A novel method for the measurement of hepatitis C virus infectious titres using the IncuCyte ZOOM and its application to antiviral screening

Stewart, Hazel; Bartlett, C. J.; Ross‐Thriepland, Douglas; Shaw, J.C.; Griffin, Stephen; Harris, Mark

doi:10.1016/j.jviromet.2015.03.009

Cited by 29 publications

(43 citation statements)

References 28 publications

(41 reference statements)

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“…We initially established a high throughput fluorescence detection system to monitor the expression of SV40 T-antigens following virus infection using an Incucyte ZOOM instrument, in a manner comparable to previous methods described for Hepatitis C virus measurement ( Fig. 1A-B ) (54, 55). Cells infected with SV40 were fixed, then permeabilised and immunostained to detect T-antigens, prior to automated imaging and analysis to determine the number of T-antigen positive cells.…”

Section: Resultsmentioning

confidence: 99%

A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

Dobson

Mankouri

Whitehouse

2019

Preprint

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19Following internalisation viruses have to escape the endocytic pathway and deliver their 20 genomes to initiate replication. Members of the Polyomaviridae transit through the 21 endolysosomal network and through the endoplasmic reticulum (ER), from which heavily 22 degraded capsids escape into the cytoplasm prior to nuclear entry. Acidification of 23 endosomes and ER entry are essential in the lifecycle of polyomaviruses, however many 24 mechanistic requirements are yet to be elucidated. Alteration of endocytic pH relies upon the 25 activity of ion channels. Using two polyomaviruses with differing capsid architecture, namely 26 Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we firstly describe methods 27 to rapidly quantify infection using an IncuCyte ZOOM instrument, prior to investigating the 28 role of K + and Ca 2+ channels during early stages of infection. Broad spectrum inhibitors 29 identified that MCPyV, but not SV40, is sensitive to K + channel modulation. In contrast, both 30 viruses are restricted by the broad spectrum Ca 2+ channel inhibitor verapamil, however 31 specific targeting of transient or long lasting Ca 2+ channel subfamilies had no detrimental 32 effect. Further investigation revealed that tetrandrine blockage of two-pore channels (TPCs), 33the activity of which is essential for endolysosomal-ER fusion, ablates infectivity of both 34 MCPyV and SV40 by preventing disassembly of the capsid, which is required for the 35 exposure of minor capsid protein nuclear signals necessary for nuclear transport. This study 36 therefore identifies a novel target to restrict the entry of polyomaviruses. 37 IMPORTANCE 38Polyomaviruses establish ubiquitous, asymptomatic infection in their host. However, in the 39 immunocompromised these viruses can cause a range of potentially fatal diseases. Through 40 the use of SV40 and MCPyV, two polyomaviruses with different capsid organisation, we 41 have investigated the role of ion channels during infection. Here, we show that Ca 2+ channel 42 activity is essential for both polyomaviruses and that MCPyV is also sensitive to K + channel 43 blockage, highlighting new mechanistic requirements of ion channels during polyomavirus 44 infection. In particular, tetrandrine blockage of endolysosomal-ER fusion is highlighted as an 45 essential modulator of both SV40 and MCPyV. Given that the role of ion channels in disease 46 have been well characterised, there is a large panel of clinically available therapeutics that Polyomaviruses (PyVs) are small double stranded DNA viruses that establish persistent 51 infections in their hosts. Whilst infections are generally asymptomatic, PyVs can cause 52 severe disease in the immunosuppressed. Common examples include BKPyV-associated 53 nephropathy and haemorrhagic cystitis, and JCPyV-induced progressive multifocal 54 leukoencephalopathy (PML) (1-3).55 In ~80% of Merkel cell carcinoma (MCC) cases, Merkel cell PyV (MCPyV) infection, clonal 56 integration and UV-mediated mutation of the viral genome occur prior to tumour c...

