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

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Sep 4;24(10):2573-2580.e4.
doi: 10.1016/j.celrep.2018.08.008.

A Chimeric Lloviu Virus Minigenome System Reveals that the Bat-Derived Filovirus Replicates More Similarly to Ebolaviruses than Marburgviruses

Whitney A Manhart  1 , Jennifer R Pacheco  1 , Adam J Hume  1 , Tessa N Cressey  1 , Laure R Deflubé  1 , Elke Mühlberger  2
Affiliations

A Chimeric Lloviu Virus Minigenome System Reveals that the Bat-Derived Filovirus Replicates More Similarly to Ebolaviruses than Marburgviruses

Whitney A Manhart et al. Cell Rep. .

Abstract

Recently, traces of zoonotic viruses have been discovered in bats and other species around the world, but despite repeated attempts, full viral genomes have not been rescued. The absence of critical genetic sequences from these viruses and the difficulties to isolate infectious virus from specimens prevent research on their pathogenic potential for humans. One example of these zoonotic pathogens is Lloviu virus (LLOV), a filovirus that is closely related to Ebola virus. Here, we established LLOV minigenome systems based on sequence complementation from other filoviruses. Our results show that the LLOV replication and transcription mechanisms are, in general, more similar to ebolaviruses than to marburgviruses. We also show that a single nucleotide at the 3' genome end determines species specificity of the LLOV polymerase. The data obtained here will be instrumental for the rescue of infectious LLOV clones for pathogenesis studies.

Keywords: Ebola virus; Lloviu virus; Marburg virus; RNA-dependent RNA polymerase; emerging viruses; filoviruses; minigenomes; replication and transcription.

PubMed Disclaimer

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Chimeric LLOV Minigenomes Are Recognized by the Replication Complexes of Other Filoviruses
(A) Scheme of the LLOV genome. Solid black lines indicate known sequences, and dotted lines indicate missing sequences. Light gray boxes indicate the leader (le) and trailer (tr). Dark gray boxes indicate non-coding regions flanking the open reading frames (ORFs). ORFs are shown as white boxes. Gene start signals are illustrated as green triangles and gene end signals as red bars. (B) 3’ genome ends of filovirus species. Shown are the sequences of the leaders and NP gene start signals (GS). BDBV, Bundibugyo virus; SUDV, Sudan virus; TAFV, Tai Forest virus. (C) Schemes of chimeric minigenomes. A reporter gene is flanked by the 3’ leader and non-coding region (NCR) of the LLOV NP gene and the 5’ NCR of the L gene and trailer of EBOV, RESTV, or MARV. (D) BSRT7/5 cell were transfected with EGFP-expressing LLOV minigenomes along with the system components of EBOV, RESTV, or MARV, respectively. Authentic filovirus minigenomes were transfected with their corresponding system components as positive controls. Lsynth ‒ indicates the negative control, in which a catalytically inactive L was used. Minigenome activity was visualized 2 DPT. Experiment was performed three times. Representative results are shown.
Figure 2.
Figure 2.. LLOV Minigenomes Are Accepted as Templates by LLOV Replication Complex
(A) HEK293T cells were transfected with EGFP-expressing LLOV minigenomes along with LLOV system components. As a negative control, polymerase L was omitted (–L). Minigenome activity was visualized 3 DPT. Experiment was performed three times. Representative results are shown. (B) HEK293T cells were transfected with firefly luciferase-expressing LLOV minigenomes along with LLOV system components and pMIR β-gal plasmid for normalization. Cells were harvested for analysis 3 DPT. Data represent the means of three independent experiments, and error bars represent ± SEM. (C) EBOV, RESTV, or MARV EGFP-expressing minigenomes were transfected into HEK293T cells along with LLOV system components. Mini-genome activity was visualized 3 DPT. Experiment was performed three times. Representative results are shown. (D) U2OS cells were transfected with EGFP-expressing minigenome 3L5E along with LLOV system components. Cells were fixed at 3 DPT, and EGFP expression was visualized by confocal microscopy. Viral inclusions are marked by arrows. Nuclei are stained with DAPI. As a negative control, cells were transfected with pCAGGS-EGFP. Experiment was performed three times. Representative results are shown. Scale bars are 20 μm.
Figure 3.
Figure 3.. LLOV Requires VP30 for Transcription
EGFP-expressing 3L5E (A), 3L5R (B), or 3L5M (C) minigenomes were transfected into HEK293T cells along with LLOV system components, including LLOV VP30. As a negative control, polymerase L was omitted (–L). LLOV VP30 was either omitted (–VP30) or replaced with the VP30 proteins of EBOV, RESTV, or MARV. Minigenome activity was visualized 3 DPT. Experiment was repeated three times, and representative fluorescence images are shown.
Figure 4.
Figure 4.. LLOV Replication Complex Does Not Recognize MARV-like 3′ Leader Sequence
(A) 3’ terminal nucleotides of filovirus genomes. The first three nucleotides of the MARV leader sequence (UCU, highlighted) were introduced into the LLOV leader. (B–D) HEK293T cells were transfected with EGFP-expressing LLOV minigenomes along with LLOV (B), MARV (C), or EBOV (D) system components. As a negative control, polymerase L was omitted (–L). Minigenome activity was visualized 3 DPT. Experiment was repeated three times. Representative images are shown. (E) HEK293T cells were transfected with firefly luciferase-expressing LLOV minigenomes along with the LLOV system components and pMIR β-gal plasmid for normalization. Cells were harvested for analysis 3 DPT. Data represent the means of three independent experiments, and error bars represent ± SEM.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albariño CG, Uebelhoer LS, Vincent JP, Khristova ML, Chakrabarti AK, McElroy A, Nichol ST, and Towner JS (2013). Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate. Virology 446, 230–237. - PMC - PubMed
    1. Amarasinghe GK, Aréchiga Ceballos NG, Banyard AC, Basler CF, Bavari S, Bennett AJ, Blasdell KR, Briese T, Bukreyev A, Cai Y, et al. (2018). Taxonomy of the order Mononegavirales: update 2018. Arch. Virol 163, 2283–2294. - PMC - PubMed
    1. Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, Soropogui B, Sow MS, Keïta S, De Clerck H, et al. (2014). Emergence of Zaire Ebola virus disease in Guinea. N. Engl. J. Med 371, 1418–1425. - PubMed
    1. Bausch DG (2017). West Africa 2013 Ebola: from virus outbreak to humanitarian crisis. Curr. Top. Microbiol. Immunol 411, 63–92. - PubMed
    1. Boehmann Y, Enterlein S, Randolf A, and Mühlberger E (2005). A reconstituted replication and transcription system for Ebola virus Reston and comparison with Ebola virus Zaire. Virology 332, 406–417. - PubMed

Publication types

Substances