FMDV replicons encoding green fluorescent protein are replication competent

Tulloch, Fiona; Pathania, Uday; Luke, Garry A.; Nicholson, John; Stonehouse, Nicola J.; Rowlands, David J.; Jackson, Terry; Tuthill, Toby; Haas, Juergen; Lamond, Angus I.; Ryan, Martin

doi:10.1016/j.jviromet.2014.08.020

Cited by 34 publications

(46 citation statements)

References 18 publications

(23 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…as a result of input translation only). Levels of GFP expression in GFP-pac-GNN replicon-transfected cells were similar to those with a second replication-deficient replicon, GFP-pac-D3D (Tulloch et al, 2014), with a large deletion in the 3D pol gene (data not shown).…”

Section: Resultsmentioning

confidence: 56%

“…Development and use of the FMDV pGFP-pac-WT replicon construct has been described previously (Forrest et al, 2014;Tulloch et al, 2014). The replication-defective FMDV replicon, pGFP-pac-GNN, bearing a inactivating point mutation to the 3D pol active site was generated by standard PCR mutagenesis (Herod et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

“…Details of transcription of pGFPpac replicons (Tulloch et al, 2014) are described elsewhere (Bentham et al, 2012;Herod et al, 2015). RNA was purified using a Zymogen RNA clean and concentrate kit according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…+ve, Positive. Tulloch et al, 2014). After transfection with replicon RNA, the cells were maintained within an IncuCyte housed at 37 C in a humidified 5 % CO 2 incubator.…”

Section: Methodsmentioning

confidence: 99%

“…Replication-deficient constructs have a GNN mutation in the active site of 3D pol (pGFP-pac-GNN). Levels of GFP expression over time can be measured using an IncuCyte Dual Colour ZOOM FLR (Forrest et al, 2014;Herod et al, 2015;Tulloch et al, 2014). Replicons have also been used to study the replication of a number of other viruses.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

Loundras

Herod

Harris

et al. 2016

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

Foot-and-mouth disease virus (FMDV) causes economically damaging infections of clovenhooved animals, with outbreaks resulting in large financial losses to the agricultural industry. Due to the highly contagious nature of FMDV, research with infectious virus is restricted to a limited number of key facilities worldwide. FMDV sub-genomic replicons are therefore important tools for the study of viral translation and genome replication. The type III phosphatidylinositol-4-kinases (PI4Ks) are a family of enzymes that plays a key role in the production of replication complexes (viral factories) of a number of positive-sense RNA viruses and represents a potential target for novel pan-viral therapeutics. Here, we investigated whether type III PI4Ks also play a role in the FMDV life cycle, using a combination of FMDV sub-genomic replicons and bicistronic internal ribosome entry site (IRES)-containing reporter plasmids. We demonstrated that replication of the FMDV replicon was unaffected by inhibitors of either PI4KIIIa or PI4KIIIb. However, PIK93, an inhibitor previously demonstrated to target PI4KIIIb, did inhibit IRES-mediated protein translation. Consistent with this, cells transfected with FMDV replicons did not exhibit elevated levels of phosphatidylinositol-4-phosphate lipids. These results are therefore supportive of the hypothesis that FMDV genome replication does not require type III PI4K activity and does not activate these kinases.

show abstract

Section: Resultsmentioning

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…+ve, Positive. Tulloch et al, 2014). After transfection with replicon RNA, the cells were maintained within an IncuCyte housed at 37 C in a humidified 5 % CO 2 incubator.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

Loundras

Herod

Harris

et al. 2016

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

show abstract

Relevance of the N-terminal and major hydrophobic domains of non-structural protein 3A in the replicative process of a DNA-launched foot-and-mouth disease virus replicon

et al. 2018

View full text Add to dashboard Cite

A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls. Alanine substitution of the 17 aa present in the central HR, on the other hand, resulted in complete inhibition of luciferase activity and in the accumulation of the mutated 3A within the cell nucleus according to immunofluorescence analysis. Our results suggest that both the aa sequence around the putatively exposed hydrophilic E20 residue at the N-terminus of the protein and the hydrophobic tract located between aa 59 and 76 are of major relevance for maintaining the functionality of the 3A protein and preventing its mislocalization into the cell nucleus.

show abstract

Viral replicons as valuable tools for drug discovery

Hannemann¹

2020

Drug Discovery Today

View full text Add to dashboard Cite

RNA viruses can cause severe diseases such as dengue, Lassa, chikungunya and Ebola. Many of these viruses can only be propagated under high containment levels, necessitating the development of low containment surrogate systems such as subgenomic replicons and minigenome systems. Replicons are self-amplifying recombinant RNA molecules expressing proteins sufficient for their own replication but which do not produce infectious virions. Replicons can persist in cells and are passed on during cell division, enabling quick, efficient and high-throughput testing of drug candidates that act on viral transcription, translation and replication. This review will explore the history and potential for drug discovery of hepatitis C virus, dengue virus, respiratory syncytial virus, Ebola virus and norovirus replicon and minigenome systems.

show abstract

FMDV replicons encoding green fluorescent protein are replication competent

Abstract: HighlightsFMDV replication can be studied outwith high disease secure facilities.FMDV replicon genomes encoding GFP are replication competent.These FMDV replicon systems can be used to study replication by live-cell imaging/image analyses.

Cited by 34 publications

References 18 publications

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

Relevance of the N-terminal and major hydrophobic domains of non-structural protein 3A in the replicative process of a DNA-launched foot-and-mouth disease virus replicon

Viral replicons as valuable tools for drug discovery

Contact Info

Product

Resources

About