Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere

Here we report the discovery of two Tupanvirus strains, the longest tailed Mimiviridae members isolated in amoebae. Their genomes are 1.44–1.51 Mb linear double-strand DNA coding for 1276–1425 predicted proteins. Tupanviruses share the same ancestors with mimivirus lineages and these giant viruses present the largest translational apparatus within the known virosphere, with up to 70 tRNA, 20 aaRS, 11 factors for all translation steps, and factors related to tRNA/mRNA maturation and ribosome protein modification. Moreover, two sequences with significant similarity to intronic regions of 18 S rRNA genes are encoded by the tupanviruses and highly expressed. In this translation-associated gene set, only the ribosome is lacking. At high multiplicity of infections, tupanvirus is also cytotoxic and causes a severe shutdown of ribosomal RNA and a progressive degradation of the nucleus in host and non-host cells. The analysis of tupanviruses constitutes a new step toward understanding the evolution of giant viruses.

one copy which is located in an intronic region next to a self-splicing group I intron endonuclease. Lineage C presented two copies of the 18S rRNA sequence: copy 1 was disposed in a similar pattern of the copies presented in both tupanviruses (in intergenic regions) and copy 2 showed a most similar pattern to the unique copies of lineages B and C (in an intronic region also next to a self-splicing group I intron endonuclease) ( Fig. 6 A-E). Phylogenetic analyses suggested that 18S rRNA copies 1 and 2 of mimiviruses lineage C had separate and different origins. However, Tupanvirus 18S rRNA copy 1 and 2 seem to be related to mimiviruses lineage A and B (single copy) and fungi mitochondrial 18S rRNA intronic region (Fig. 6F). We also observed the presence of three or four copies of the 18S-like sequences in some Chlorella virus, which were all phylogenetically inter-related (Fig. 6F). Analyses involving FISH and qPCR of the intronic 18S ribosomal region in tupanvirus soda lake demonstrated that, although these 18S rRNA copies are located in intergenic regions, they are highly expressed during the entire infection, especially in intermediate and later phases (6 and 12 hours post infection) (Supplementay Fig. 8).

Profile of infectiveness of Tupanvirus
Tupanvirus was first isolated in both Acanthamoeba castellanii and Vermamoeba vermiformis, suggesting a broader host-range compared to other previously described amoebal giant viruses. In light of this, we tested the infectiveness of the new isolate on a large panel of protozoa. Four distinct infectiveness profiles were observed (Supplementary Table 1). Cytopathic effect (CPE), increase of viral titer, and genome replication were observed in A. castellanii, A. polyphaga, A. sp E4, A. griffini, V. vermiformis, Dysctiostelium discoideum, and Willartia magna, characterizing a productive cycle wherein these hosts were permissive to Tupanvirus. An abortive cycle was observed in Acanthamoeba sp michelline and A. royreba, wherein CPE and Tupanvirus genome replication were observed, but there was no particle formation since the viral titer did not increase. Trichomonas tenax were completely refractory to Tupanvirus, since CPE, genome replication and increase of viral titer were not observed. The fourth profile was observed in RAW247, THP-1 cells and in Tetrahymena hyperangularis, a ravenous free-living protist, wherein Tupanvirus was able to induce a cytopathic effect, but neither an increase of viral titer nor genome replication were observed, thus we concluded that TPV was toxic to the susceptible cell -an unprecedented profile among amoebal giant viruses.

Tupanvirus modulates a non-host organism
Tetrahymena sp was susceptible to Tupanvirus, which caused several effects ( Fig. 7G;   Fig. 8); however it could not replicate within the protist. In light of this, we hypothesized that the reduction of physiological activity of a (non-host) predator could increase the virus' fitness. We put it to the test by performing in vitro simulations, wherein Acanthamoeba castellanii (AC) and Tetrahymena sp cells were put together and infected at M.O.I. of 10 (Tupanvirus or APMV), and observed over 12 days. Input of fresh medium and permissive host (AC) was done at days four and eight post infection. After the input, Tupanvirus reached higher titers while APMV was extinguished, since the latter was not able to modulate the predator organism, unlike Tupanvirus (Fig. 8N). When we induced a previous ribosomal RNA shutdown in Tetrahymena with geneticin, APMV was able to survive until the last day of observation, similar to tupanvirus. This indicates that viral particles' toxicity confers an advantage in a habitat of intense competition where giant viruses are abundant (e.g. water environment), favoring the encounter between the virus and a permissive host.
Altogether, this data suggest that viral particles can act as active "non-alive" players favoring viral progeny maintenance, in a distinct way of their canonical role of transmitting genetic information. Considering that Tupanvirus is sister group of mimivirus, such modulation mechanism could be an ancient inheritance from an ancestor of Mimivirus or Mimiviridae. This new mechanism demonstrates that selective pressures over virion content go beyond the metastability properties in early steps of host infection, and can act as unexpected players to protect the rest of the viral progeny against predation.

rRNA shutdown does not seems to be related to ribophagy
The cytotoxic phenotype caused by Tupanvirus in host (A. castellanii) and non-host cells (Tetrahymena) is a circumstance never previously described and seems to be related to the shutting down of host ribosomal amounts ( Fig. 7C; Fig. 8B,C,D,K,L). In a first moment, we believed that the autophagy might be related to Tupanvirus infection.   Tupanvirus soda lake. The sequence of the glutaminyl-tRNA synthetase was split into short fragments and blasted against the NR database. Best hit was selected and integrated in a circular gene data image. Taxonomic origins are colored in blue for eukaryote, green for bacteria and orange for viruses.

Supplementary Figure 6: Genomic environment and best-hit analyses of
Tupanvirus soda lake 18S rRNA intronic regions. Genomic environment of copy 1 (a) and copy 2 (b). The 100 best hits for copies 1 (c) and 2 (d).