Background
Rotaviruses are the major cause of severe diarrhea in children and young animals worldwide. As a members of the family
Reoviridae, they have a genome of 11 segments of double-stranded RNA (dsRNA) enclosed in three protein layers, forming infectious triple-layered particles (TLP) [
1]. During, or just after entering the cell's cytoplasm, the outer capsid, composed of VP4 and VP7, is released, yielding transcriptionally active double-layered particles (DLP). The produced viral transcripts direct the synthesis of viral proteins and serve as templates for the synthesis of negative-RNA strands to form the genomic dsRNA. During the replication cycle of rotavirus electron-dense cytoplasmic inclusions, named viroplasms, are formed [
2]. Such cytoplasmic inclusions are observed during infection with a number of animal viruses [
3], including reoviruses, as other members of the
Reoviridae family [
4].
In rotaviruses two non-structural proteins, NSP2 and NSP5, have been shown to be sufficient to form membrane-free cytoplasmic inclusions, which are known as viroplasms-like structures [
5].
In vivo immunofluorescence visualization of viroplasms shows they are heterogeneous in size [
6,
7]. It is in these structures where the synthesis of dsRNA and its packaging into pre-virion core particles take place [
8]. Besides NSP2 and NSP5, other viral proteins accumulate in viroplasms - namely VP1, VP2, VP3, VP6, and NSP6 [
7,
9‐
11]. The key role of NSP2 and NSP5 proteins in the formation of viroplasms has been demonstrated by knocking-down their expression by RNA interference, which results in the inhibition of viroplasm formation, genome replication, virion assembly, and a general decrease of viral protein synthesis [
7,
8,
12]. Viroplasm formation has been studied using electron or fluorescence microscopy [
6,
13‐
15], however, despite their importance in the replication cycle of rotavirus, little is know about their dynamics of formation. The observation that bromouridine-labeled RNA localizes to viroplasms suggested that the viral transcripts are synthesized within viroplasms, which led to the hypothesis that the entering viral particles could serve as points of nucleation for the formation of viroplasms [
8]. In this work, the dynamics of viroplasm formation in MA104 cells infected with rotavirus strain RRV was studied as a function of time and multiplicity of infection (MOI). Using fluorescently labeled purified rotavirus particles; we showed that the incoming TLPs do not seem to be involved in the formation of viroplasms.
Discussion
The formation of viroplasms has been previously studied using electron and fluorescence microscopy, however, those studies have focused only on late (4 to 24 hpi) stages of infection [
6,
13,
15]. Only Eichwald et al [
14] have studied earlier stages of viroplasm formation, and in their work, following the expression of an NSP2 protein fused to EGFP in rotavirus SA-11 infected cells, they observed that the total number of viroplasms decreased with time, with a concomitant increase in their size, starting at 6 hpi. This observation was interpreted as fusion events between smaller viroplasms. Similar results were reported by Cabral-Romero and Padilla-Noriega [
15] using the strain SA-11 in BSC1 cells, although at even later (10 hpi) stages of infection. Comparing the formation of viroplasms between SA-11 and OSU rotavirus strains, Campagna et al. [
6] observed that the viroplasms formed in OSU infected cells did not increase in size as readily as those formed during infection with SA-11. In this work, after infection with rotavirus strain RRV, using different MOI's, an increase in the number of viroplasms and in the amount of the NSP2 protein was observed. The size of viroplasms was observed to increase when higher MOI's were used.
There are several possibilities to explain the discrepancies reported. First, the decrease in the number of viroplasms was observed only during infection with strain SA-11 [
14,
15], but not with strains OSU [
12], and RRV (this work). It is known that some viral functions (receptor specificity, plaque formation, extraintestinal spread, IRF3 degradation, etc) may vary among different rotavirus strains [
25‐
28] what opens the possibility that there could also be strain-specific differences for viroplasm formation. In fact, an impaired phosphorylation of NSP5 affected differently the morphogenesis of viroplasms in cells infected with either SA-11 or OSU rotavirus strains [
6]. The differences observed between our studies and those of other groups could also arise from the different methodologies used to detect viroplasms. While in our case the newly synthesized rotavirus proteins were immunodetected and analyzed in 400 cells, in the study by Eichwald et al. [
14] the identification of viroplasms was based on the detection of NSP2-EGFP or NSP5-EGFP fusion proteins in 20 cells. It is possible that the large amount of recombinant fusion proteins that accumulated in the cytoplasm of transfected cells before rotavirus infection could change the kinetics of viroplasm formation, since upon rotavirus infection a rapid redistribution of the EGFP - proteins was observed. It was not possible to compare the exact number of viroplasms obtained in that study, since the MOI that was used to infect the transfected MA104 cells was not mentioned.
