Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Atypon full text link Atypon Free PMC article
Full text links

Actions

.2016 Oct 14;90(21):10039-10047.
doi: 10.1128/JVI.01319-16. Print 2016 Nov 1.

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

Affiliations

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

Yael Fridmann-Sirkis et al. J Virol..

Abstract

The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses.IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Mimivirus factories within host cells and following isolation. (A to C) Amoeba cells infected with mimivirus at 3 successive postinfection (p.i.) time points (4 h [A], 5.5 h [B], and 7 h [C]) were stained with antibodies against mature virions (red) and counterstained with DAPI (blue). Cell and nucleus contours were derived from differential interference contrast (DIC) micrographs. (D to F) SEM images of isolated viral factories at 4 h (D), 5.5 h (E), and 7 h (F) p.i. Both SEM and fluorescence studies revealed the coalescence of initial viral replication centers into a single viral factory. Insets in panels E and F show stargate structures. (G) Low-magnification SEM micrograph depicting a large field of isolated factories at 7 h p.i. The micrograph reveals that the factories are essentially free from large host components. (H) Purified virions appear pure when surveyed by low-magnification TEM. Scale bars: panels A to C, 5 μm; panel D, 200 nm; panels E to F, 1 μm; panel G, 10 μm; panel H, 500 nm.
FIG 2
FIG 2
Isolated viral factories contain the core protein L410. (A) Western blots of total cell lysates at different postinfection time points were incubated with anti-core protein (L410) antibodies. L410 was detected only at later stages of infection, matching our mass spectroscopy findings. (B) Western blots of purified viral factories were incubated with anti-L410 antibodies. L410 was detected in viral factories, as well as in the virion, only at 7 h p.i., thus further substantiating our MS results. (C) Immunolabeling of cryopreserved cells at 8 h p.i. with rabbit anti-L410 antibodies, followed by exposure to gold-conjugated secondary antibody, revealed that the core protein is present in the viral factory as well as in the assembling virions. Scale bar: 1 μm.
FIG 3
FIG 3
Eukaryotic translation initiation factor 4E-like protein L496 and ribosomes are not incorporated into the viral factories. (A) Western blots of total cell lysates at different postinfection time points were incubated with anti-4E-like protein (L496) antibodies. L496 was detected only in the total cell lysates and not in the factories, as indeed indicated by our MS findings. (B) TEM of intracellular replication centers at 4 h p.i. in the process of coalescing into a single viral factory. The micrograph reveals ribosomes (dark dots with diameters of ∼25 to ∼30 nm) that surround the replication centers but are not incorporated into these organelles. Rc and m, replication centers and mitochondria, respectively. Asterisks represent the cores of the original infecting virus (26). Scale bar: 500 nm.
FIG 4
FIG 4
Venn diagrams depicting protein content in mimivirus factories and in mature virions. (A) Proteins detected in isolated viral factories at 4, 5.5, and 7 h p.i. (blue, yellow, and green circles, respectively). The diagram demonstrates that the compositions of viral factories along the infection cycle differ substantially, thus underscoring the dynamic nature of these virus-generated organelles. (B) Relations between proteins detected in mature virions and proteins present in viral factories at 4 h (magenta and blue circles, respectively). (C) Relations between proteins detected in viral factories at 7 h and proteins found in mature virions (green and magenta circles, respectively). This diagram reveals that many proteins were present in the factories but not included in mature virions, implying that these proteins act as part of a production line, on par with the notion that viral factories represent intracellular organelles where vigorous viral assembly occurs.
FIG 5
FIG 5
Dynamic composition of the mimivirus cytoplasmic factories. (A1 to A3) Distribution of three helicases in viral factories and mature viral particles. (B1 to B3) Distribution of DNA repair proteins in viral factories and in mature viral particles. (C1 to C3) Distribution of DNA-replication-related proteins. (D1 to D3) Distribution of protein-degradation-related proteins. Intensities represent relative protein amounts. The figure highlights the fact that the relative amounts of proteins belonging to similar functional groups in viral factories substantially differ at progressive postinfection time points, as well as in mature virions.
FIG 6
FIG 6
Genomic organization of proteins detected in viral factories and mature virions. Our proteomic analyses indicate that the majority of proteins found in the viral factories of mimivirus are encoded by genes located in the center of the mimivirus chromosome. This observation is consistent with previous findings according to which mimivirus virions propagated under axenic conditions undergo substantial reduction of their genome that specifically occurs at the extremities of the genome and yet is not detrimental to mimivirus infectivity (35). CDSs (coding DNA sequences) are represented as blue arrowheads. Proteins detected in VFs at 4, 5.5, and 7 h p.i. are labeled in blue, purple, and maroon rectangles, respectively. Proteins found in mature mimivirus particles are labeled in green. The linear mimivirus genome is depicted as a circle starting from the site indicated by an arrow. The figure was constructed with the CGView Comparison Tool (CCT) (46).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. de Castro IF, Volonte L, Risco C. 2013. Virus factories: biogenesis and structural design. Cell Microbiol 15:24–34. - PMC - PubMed
    1. den Boon JA, Ahlquist P. 2010. Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu Rev Microbiol 64:241–256. doi: 10.1146/annurev.micro.112408.134012. - DOI - PubMed
    1. Netherton C, Wileman T. 2011. Virus factories, double membrane vesicles and viroplasm generated in animal cells. Curr Opin Virol 1:381–387. doi: 10.1016/j.coviro.2011.09.008. - DOI - PMC - PubMed
    1. Novoa RR, Calderita G, Arranz R, Fontana J, Granzow H, Risco C. 2005. Virus factories: associations of cell organelles for viral replication and morphogenesis. Biol Cell 97:147–172. doi: 10.1042/BC20040058. - DOI - PMC - PubMed
    1. Abergel C, Legendre M, Claverie JM. 2015. The rapidly expanding universe of giant viruses: Mimivirus, Pandoravirus, Pithovirus and Mollivirus. FEMS Microbiol Rev 39:779–796. doi: 10.1093/femsre/fuv037. - DOI - PubMed

LinkOut - more resources

Full text links
Atypon full text link Atypon Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp