Master’s Thesis ProposalA Cell Based Assay for the Discovery of West Nile Virus Protease Inhibitors Jason Machula Wolkowicz Lab April 2012
Aims:The goal of this project is to develop an assay to monitor the activity of West Nile Virus (WNV) protease inHEK293T cells. We have previously developed an assay for HIV-1 protease in a Gal4 fusion context in non-adherent T-cells1. This assay exploits the autocatalytic properties of HIV-1 protease (PR), and Gal4, atranscription factor that is only active when the terminal DNA binding and Trans-activating domain(DBD, TAD)are linked1–3. This assay for WNV will provide a platform for the high-throughput screening for novel inhibitorsof WNV protease utilizing flow cytometryand/or plate reader based technologies. In addition it will provide theproof of concept for the utility of the Gal4 protease fusion system,for a different viral type of protease, which, incontrast to HIV-1, requires a cofactor for full activity. Moreover, the assay will be developed in adherent cellsrather than in T-cells. While the main goal of the assay is to drastically facilitate drug discovery it could be usedto study the interaction of protease, cofactor, host and other viral factors required for protease activity.Background:WNV, a member of the Flaviviridae,is a (+) single stranded RNA virus with a genome of about 11Kb encased in anicosahedral capsid surrounded by an envelope. Viral entry occurs via endocytosis facilitated by receptor binding.The capsid is released into the cytoplasm following membrane fusion. The entire genome consists of one 3300amino acid open reading frame and can serve as mRNA. Upon translation of the single open reading frame it isthen processed by both viral and cellular proteases into structural and non-structural proteins (Figure 1)4–7.WNV infections are usually asymptomatic however infection can be febrile, or even neuroinvasive causingmeningitis or encephalitis8. WNV infection has been found in a wide variety of mammals and even in somereptiles9. These hosts are considered dead-end hosts although birds are an amplifying host providing viral loadshigh enough to be transmitted by mosquitos, which is the vector responsible for human infection10,11. WNV wasfirst identified in 1937 in Uganda and was first reported in the US in 199912,13. By 2004 it had spread throughoutthe continental US and has since been found in Alaska, Hawaii, and Canada14. It was originally found to bespread by only one type of mosquito however it is now spread by three different known species in the USalone15.Overwintering mosquitos have also been found that continue to carry viral loads through the winter16–18.The wide variety of hosts from mammals, including rodents, humans, and killer whales, to birds, combined withthe processive RNA-dependent RNA polymerase prone to errors provide an environment for the emergence ofnew strains of WNV7,19,20. The lack of effective vaccines or FDA approved inhibitors against WNV reiterates theneed for new methods that facilitate the discovery of antivirals against WNV21. The assay described in thisproposal will provide a platform for the high throughput screening of random peptide or compound libraries forthe discovery of novel WNV protease inhibitors.
Figure 1.Genome, Polyprotein Precursor, and Mature Processed Proteins of WNV.(Murray KO, Mertens E, Desprès P. WestNile virus and its emergence in the United States of America.Veterinary Research. 2010;41(6):67.)The assay is based on the inducible expression of the viral proteaseas a fusion within the DBD and TADof Gal4,and the activation of the reporter green fluorescent protein (GFP) driven by an Upstream Activation Sequence(UAS) which serves as the promotor for Gal41. Gal4 is a well characterized cis-acting transcription factor fromyeast with two required domains, the DBD binds to the UAS and the TAD activates expression of downstreamgenes. The two required domains can be spatially separated however they must be present in a singlepolypeptide to remain active2,3. The engineered fusion, where the viral protease is inserted between the Gal4domains, will be cleaved by protease if activethuspreventing the induction of GFP driven by the UAS. Inductionof GFP, via the UAS thus occurs only when protease is inhibited, acting as a biosensor for protease activity1. Toavoid possible cytotoxic effects of protease expression, the Gal4/protease fusion will be expressed in an off/oninducible manner regulated by a Tetracycline Responsive Element (TRE). When Tetracycline (Tet) or Doxycycline(Dox) is present it will bind to and change conformation of the reverse tetracycline trans-activator (rtTA)allowing it to bind to TRE and activate transcription22. Without Tet or Dox, the Gal4/protease fusion will not beexpressed (Figure 2).
Figure 2: Assay Overview. A. Wild type Gal4 as control, no Dox. Without Dox, rtTA cannot bind to the Tet-responsiveelement (TRE) thus Gal4 is not expressed and, consequently, neither is GFP. B. In the presence of Dox (blue diamond), Gal4is expressed and binds the Upstream Activating Sequence (5xUAS), activating GFP expression. C. The PR/Gal4 fusion-basedsystem. In the presence of Dox PR/Gal4 is expressed; however, its catalytic activity results in the separation of the Gal4domains and thus, no GFP expression. D. Same scenario as in C but in the presence of Protease inhibitors (yellow circles).PR/Gal4 fusion remains intact, resulting in the induction of GFP expression.WNV like HIV rely on the processing of their proteome however the WNV protease, like other flaviviralproteases, requires a cofactor for activity1,23,24. In order for members of the Flaviviridaeto become infective thecofactor NS2B is autocatalytically cleaved from the protease NS3 (Figure 1)25–27. The boundary between NS2Band NS3 and the NS2B/NS3 protease are thus an attractive target for the discovery of protease inhibitors orcompetitors because this site is only cleaved by viral,not host, proteases, as is the case with other NSproteins21,26,28,11. The assay,previously developed in T-cells for HIV, is being adapted to adherent mammaliancells, specifically Human Embryonic Kidney (HEK) 293T cells, providing a better model for flaviviral infections.Experimental Design:Many studies have been performed regarding the analysis of the structure and functions of the NS3 proteaseand its cofactor (NS2B). It has been shown that only the central hydrophilic domain of cofactor NS2B is requiredfor proteolytic activity of NS3 and that the N-terminal NS3 protease domain does not require the C-terminal NS3helicase domain for autolytic activity23,25,28,11,29–32. Shiryaev et al.have built a construct with the required portionof NS2B linked by a GGGGSGGGG linker to the protease portion of NS333. This construct was generouslysupplied as the source of WNV protease. The construct provided contains a K48A mutated cleavage siterendering the site un-cleavable (Figure 3). Many other flaviviral protease studies use the same constructs, asthey allow the study of the activity of the entire NS2B/NS3 protein avoiding cleavage at the NS2B/NS3 boundary.