In this work, studying the kinetics of viroplasm formation during the infection of strain RRV, we observed an increase in both the number and size of viroplasms with time and this increment was dependent on the MOI used. At high MOI's (2.5 and 10) the increase correlated with the amount of NSP2 protein detected at a given time point, while at lower MOI's (0.1 and 0.5), the smaller increase in NSP2 protein correlated with a less variable viroplasm size. It is possible that when a critical concentration of NSP2 and NSP5 is reached, and as other viral proteins accumulate, viroplasms start to form, first as small entities, and then becoming larger at later stages of the replication cycle. Although, it is not possible to determine if the increase in size is caused by fusion of smaller viroplasms or by addition of newly produced rotavirus proteins to small viroplasms, our observations are more consistent with the idea that new small viroplasms are generated constantly during the replication cycle, since even at later stages of infection a large proportion of small viroplasms was observed. It remains to be determined if the small viroplasms, presumably generated by the aggregation of NSP2 and NSP5 require an additional priming signal, or if it is only the concentration of free NSP2 and NSP5 what dictates the formation of a new viroplasms.
The mechanism of viroplasms formation and its protein content is unknown. The fact that viroplasms are sites for rotavirus transcription at late stages of infection (8.5 hpi) led to the so far unproven hypothesis that incoming DLP's serve as focal points of viroplasm assembly [
8]. In this work we tested this hypothesis by visualization of incoming viral particles and by analyzing their colocalization with newly formed viroplasms. Only 2 out of 117 CY5-conjugated viral particles observed in 31 cells colocalized with viroplasms, suggesting that the entering virus particles do not serve as focal points for accumulation of the newly synthesized proteins into viroplasms.
In addition, if the entering virus particles served as focal point for viroplasm formation, the number of viroplasms at early times of infection should correspond to the estimated number of infectious viral particles that entered the cell. However, a correlation between the number of viroplasms detected at early times post-infection and the expected number of infectious particles entering the cells, according to the Poisson distribution (Table
1), was not observed (Figure
2). At low MOI's, when 95% and 77% of infected cells are expected to be infected with only 1 viral particle (with MOI's of 0.1 and 0.5 respectively), there were more viroplasms per cell [1.6 and 3.1 for a MOI of 0.1 and 2.4 and 4.1 for an MOI of 0.5 (2 and 4 hpi respectively)], while at an MOI of 10, when 87% of the cells are expected to be infected with 7 or more infectious viral particles, only 4.2 and 7 viroplasms were observed at 2 and 4 hours post-infection (Figure
2). These results suggest that at the onset of infection the entering viral particles do not serve as nucleation centers for the formation of viroplasms as suggested [
8]. The fact that the plasmid expression of NSP2 and NSP5 proteins alone, in absence of infectious virus, are able to form viroplasm-like structures also supports this conclusion.
Table 1
Theoretical percentage of cells infected with a given number of viral particles at different multiplicities of infection, as determined by the Poisson distribution, with 100% being all infected cells.
1 | 95.1* | 77.1 | 22.4 | 0.05 |
2 | 4.7 | 19.3 | 27.9 | 0.2 |
3 | 0.2 | 3.2 | 23.3 | 0.8 |
4 | 0.003 | 0.4 | 14.6 | 1.9 |
5 | 0† | 0.04 | 7.3 | 3.8 |
6 | 0† | 0.003 | 3.0 | 6.3 |
7 | 0† | 0.0002 | 1.1 | 9.0 |
8 | 0† | 0† | 0.3 | 11.3 |
9 | 0† | 0† | 0.09 | 12.5 |
10 | 0† | 0† | 0.02 | 12.5 |
11 | 0† | 0† | 0.005 | 11.4 |
12 | 0† | 0† | 0.001 | 9.5 |
13 | 0† | 0† | 0.0002 | 7.3 |
14 | 0† | 0† | 0† | 5.2 |
≥15 | 0† | 0† | 0† | 8.3 |
% of total cells infected | 9.5 | 39.3 | 91.8 | 99.8 |
Recently it was suggested that rotavirus viroplasms could interact with microtubules [
15]. NSP2 was also shown to interact with tubulin, inducing the collapse of the microtubule network, and viroplasms were shown to colocalize with tubulin granules [
29]. Similar interaction of reovirus viral inclusion bodies with microtubules [
30] suggests the possibility that tubulin could have a more general role in the replication cycle of viruses of the
Reoviridae family.
Although viroplasms play a crucial role in rotavirus replication and assembly, the factors that govern their formation and function, are still not clearly understood. The development of live cell imaging tools should provide more detailed information about these processes.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JJCT carried out study of kinetics of viroplasms formation, started analysis of fluorophore conjugated viral particles, MG carried out Cy5-TLP's: viroplasm colocalization studies, CFA: has been involved in data analysis and revising final manuscript, SL participated in designing of the study and in critical reading of manuscript, PI conceived of the study, has been involved in Cy5-TLP's: viroplasms colocalization, interpretation of results and drafted the manuscript. All authors read and approved the final manuscript.