Characterization of viroplasm formation during the early stages of rotavirus infection

Background: During rotavirus replication cycle, electron-dense cytoplasmic inclusions named viroplasms are formed, and two non-structural proteins, NSP2 and NSP5, have been shown to localize in these membrane-free structures. In these inclusions, replication of dsRNA and packaging of pre-virion particles occur. Despite the importance of viroplasms in the replication cycle of rotavirus, the information regarding their formation, and the possible sites of their nucleation during the early stages of infection is scarce. Here, we analyzed the formation of viroplasms after infection of MA104 cells with the rotavirus strain RRV, using different multiplicities of infection (MOI), and different times post-infection. The possibility that viroplasms formation is nucleated by the entering viral particles was investigated using fluorescently labeled purified rotavirus particles. Results: The immunofluorescent detection of viroplasms, using antibodies specific to NSP2 showed that both the number and size of viroplasms increased during infection, and depend on the MOI used. Small-size viroplasms predominated independently of the MOI or time post-infection, although at MOI's of 2.5 and 10 the proportion of larger viroplasms increased. Purified RRV particles were successfully labeled with the Cy5 mono reactive dye, without decrease in virus infectivity, and the labeled viruses were clearly observed by confocal microscope. PAGE gel analysis showed that most viral proteins were labeled; including the intermediate capsid protein VP6. Only 2 out of 117 Cy5-labeled virus particles colocalized with newly formed viroplasms at 4 hours post-infection. Conclusions: The results presented in this work suggest that during rotavirus infection the number and size of viroplasm increases in an MOI-dependent manner. The Cy5 in vitro labeled virus particles were not found to colocalize with newly formed viroplasms, suggesting that they are not involved in viroplasm nucleation.