Characterization of the tupanvirus cytopathic effect: a giant virus inducing cell aggregation

In order to characterize the cytopathic effect (CPE) triggered by tupanvirus, A. castellanii cells were infected at an M.O.I. of 10 and observed at up to 72 h.p.i. The cells became rounded and lost their adherence; this effect became more evident at 4 h.p.i. (Fig. 1A). Interestingly, tupanvirus exhibits a peculiar CPE that is characterized by amoebae aggregates, called “bunches.” The formation of bunches starts at approximately 6 h.p.i. and becomes more evident at 8, 12 and 16 h.p.i., when almost all the cells are seen incorporating into large bunches (Fig. 1A). From 24 h.p.i., despite the presence of bunches, cell lysis is more evident and is concomitant with the reduction of bunches size. At 32 h.p.i., the complete disaggregation of bunches was observed, and at 48–72 h.p.i. the majority of amoebae were lysed (Fig. 1A). The formation of bunches was also observed when a low M.O.I. (0.01) was used, although there was a temporal delay in bunch appearance (Suppl. Fig. 1). The CPE triggered by tupanvirus is unlike that of the other mimivirus, as well as other known giant amoebal viruses. Acanthamoeba polyphaga mimivirus, the prototype of the genus Mimivirus, triggers rounding and lysis in A. castellanii cell cultures, being a CPE commonly described for other giant viruses (M.O.I. of 10) (Suppl. Fig. 2).

Different timepoints during infection (at an M.O.I. of 10) were selected for an immunofluorescence assay (IF) and transmission/scanning electron microscopy (TEM and SEM). IF assays using anti-tupan antibody revealed that at 0 and 1 h.p.i. tupanvirus particles appeared attached to amoebas (Fig. 2), even after the cell washing step, suggesting virion fibrils early interactions with host-cells, as demonstrated for other mimiviruses19. VFs start to be detected by IF in amoebas’ cytoplasm using anti-tupanvirus whole-particle antibodies at 12 h.p.i. (Fig. 2). Curiously, even at high a M.O.I. it was not possible to observe a synchronous infection of all cells, something that was peculiarity related to tupanvirus and that needs further investigation. Remarkably, we observed amoebas with and without VFs or viral particles in the cytoplasm by IF, especially at 12 h.p.i., suggesting that early bunches are formed by the association of cells at different stages of the viral replication cycle (Fig. 2). Even at 16 h.p.i., when the viral titer reached its plateau (Fig. 1B) and the significant majority of cells already presented viral factories and particles (Fig. 2), we were able to find a very few cells with no detectable signs of tupanvirus by IF. By SEM (Fig. 3A) and TEM (Fig. 3B), it was possible at 16 h.p.i. to observe bunches of amoebas with damaged cells filled with or releasing tupanvirus particles, which were associating with cells with an intact plasma membrane and only a few particles inside them (Fig. 3).

Figure 2 Characterization of the tupanvirus replication cycle in A. castellanii by IF. A. castellanii monolayer was infected by tupanvirus at an M.O.I. of 10 and visualized by IF. At early timepoints, tupanvirus particles are observed attaching to the amoebae surfaces, even after th cell washing step was done. The VF forms between 8 and 12 h.p.i. At 16 and 24 h.p.i. the A. castellanii cytoplasm is filled with viral particles and cell lysis is observed at 24 h.p.i. (at which time the cytoskeleton of lysed amoebas is visualized, although these cells remain adhered). The viral particles are in green (anti-tupan particle antibody) and amoeba cytoskeleton in red (stained by Evans Blue). Scale bar, 100 µm. Full size image

Figure 3 Detailed images of a 16 h.p.i. bunch. The bunches induced by tupanvirus in A. castellanii cells at different stages of infection were analyzed by: (A) Scanning electron microscopy and (B) transmission electron microscopy. White arrows indicate some tupanvirus particles being released from damaged amoebas. Scale bars, 2 µm and 10 µm. Full size image

