Identification Of Cellular Proteins Modified In Response To Rotavirus Infection By Proteomics

Rotavirus is the primary cause of acute dehydrating diarrhea in infants and young children worldwide. MA104 cells are most usually used in investigating rotavirus infectious biology in vitro. Some limited attempts have been made to characterize the mRNA expression stations after rotavirus infection by RT-PCR, Northern blotting and cDNA microarray. However, cDNA does not equal to its final product, the direct performer of cell life, protein. In order to reveal the mechanisms in rotavirus-host cell interactions, we analyzed the protein expression profiles of mock and rotavirus infected MA104 cells in a proteomic way. First of all, we established a rotavirus infection model on MA104 cells. We tested the infectious abilities, replication abilities, apoptosis and necrosis inducing abilities of two standard rotavirus strains: human rotavirus Wa strain which is SA-independent and simian rotavirus SA11 strain which is SA-dependent, and found when infected at moi=2(for Wa strain) or moi=8(for SA11 strain), most of MA104 cells could be infected with intact cell shape and little apoptosis or necrosis in 12hs post infection, thus suitable for proteomic analyse.To ananlyse the changes in cell protein profiles between mock and rotavirus infected host cell, we carried out two-dimensional electrophoresis using 13cm pH3-11NL IPG strips for IEF. Computer analysis of the gel detected about 773 and 798 protein spots in mock and human rotavirus Wa infected MA104 cells, compared to 1230 and 1312 spots for mock and simian rotavirus SA11 infection. By comparing the 2D-E maps, we found 14 protein sports obviously up-expressed and 37 spots remarkably down-regulated 12h post human rotavirus Wa infection, with 24 spots up-expressed and 26 down-regulated for simian rotavirus SA11. Between the two group of the up-expressed spots caused by the two rotavirus strains, only two different spots each got similar PI and molecular weight, indicating that they might possibly represent the same proteins. Because it was reported that rotavirus infection caused globly cell proteins down-regulation, up-regulated ones might be more meaningful, and were chosen for further identification.The up-regulated proteins after rotavirus infection were identified by MALDI-TOF-MS and database (NCBInr) searching using mascot. In human rotavirus Wa infection group 7 out of 14 were successfully identified with two spots reprenting the same protein HSP27. Otherr positively identified spots represented an unnamed protein product homolog to zeta-crystalline ( gi|90085272 ) , hypothetic protein ( gi|114583139 ) , hyaluronan-mediated motility receptor (RHAMM), rotavirus NSP3 and keratin 8. In simian rotavirus SA11 infection group only 4 out of 24 proteins were positively identified, which were rotavirus NSP3, plectin 1 isoform 6, protein complex containing cortactin-binding protein 2 and leucine-zipper-like transcription regulator 1 isoform 1, protein complex containing KRT8 protein and reticulocalbin 1. These proteins might associate with host proteins inhibition, signal transmission, metabolism, virus replication and transportation as well as cell differentiation, indicating the remolding of host cell by rotavirus to establish infection.To test the reliability of the MALDI-TOF-MS results, we compared the HSP27 expression between mock and rotavirus infected cells by western blot. We found that HSP27 was obviously elevated in MA104 cells 12 hours after infected by human rotavirus Wa strain, coordinating to the results of MALDI-TOF-MS. However, we found HSP27 was also elevated in simian rotavirus SA11 infected MA104 cells, for the protein spots responding to HSP27 in SA11 infected MA104 cells probably failed to be indentified by MALDI-TOF-MS. We also found that the HSP27 level was much lower in a less susceptible cell line HT-29, and neither of the rotavirus strains up-regulated its expression, showing that the up-regulation of HSP27 in rotavirus infection might be host cell specific, implying its special role in rotavirus infection. Other proteins are waiting for further confirming.