Fig. 6: Virophage halts Tupanvirus spread and protects the host cell (amoebas) population from lysis.

a–c Dose-response of giant virus to virophage. A. castellanii cells were coinfected with APMV (a) or Tupanvirus Deep Ocean (b, c) at different MOIs (1, 0.1 and 0.01) and increasing MOIs (0.1, 1 and 10) of the virophage mixture containing mutant Guarani (a, c) or Zamilon (b). The Tupanvirus titer in the supernatant was then measured by end-point dilution after complete lysis of cells or 5 days p.i. in the absence of cell lysis. c Higher concentrations of virophage cause a systematic fall in Tupanvirus titer to undetectable levels. We interpreted these observations as the results of a phenomenon of elimination induced by virophage. This phenomenon was not observed when Tupanvirus was challenged with Zamilon (b) nor when APMV was challenged with the virophage mixture that contains mutant Guarani (a). d Analyses of host cell survival in presence and absence of mutant Guarani infection. A. castellanii cells in PYG medium were coinfected with Tupanvirus Deep Ocean at different MOIs (1, 0.1 and 0.01) and virophage mixture of wild-type and mutant Guarani at an MOI of 10. Cell densities were then monitored by microscopy count from 0 h to 96 h p.i. Uninfected amoebas were used as controls. In the absence of virophage, regardless of Tupanvirus MOI, the virus lyses the entire host cell population at 96 h p.i. approximatively. The presence of virophage dramatically modifies the lysis of amoebas at low MOIs of the Tupanvirus. The virophage protects a portion of host cells from lysis at MOIs of 0.1 and 0.01 of Tupanvirus. At an MOI of 0.01 of Tupanvirus, the phenotype of the amoebae population seems similar to that of uninfected cells in presence of mutant Guarani. e Histograms depicting the genome replication of Guarani with Tupanvirus Deep Ocean at different MOIs (1, 0.1 and 0.01). DNA quantification was done by Real-time PCR and analyzed as described above. Figures, in (b–d), are listed according to the MOI of Tupanvirus. Error bars, standard deviation (n = 3 biologically independent experiments). N.D.: Not detectable. TDO: Tupanvirus Deep Ocean.