Early amoebal bunches reform after mechanical separation of cells

Another phenomenon that drew our attention was the occurrence of bunch reformation after mechanical breakdown (Fig. 4). Initially, we observed that early and late bunches could be easily disaggregated by homogenization using a pipette or by vortexing. However, 30 min after disaggregation, cells from early bunches (12 h.p.i.) were attracted to each other, forming large clusters of cells and assuming an appearance similar to early bunches before cell separation. In contrast, reformation of late bunches (24 h.p.i.) was not observed. In addition, using light microscopy we observed that some cells belonging to late bunches were larger in size, which was especially obvious after the disaggregation procedure (Fig. 4). The lack of bunch reformation at late timepoints indicates that at 24 h.p.i., cells were already killed by the viral infection, and a simple homogenization was able to permanently disaggregate the cells that formed late bunches. To confirm this hypothesis, we performed SEM on late bunches. Interestingly, we observed cells with the plasma membranes almost completely degraded and filled with intricate networks of tupanvirus particles and cell debris (Fig. 5).

Figure 4 Early bunches re-aggregate after disassociation. A. castellanii cells infected with tupanvirus. Early (12 h.p.i.) and late (24 h.p.i.) bunches are shown, which were mechanically homogenized for cell disaggregation. Thirty minutes later, the cells were analyzed. (A) Re-aggregation of the early bunches 30 min after disassociation. (B) Re-aggregation of late bunches was not observed. Scale bar, 200 µm. Full size image

Figure 5 Late bunches are composed of highly damaged dead A. castellanii cells. A. castellanii cells shown intensely damaged at 24 h.p.i., as observed by SEM. Scale bars, 5 µm and 10 µm. Full size image

Taken together, these results indicate that tupanvirus promotes the formation of large aggregates of amoebas, which are able to re-aggregate as long as cells are still alive. These findings motivated us to investigate possible factors that may interfere in bunch formation and the biological relevance of tupanvirus-promoted bunches.

Amoebal-bunch formation correlates with increased mRNA levels of mannose binding protein genes

Acanthamoeba spp. is widely distributed in the environment and can cause human diseases, mainly associated with immunocompromised patients and contact lenses wearers. Furthermore, Acanthamoeba spp. has a relevant role in ecosystems, due to its ability to act as a host for microbial pathogens20,21,22,23. Mannose receptor proteins are well known for promoting interactions between amoebas and other cells. In this way, previous studies revealed that mannose-binding protein (MBP) present on the surface of Acanthamoeba is one of the major virulence proteins and may be essential for pathogenesis and invasion of host cells11,15,24 demonstrated that the potential of Acanthamoeba pathogenicity is directly dependent on the level of MBP24. In addition, previous studies showed that A. castellanii treated with mannose had reduced adhesion and cytotoxicity, strongly suggesting that its pathogenicity is associated with MBP11,25. Interestingly, after an in-depth analysis of the tupanvirus soda lake genome, we found that the virus has an 525 bp MBP gene. The domain involved in mannose recognition contains a three-fold internal repeat, and the consensus sequence motif is QXDXNXVXY. A previous study has suggested that the QDN subdomains may be essential for functionality of the protein. In addition, the importance of Y in subdomain of the MBP-mannose interaction was demonstrated26.

This finding led us to investigate the MBP viral and cellular expression during the replication of tupanvirus and the possible effects of free mannose on the formation of bunches. An analysis of cellular MBP gene expression in uninfected A. castellanii cells showed a basal level of MBP transcripts, meaning it is likely essential for cellular processes (Fig. 6A). However, during tupanvirus infection, the levels of cellular MBP transcripts significantly increased (p = 0.0001) at earlier times during infection (1, 2 and 4 h.p.i.). The increase of cellular MBP gene expression induced by tupanvirus precedes bunch formation by a few hours (6 h.p.i.) (Figs 1A and 6A). Regarding tupanvirus encoding MBP, we observed a gradual increase in (or accumulation of) transcripts through the infection time-course. High levels of viral MBP transcripts were observed from 4 until 8 h.p.i (Fig. 6B). Therefore, our data indicate that the expression of cellular and viral MBP genes is induced during tupanvirus infection, suggesting a possible relevance of these genes in the viral replication cycle (Fig. 6).