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Section: Resultsmentioning

confidence: 99%

A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

Dobson

Mankouri

Whitehouse

2019

Preprint

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“…PIK93 (1-5 µM), or CMPD (3) or (7) (0.5-20 µM), was added to the cells 2 h prior to transfection, or at the time of transfection, respectively, and replaced once the transfection mix was added to the cells. Electroporation of SGR-Luc-GFP JFH-1 RNA into Huh 7.5 cells was undertaken as described by Ross-Thriepland et al (2015) and Stewart et al (2015). ; statistical analysis was performed using a two-tailed unpaired t-test (*P<0.05, **P<0.01).…”

Section: Methodsmentioning

confidence: 99%

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

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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.

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“…Huh7 cells were cultured and propagated as described previously. Secreted infectivity was measured as described 28 . Briefly, 1 µg of linearised HCV constructs pJFH1 29 , pJ4JFH1 30 , or pS52-JFH1 31 was used to perform in vitro transcription (RiboMax express, Promega) following the manufacturer's protocol.…”

Section: Virus Secretion Inhibition Assaysmentioning

confidence: 99%

“…Cells were washed in PBS and imaged using phase and red channels (IncuCyte ZOOM). Infected cells positive for NS5A expression were quantified using IncuCyte parameters previously described 28 , normalised to DMSO control and nonlinear regression fitted using Prism 6 (GraphPad) to determine EC 50 /CC 50 .…”

Section: Virus Secretion Inhibition Assaysmentioning

confidence: 99%

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy

Shaw

Gosein

Kalita³

et al. 2018

Preprint

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29 Brenner Building, St James' University Hospital, Leeds LS9 7TF. Telephone: (+44)113 30 3438637 31 Dr Richard Foster (R.Foster@leeds.ac.uk), School of Chemistry, Faculty of Maths 32 and Physical Sciences and Astbury Centre for Structural Molecular Biology, University of 33 Leeds, Leeds, LS2 9JT. Telephone: (+44)113 3435759 34 35 † Current AddressAbstract 46 Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion 47 channels, or "viroporins", contrasting the success of channel blocking drugs in other 48 areas of medicine. Although resistance arose to these prototypic adamantane inhibitors 49 of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and 50 economically important viruses are now recognised to encode essential viroporins 51 providing potential targets for modern drug discovery. 52 We describe the first rationally designed viroporin inhibitor with a comprehensive 53 structure-activity relationship (SAR). This step-change in understanding not only 54 revealed a second biological function for the p7 viroporin from hepatitis C virus 55 (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound 56 that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of 57 HCV antiviral targeting both the spread and establishment of infection, as well as a 58 precedent for future viroporin-targeted drug discovery. 59 60 149 words 61 62 63 MAIN TEXT 64 65 66 Hepatitis C virus (HCV) represents a global clinical challenge as a major cause of chronic 67 liver disease, with severe complications including cirrhosis, liver failure and primary liver 68 cancers (hepatocellular-and intrahepatic cholangio-carcinomas (HCC, iCCA)). Acute 69 infection is predominantly asymptomatic which, combined with limited awareness and 70 population screening, means that liver disease is often advanced upon diagnosis. WHO 71 estimates put the total number of deaths due to HCV infection in 2015 at more than 400 72 000, with ~1.75 million new infections annually. 73 HCV antiviral therapy, originally comprising recombinant type 1 interferon (IFN) 74 combined with the guanosine analogue ribavirin, has been revolutionised by new direct-75 acting antivirals (DAA). DAA are an unprecedented drug development success, capable of 76 achieving high rates of cure with favourable toxicity profiles enabling their use in patients 77 with advanced disease(1). Current DAA target three proteins within the viral replicase 78 (NS3/4A protease, NS5A and the NS5B RNA-dependent RNA Polymerase (RdRP)), with 79 drug combinations available for treating each of the eight viral genotypes. 80 However, the absence of an HCV vaccine, or other means of prophylaxis, makes DAA-81 based eradication strategies proposed for ~71 million chronically infected individuals 82 immensely challenging. DAA availability remains limited by cost, coincident with poor 83diagnostic rates and rapidly increasing burden in low/middle income countries (LMIC). 84 Resistant viral ...

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A novel method for the measurement of hepatitis C virus infectious titres using the IncuCyte ZOOM and its application to antiviral screening

Cited by 29 publications

References 28 publications

A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

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

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy

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