in viroplasms and its expression is imperative for viral cycle progress. During infection three isoforms of NSP5 can be observed by SDS-PAGE (26, 28 and 33-35kDa) and previous reports suggested that they differ in their phosphorylation patterns. In this study we obtained NSP5 from infected cells and by mass spectrometry we were able to identify nine phosphorylation sites. We detected that in all the isoforms the same residues can be found either phosphorylated or unmodified. Quantitative analysis showed that the 28kDa isoform has a higher phosphorylation level than the 26kDa isoform suggesting that migration properties depend on the total number of phosphorylated residues. Moreover, we identified two not previously described modifications for this protein: an N-acetylation in Serine-2 and an intramolecular disulfide bond in a highly conserved motif, CXXC which is located between two charged alpha-helix motifs.

Uptake, tissue distribution and accumulation of microcystin-RR in Corydoras paleatus, Jenynsia multidentata and Odontesthes bonariensis. A field and laboratory study.

The uptake and accumulation of microcystin-RR (MC-RR) in fish was investigated under laboratory conditions and in wild fish. Jenynsia multidentata and Corydoras paleatus were exposed for 24h to 50mug/L MC-RR dissolved in water. After exposure, liver, gill, brain, intestine, gall bladder, blood and muscle were analyzed for MC-RR by HPLC and analysis confirmed by LC-ESI-TOF-MS spectrometry. Furthermore, wild individuals of Odontesthes bonariensis were sampled from the eutrophic, cyanobacteria-containing San Roque reservoir, and analyzed for the presence of MC-RR in liver, gill, intestine, and muscle. MC-RR was found in liver, gills, and muscle of all exposed and wild fish, while in C. paleatus MC-RR was also present in the intestine. Moreover, we found presence of MC-RR in brain of J. multidentata. Results indicate that MC-RR uptake might occur at two different organs: intestine and gills, through either feeding (including drinking) or respiratory activities. This suggests that MC-RR is taken into the blood stream after absorption, and distributed to different tissues. The liver showed the major bioaccumulation of MC-RR in both experimentally exposed and wild individuals, with muscle of wild fish showing relative high amounts of this toxin in comparison with those exposed in the laboratory; though MC-RR was present in muscle of fish exposed for 24h. The amount of MC-RR in muscle of O. bonariensis exceeded the value suggested by WHO to be safe, thus causing a health risk to persons consuming fish as a result of chronic exposure to microcystin. Gills also showed bioaccumulation of MC-RR, raising questions on the mechanism involved in the possible uptake of MC-RR through gills as well as on its accumulation in this organ. Although MC-LR has been reported in brain of fish, this is the first report confirming the presence of MC-RR in this organ, which means that both toxins are able to cross the blood-brain barrier. These findings also raise questions on the probable neurotoxicity of microcystins.