Figure 6 Tupanvirus upregulates the expression of viral and cellular mannose binding protein (MBP) genes. A. castellanii cells were infected with tupanvirus and collected at 1, 2, 4, 6 and 8 h.p.i. and MBP mRNA levels were measured by qPCR: cellular (A) and viral (B) genes with and without the presence of mannose. The data were calculated using the 2−(Δct) method and are shown as the standard deviation of two independent biological assays. Error bars indicate SDs. The statistical significance was calculated using a two-tailed 2-way ANOVA test, Tukey’s range test, using GraphPad Prism. ****p < 0.0001. Full size image

Interestingly, orthologous MBP genes were found in the genome of other members of the Mimiviridae family, such as in the prototype APMV. Phylogenetic analysis of the amino acid sequence of viral MBP showed that tupanviruses forms an out-grouping among amoebal-mimiviruses (Suppl. Fig. 3A). Considering APMV does not induces the formation of bunches in infected amoebas, an analysis was performed to determine whether APMV was able to induce the expression of cellular MBP, as well as whether this virus express its own MBP gene throughout the time-course of infection. We observed that APMV induced a decrease in cellular MBP gene expression, even when compared to uninfected cells, in contrast to tupanvirus soda lake, which significantly increases cellular MBP gene expression which may be related to the induction of bunch formation (Suppl. Fig. 4B).

The expression of APMV MBP gene transcripts was also analyzed during the time-course of APMV infection in A. castellanii, revealing that this gene was significantly expressed at 6 and 8 h.p.i. (Suppl. Fig. 4A). However, an analysis of the APMV MBP amino acid sequence revealed polymorphisms in all three of the repeats that compose the catalytic site of the protein (the mannose-binding domain), more specifically in the QDN subdomains (Suppl. Fig. 3B), which have been associated with a loss of MBP function26. Polymorphisms in the Y amino acids have also been observed in the first repeat of the APMV MBP catalytic site (Suppl. Fig. 3B), which has also been associated with a loss of MBP function26. In contrast, tupanvirus MBP has no polymorphisms in any of the repeats in the catalytic site, when compared to the functional protein previously studied (Suppl. Fig. 3B)26. In summary, mutations in the mannose-binding domains of the APMV MBP gene could indicate that this protein is not able to bind to mannose, although more studies into structural level are needed to confirm this hypothesis.

Previous work have demonstrated that free-mannose negatively affects Acanthamoeba adhesion to cell surfaces and interferes with amoeba pathogenicity16,25. Therefore, the impact of free-mannose on cellular and viral MBP gene expression was here evaluated in uninfected and tupanvirus-infected cells. Our results showed that free mannose negatively affects the expression of the cellular MBP gene, maintaining it at close to basal levels (Fig. 6A). The same negative effect was observed in relation to the expression of the tupanvirus MBP gene (Fig. 6B). Considering these results, the effect of free mannose on the cytopathic effect of tupanvirus was evaluated as well. Interestingly, when Acanthamoeba was pre-treated with free-mannose, there was a gradual inhibition of bunch formation in a dose-dependent manner (Fig. 7), suggesting that formation of amoebal-bunches correlates with the suppression of viral and cellular MBP genes (Fig. 7).

Figure 7 Free-mannose affects tupanvirus-induced bunch formation in a dose-dependent manner. A. castellanii cells were treated with free-mannose at concentrations ranging from 25 to 1000 mM in PAS solution, and then infected with tupanvirus (M.O.I. of 10). Eight hours post-infection, cells were observed by optical microscopy. As controls, A. castellanii cells cultivated in PAS medium without mannose and uninfected cells were cultured with 600 mM mannose. Bar graphs represent the number of bunches found per ml in different concentrations of mannose. Error bars indicate SDs. The treatment of amoebas with 600 mM mannose did not affect the replication of tupanviruses in amoebas and did not induced cytotoxic effects in uninfected A. castellanii. Scale bar, 200 µm. Full size image

Previous studies have shown that Acanthamoeba MBP is itself a glycoprotein containing mannose15, which may be one of the factors that explains the formation of the bunches in cells infected by tupanvirus, since this virus induces the expression of the MBP gene. Thus, the induction of mannose receptor genes in the tupanvirus replication cycle may be important for the optimization of the formation of bunches, since the interaction between amoebas may occur through interactions among their receptors.

