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. 2017 Apr 21;8:15087.
doi: 10.1038/ncomms15087.

Noumeavirus Replication Relies on a Transient Remote Control of the Host Nucleus

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Free PMC article

Noumeavirus Replication Relies on a Transient Remote Control of the Host Nucleus

Elisabeth Fabre et al. Nat Commun. .
Free PMC article

Abstract

Acanthamoeba are infected by a remarkable diversity of large dsDNA viruses, the infectious cycles of which have been characterized using genomics, transcriptomics and electron microscopy. Given their gene content and the persistence of the host nucleus throughout their infectious cycle, the Marseilleviridae were initially assumed to fully replicate in the cytoplasm. Unexpectedly, we find that their virions do not incorporate the virus-encoded transcription machinery, making their replication nucleus-dependent. However, instead of delivering their DNA to the nucleus, the Marseilleviridae initiate their replication by transiently recruiting the nuclear transcription machinery to their cytoplasmic viral factory. The nucleus recovers its integrity after becoming leaky at an early stage. This work highlights the importance of virion proteomic analyses to complement genome sequencing in the elucidation of the replication scheme and evolution of large dsDNA viruses.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Ultrathin-section TEM imaging of Noumeavirus-infected Acanthamoeba cells.
(a) 10 min pi, virions have been engulfed and are in vacuoles. (b) 20 min pi, virions in vacuoles exhibit holes (black arrows) in their external translucent shell and appear more spherical. (c) 30 min pi, virions have entirely lost their external shell and appear as spherical electron-dense nucleoids, which can be seen in vacuoles as well as in the cell cytoplasm (and Fig. 9, black arrows). Scale bar, 100 nm. (d) 1 h pi infected cells. Electron-dense tubular structures (black arrowheads) appear in the cell cytoplasm. (e) 4 h pi viral factories (VF) settle in the cell cytoplasm next to the nucleus and the cell organelles are pushed at their periphery (scale bar, 2 μm). The new virions are assembled and the electron-dense mature (white arrows) and immature (white arrowheads) virions are scattered in the same VF. Inset: immature and mature virions inside the VF along with tubular structures (balck arrowheads). Scale bar, 200 nm. (f) 7 h pi, the newly synthesized virions are gathered into vacuoles inside the cell cytoplasm before being released in the extracellular environment (scale bar, 500 nm).
Figure 2
Figure 2. Distribution of the best-matching homologues to Noumeavirus predicted proteins.
Best-matching homologous proteins were determined using BLASTP (E-value <10–5) against the non-redundant (NR) database at the National Center for Biotechnology Information.
Figure 3
Figure 3. Phylogenetic analyses of Marseilleviridae.
(a) Maximum likelihood phylogenetic tree of the DNA polymerase of Marseilleviridae build using RaxML. Invertebrate iridescent virus 3 DNA was used as the outgroup. Numbers at the nodes indicate per cent bootstrap values. (b) Maximum likelihood phylogenetic tree based on the concatenated alignment of the 21 orthologous genes shared by Marseilleviridae and Iridovirus invertebrate iridescent virus 3.
Figure 4
Figure 4. Distribution of palindromic sequences in the genomes of three Marseilleviridae.
(a,c,e) Distributions of palindromic motifs observed at each position relative to the start codon (grey curve and inset arrow). The signal was deconvoluted in two parts depending on whether the gene upstream of each start codon was in the same orientation (red curve and Inset arrows) or in the reverse orientation (blue curve and inset arrows). (b,d,f) Distributions of palindromic motifs observed at each position relative to the stop codon (grey curve and inset arrow). The signal was deconvoluted in two parts depending on whether the gene downstream of each stop codon is in the same orientation (red curve and inset arrows) or in the reverse orientation (blue curve and Inset arrows). The enrichment seen at the start appears mostly due to palindromic signals associated with the upstream gene. Regardless of the downstream gene, the observed enrichment of palindromic signals is highly correlated to the presence of an upstream stop codon.
Figure 5
Figure 5. 3′ UTR hairpin sequence and structure as produced by RNAfold.
The cleavage site of the NMV_1 mRNA is indicated by an arrow.
Figure 6
Figure 6. Proteomic analyses of Noumeavirus and Melbournevirus virion compositions.
Comparison of the abundances of host encoded (red) and virally encoded orthologous proteins (blue) in Noumeavirus and Melbournevirus. Higher values correspond to lower protein abundances. The Pearson correlation coefficient only refers to the relative abundances of viral proteins (P-value computed using the Pearson's product moment correlation test).
Figure 7
Figure 7. Fluorescence study of A. castellanii cell nucleus during Noumeavirus infection.
Fluorescence images (scale bar, 10 μm) of the nucleus (N) in non infected (a) and cells infected by Noumeavirus expressing the nuclear ML135-GFP at (d) 30 min pi, (g) 1 h pi and (j) 4 h pi. Non-infected cells (b) and infected cells expressing the nucleolar ML216-GFP at (e) 30 min pi, (h) 1 h pi and (k) 4 h pi. Non-infected cells (c) and infected cells expressing the nuclear GFP-SUMO at (f) 30 min pi, (i) 1 h pi and (l) 4 h pi. DAPI staining remains at the nucleus all along the infection but the intense staining of the late VF makes it barely visible at the nucleus 4 h pi (jl). The GFP staining located in the nucleus of uninfected cells progressively spreads out of the nucleus (df) before filling the cytoplasm (gi). After 4 h, the GFP florescence is back into the nucleus and the DAPI staining is intense in the viral factories (jl).
Figure 8
Figure 8. Observation of A. castellanii cells expressing ML135-GFP infected by Noumeavirus between 2 and 3 h pi.
Cells were observed by interference contrast (DIC; scale bar, 10 μm) (a,d,g). DAPI-stained early nucleoids (b, white arrows) and developing viral factories (VF, e,h) produce intense fluorescence masking the DAPI staining at the nucleus (N). GFP staining co-localizes with the viral nucleoids (c, white arrows) and the early VF (f, white arrows). (i) In contrast with the strongly fluorescent nucleus, the mature VF is barely GFP stained.
Figure 9
Figure 9. Ultrathin-section TEM images of nuclei of A. castellanii cells undergoing a Noumeavirus infection.
Noumeavirus infection of (a) non-infected cells, the nucleolus (Nu) in the nucleus (N) is electron-dense and compact. (b) In cells infected by Noumeavirus at 30 min pi, the nucleus is drastically modified with many circumvolutions and the nucleolus appears disorganized and less electron-dense. Electron-dense nucleoids can be seen in the cytoplasm (black arrows and Fig. 8, white arrows) (c) 1 h pi the nucleus is still deformed and nucleoids are still visible. (d) 4 h pi the nucleus recovered its spherical shape with electron-dense and compact nucleolus and coexists with viral factories (VF). Scale bar, 1 μm.

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