pubmed.ncbi.nlm.nih.gov

A giant virus infecting the amoeboflagellate Naegleria - PubMed

  • ️Mon Jan 01 2024

A giant virus infecting the amoeboflagellate Naegleria

Patrick Arthofer et al. Nat Commun. 2024.

Abstract

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

© 2024. The Author(s).

PubMed Disclaimer

Conflict of interest statement

S.R. acts as advisor for Cryomodultech, e.U., companies registered in Vienna No. FN460571. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The Catovirus naegleriensis (Naegleriavirus) genome, phylogeny, and translation modules.

a The linear double stranded DNA genome with 1000 predicted CDS. From outer to inner track: blue, putative host-derived genes; gray, black 480 and 520 CDS on the two strands, respectively; cyan, proteins detected in the virion; gray links represent homologous CDS. b Maximum likelihood tree constructed with three Nucleocitoviricota core genes, DNA polymerase family B, A18-like helicase, and poxvirus late transcription factor VLTF3. The tree is rooted with the Asfarviridae as an outgroup. The full version of the tree is available as Supplementary data set 2. c Circular representation of homologs shared between the Naegleriavirus (NiV) genome and the two contigs of the Catovirus CTV1 MAG; colored links depict bi-directional best BLAST hits. d Translation-related genes found in Naegleriavirus compared to other Klosneuviruses. Numbers in the boxes indicate the number of homologs. The relationship of the viruses is indicated as a schematic representation of the phylogenetic tree in panel b.

Fig. 2
Fig. 2. The Naegleriavirus genome and its virion proteome.

a Taxonomic classification of 1000 predicted NiV genes based on similarity to viruses and cellular organisms using BLASTp against the NCBI nr database. b Functional categorization of NiV genes based on an automated PROKKA annotation, InterproScan, eggNOG, and manual curation. The percentages of genes/proteins in the genome/proteome for each COG category are depicted. Annotation details are available in Supplementary Data Sets 1, 3.

Fig. 3
Fig. 3. Naegleriavirus genes potentially acquired from Naegleria hosts.

Approximated maximum-likelihood phylogenetic trees are shown for genes encoding an a ATPase-domain containing protein, b Bax 1 inhibition factor 1, c mitochondrial chaperone, d SNARE coiled-coil domain-containing protein, and e HSP70. Bootstrap values lower than 0.8 are depicted as gray circles. Only the relevant subtrees, including Naegleriavirus genes are shown; full versions of the trees are available as Supplementary Files.

Fig. 4
Fig. 4. The Naegleriavirus replication cycle in Naegleria clarki.

The infection was performed at a multiplicity of infection (MOI) of 10. Fluorescence in situ hybridization (FISH) images are shown. The host cell is depicted in magenta (oligonucleotide probe Nag1088; Supplementary Table 3), with nucleic acid staining by DAPI in cyan. a An uninfected N. clarki trophozoite. b An ameba cell 1 hour post viral infection; small DAPI-stained structures start to accumulate in the cytoplasm. c 4 hpi, intermediate stages of the viral factory are visible. d 8 hpi, the major viral factory and mature viral particles accumulating in the host cytoplasm can be seen. N = nucleus. VF = viral factory. V = virion. Scale bar = 5 µm.

Fig. 5
Fig. 5. The ultrastructure of Naegleriavirus virions.

Transmission electron microscopy of cryoimmobilized samples processed by freeze substitution or chemically fixed at room temperature (indicated by *). a Medial section of a virion in the host cytoplasm exhibiting the viral core surrounded by membranes, the capsid shell, and a fiber shell. The NiV particle structure is notably similar to that of mimiviruses. b Medial section of virions showing open and closed stargates (arrowheads). c Virion within a host cell phagosome devoid of its viral core, and membranous structures. Note a well-visible fiber layer covering the electron-dense capsid shell of the virion. d Vertex with starfish-shaped edges (arrowheads). e A lateral section showing a triangular profile of the icosahedral capsid shell surrounded by the fiber layer. All scale bars: 500 nm. CS = capsid shell. CW = core wall. AM = proposed additional membrane. FL = fiber layer. ICS = inner capsid shell. IM = inner membrane. VC = viral core. SGC = stargate closed. SGO = stargate opened. Terminology is based on,.

Fig. 6
Fig. 6. Features of the Naegleriavirus replication cycle.

Transmission electron microscopy of cryoimmobilized samples processed by freeze substitution or chemically fixed at room temperature (indicated by *). Numbers indicate hours post-infection (hpi). a N. clarki trophozoite forming a phagocytic cup and containing two virions in the phagosome (boxed rectangle); potentially phagocytosed virion marked with an arrowhead. b Open stargate (black arrow head) in the upper virion. Virion underneath displays the beginning of the stargate opening; the extra membrane sac can be seen. c Fusion of a NiV inner membrane with a phagosome membrane (black arrow head). d Two virions devoid of viral cores enclosed by a multilamellar structure within a phagosome. e Fully grown and productive viral factory. f Infected ameba showing a large viral factory and virions accumulating in the host cytoplasm. Membrane-enclosed virions suggest ongoing phagocytosis (white arrowheads). ELS = extracellular lamellar structures. EMS = extra membrane sac. MF = membrane fusion. MLS = multilamellar structure. N = nucleus. NL = nucleolus. PC = phagocytic cup. SG = star gate. VC = viral core. VF = viral factory.

Fig. 7
Fig. 7. The tested host range of Naegleriavirus.

An 18 S rRNA-based maximum likelihood tree is depicted to visualize the evolutionary relationships of amoebae. The phylogenetic positioning of Naegleria americana was inferred from Wang et al. and is shown in gray. The color-coded amoebae indicate whether the host was lysed post NiV infection or not.

Fig. 8
Fig. 8. Naegleriavirus replication at different temperatures.

NiV replication at 24 hpi, shown as fold change (FC) of NiV genome copy numbers in N. clarki (a) and N. lovaniensis (b) grown at 20, 30, or 40 °C. Fold change of ameba cell numbers of N. clarki (c) and N. lovaniensis (d) at 20, 30, and 40 °C with and without NiV at 48 hpi. MOI = 1. Statistical tests compared the viral load 24 hpi for the three setups in (a) and (b), and the final host concentration at 48 hpi to the initial concentration in (c) and (d). The (two-tailed) Welch’s t-test was used for statistical analysis; n = 3 independent replicates; p-values are depicted (rounded to the third decimal). Boxes indicate the interquartile range, the line indicates the median, whiskers indicate minimum and maximum, respectively. Additional timepoints and ameba cell counts are available as Supplementary Fig. 3.

Similar articles

References

    1. Suttle CA. Marine viruses - major players in the global ecosystem. Nat. Rev. Microbiol. 2007;5:801–812. doi: 10.1038/nrmicro1750. - DOI - PubMed
    1. La Scola B, et al. A giant virus in amoebae. Science. 2003;299:2033. doi: 10.1126/science.1081867. - DOI - PubMed
    1. Schulz F, et al. Giant viruses with an expanded complement of translation system components. Science. 2017;356:82–85. doi: 10.1126/science.aal4657. - DOI - PubMed
    1. Abrahão J, et al. Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere. Nat. Commun. 2018;9:749. doi: 10.1038/s41467-018-03168-1. - DOI - PMC - PubMed
    1. Moniruzzaman M, Martinez-Gutierrez CA, Weinheimer AR, Aylward FO. Dynamic genome evolution and complex virocell metabolism of globally-distributed giant viruses. Nat. Commun. 2020;11:1710. doi: 10.1038/s41467-020-15507-2. - DOI - PMC - PubMed

Publication types

MeSH terms