Bunch-forming cells are able to interact with uninfected cells

As previously characterized in this study, the formation of the bunches starts at approximately 6 h.p.i. and persists until at least 24 h.p.i. At 16 h.p.i., almost all the cells are infected and clustered forming large bunches (Fig. 1A). An experiment was designed in order to evaluate whether bunch-forming cells were able to interact with uninfected cells (Fig. 8A). For this, A. castellanii cells were infected with tupanviruses (at an M.O.I. of 10), and at 16 h.p.i. the bunches were collected and used to inoculate a flask containing uninfected A. castellanii cells in suspension. After incubation, the supernatant (containing bunches and eventual non-infected amoebas interacting with bunches) were removed. The cells that remained adhered to the flask (the ones that did not interact with bunches) were counted to evaluate if the bunches were able to hijack the uninfected cells (Fig. 8). In addition, the supernatant was submitted to SEM and IF, using tupanvirus-specific antibodies. APMV-infected amoebae, which do not induce bunch formation, were used as a control in order to compare whether APMV-infected cells were also able to interact with and hijack uninfected cells (Fig. 8B).

Figure 8 Tupanvirus-induced bunches may bind to and hijack uninfected cells. (A) A. castellanii cells were infected with tupanvirus at an M.O.I. of 10 and at 16 h.p.i. bunches were collected and used to inoculate flasks containing fresh, uninfected A. castellanii cells. After 30 min of incubation, the supernatant in flasks containing bunches and uninfected cells was removed. The remaining cells attached to the flask were then quantified. As a control, APMV-infected amoebae (which does not induce bunches formation) were used in parallel with tupanvirus-infected ones. (B) Remaining cells attached to flasks after incubation with tupanvirus-induced bunches or cells infected with APMV were then counted. Tupanvirus-induced bunches attached to and hijacked uninfected cells, causing a significant decrease of remaining adherent cells in the flasks, compared to APMV-infected cells. The statistical significance was calculated using a two-tailed Student’s t-test. *p < 0.05, performed using GraphPad Prism. Error bars indicate SDs. Full size image

Our results revealed that tupanvirus-induced bunches are able to bind to and hijack many uninfected cells. The number of cells remaining in the flasks after interaction with tupanvirus-induced bunches was significantly lower than those in flasks inoculated with APMV-infected cells (Fig. 8B). The interaction among tupanvirus-induced bunches and uninfected cells was also observed by SEM; it was possible to observe infected cells, which were rounded, interacting with fresh trophozoites presenting their typical pseudopods (Fig. 9). IF assays also showed bunches containing a mix of infected and uninfected cells (data not shown). Lastly, considering the ability of tupanvirus-induced bunches to bind and hijack uninfected cells, we evaluated whether such interactions could promote an increase in viral titer. For this, bunches were collected at 16 h.p.i. and inoculated in flasks containing fresh, uninfected amoebas, as previously described above. After incubation, the supernatant containing bunches attached to uninfected cells was collected and transferred to a new flask. Infectious particles in these population of cells were tittered at 0 and 36 h.p.i, revealing an of almost 2 log increase in viral titer (Suppl. Fig. 5).

Figure 9 SEM showing the interaction between uninfected A. castellanii cells and tupanvirus-induced bunches. SEM images of uninfected cells that interacted with bunches and were carried by infected cells (experiment described in Fig. 8). Scale bars, 10 µm and 20 µm. Arrows indicate infected A. castellanii cells (rounded) and asterisks indicate uninfected A. castellanii cells (most with pseudopods). Full size image

Tupanviruses has the broadest host spectrum known among amoebal giant viruses1. While most of the giant viruses, such as cedratvirus, marseilleviruses, mollivirus, pandoraviruses, mimivirus, faustovirus and kaumoebavirus, are known to be able to replicate only in a single amoeba genus, tupanvirus is able to replicate in Acanthamoeba spp., Vermoameba vermiformis, Dysctiostelium discoideum and Willartia magna1,5,6,7,8,9,27,28. The generalist profile displayed by tupanviruses may be related to the extreme aquatic environments where they were isolated (high salinity, pH and depths); within inhospitable environments there is a lower species richness and abundance, meaning there are more hosts available and a better chance of a future infection29. In this way, bunch formation induced by tupanvirus may be important to the recruitment of cells, improving the chances of viral progeny to find a new host cell that may be scarce in those environments. In addition, bunch formation can reduce the dilution effect in aquatic environments, thereby allowing the release of more particles near the host, which could also facilitate the encounter between viral particles and their hosts.