Characterization of viroplasm formation during the early stages of rotavirus infection

Abstract

BackgroundDuring 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.ResultsThe 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.ConclusionsThe 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.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

“…The kinetics of NSP1 expression were different from the major viroplasm component NSP2, which was previously analysed using the same method, cell line, virus and MOI (Carreno-Torres et al 2010). Carreno-Torres et al (2010) found a sharp increase in the level of NSP2 expression starting at 4 hpi that correlated with the logarithmic phase of viral replication and increased viroplasm number. In contrast, we found a rise in NSP1 expression starting much later than NSP2 at 9 hpi.…”

“…Others have found that in RRV-infected MA-104 cells, the expression level of the major viroplasm protein NSP2 and the number and size of viroplasms increase with MOI. At 8 hpi, a 20-fold increase in MOI (from 0.5-10) increased the expression level of NSP2 6-fold and the number of viroplasms 1.3-fold (Carreno-Torres et al 2010). In contrast, silencing the expression of viral proteins that are essential for viroplasm formation, such as NSP2, greatly reduced the formation of viroplasms and the synthesis of viral mRNAs in these inclusion bodies (Silvestri et al 2004).…”

“…Protein synthesis lags closely behind mRNA synthesis, such that typical RVA proteins, such as NSP2 (the major component of viroplasms), start to accumulate sharply at 4 hpi and reach a very high level at 8 hpi ( Carreno-Torres et al 2010). The kinetics of dsRNA and infectious virus progeny production show a 2-h delay compared to mRNA synthesis, such that a slight linear increase occurs from 3-6 hpi followed by a logarithmic increase from 6-12 hpi (Ayala-Breton et al 2009).…”

Gangliosides Have a Functional Role during Rotavirus Cell Entry

“…After transfection, cells were washed with MEM and infected with different rotavirus strains at a multiplicity of infection (MOI) of 3. Eight hours postinfection (hpi), cells were fixed with 2% paraformaldehyde, permeabilized with Triton X-100, and stained with rabbit polyclonal sera to NSP2 protein, followed by goat anti-rabbit IgG coupled to Alexa 568, as previously described (33), and with cholera toxin B subunit coupled to Alexa 488. Cell nuclei were stained with DAPI (4=,6-diamidino-2-phenylindole).…”

Molecular Characterization of the Porcine Group A Rotavirus NSP2 and NSP5/6 Genes from São Paulo State, Brazil, in 2011/12

“…NSP5 interacts with NSP2 to form cytoplasmic structures known as viroplasms, inside of which RNA replication and morphogenesis of new viral particles occur [1, 14, 15]. The interaction with NSP2 also enhances the process of NSP5 hyperphosphorylation [9, 16].…”