Figure 3. West Nile Virus Proteinase Based ConstructsThe autolytic site deficient construct with the K48A mutationwithout the helicase domain was generously provided by Sergey Shiryaev of the Burnham Institute. The wild type constructwas made by reverting the mutation with overlapping PCR. The catalytically inert mutant will be made in a similar manner(See body for details).(Shiryaev SA, Kozlov IA, Ratnikov BI, et al. Cleavage preference distinguishes the two-component NS2B–NS3 serineproteinases of Dengue and West Nile viruses. Biochemical Journal. 2007;401(3):743)Gal4/NS2B/3 based constructs were introduced into pcDNA for transient expression in HEK293T and Huh cells.In this plasmid, expression is driven by a minimal Cytomegalovirus(mCMV) promoter thus allowing forexpression in mammalian cells, at least transiently. Two main pcDNA constructs were engineered: One with theprovided protease bearing the mutated cleavage site and a second one with the wild type sequence obtained byreverting the A48K mutation present in theNS2B/3 cleavage site by overlapping PCR. These proteasesequences were then ligated into a pcDNA-mCMV-Gal4 vector cut with EcoRI and dephosphorylated using calfintestinal phosphatase (Figure 4).Figure 4.The WNV Genome Consisting of a Single Polyprotein Precursor and Derived Constructs. Theprotease NS3 and required cofactor NS2B in red within the Gal4 transcription factor.A. Also depicted the Gal4/NS2B/NS3fusion with wild-type cleavage site (green) B. Construct with mutated, non-cleavable,cleavage site (crimson). C. Gal4without a protease will be used as a positive control.
Lentiviral packaging vectors (pH) were used to make HIV based viral particles for the stable expression of assaycomponents in HEK293T cells. Virus was produced in HEK293T cells with the envelope glycoprotein from theVesicuarStomatitus Virus and the Gag, Pol, and Vpr proteins of HIV. Four different viral particles were producedin this manner. Two of them include basic assay components for development of the HEK293T cell lines; thereporter vector pH-5xUAS-GFP and the vector providing rtTA, pBMN-rtTA-i-Lyt2. The latter construct containsan internal ribosome entry site (IRES) allowing expression of two genes from the same mRNA and Lyt2, areceptor from mouse CD8a that acts as a selectable marker. Two viruses were also packaged for Gal4/NS2B/3constructs (wild type and mutant, referred to PR and PRm respectively, in Figure 6. For that purpose the Gal4fusion constructs were cut out of the pcDNA Gal4 construct with NotI and XbaI then ligated into pcDNA-Hygro,cut out with BamHI and XbaI and finally ligated into the pH-7xTRE vector.Figure 5. Retroviral Constructs for Development of HEK293T Cell LinesThe retroviral 5xUAS-GFP and rtTA constructs for the inducible system. The original rtTA construct in the HIV assaycontained an IRES-mCherry, which is replaced by the Lyt-2 receptor (mouse CD8a) to free the channel for mCherry forfuture studies.Figure 6: Retroviral Gal4/Protease Fusion Constructs. Schematics of the retroviral constructs containing the wildtype Gal4/PR (pH-TRE-PR), and mutated Gal4/PRm (pH-TRE-PRm) and Gal4 controls (pH-TRE-Gal4) used in the HIV-1 assay.Each construct contains 7xTRE, mCMV promoter, and 5’ and 3’ Long Terminal Repeats (LTR).
HEK293T cells have been successfully infected and express both 5xUAS GFP and rtTA. The next step will be toinfect these cells with pH-TRE-Gal4 fusion constructs. Selection of the cells successfully infected should bestraightforward with the mutated construct as they have all of the components required to express GFP in thepresence of Dox. This is not the case with the wild type fusion-expressing cells as they will not be distinguishableby GFP expression. In order to select these cells an inhibitor must be used. Several protease inhibitors havebeen shown to be successful in-vitro including, aprotinin, palmatine, and various peptide inhibitors4,24,30,34,35. Ifinhibitors are proven to not successfully inhibit protease, additional Gal4 fusion constructs will be needed.In order to determine whether the protease is actually being expressed and the expected GFP patternexpression is not due to non-specific factors, western blots will be performed utilizing monoclonal antibodiesagainst WNV protease and/or Gal4. Western blotting will confirm expression of the proteasewhether there isGFP expression or not. Uncleaved Gal4 fusion should be around 56 KDa and when cleaved theDBD and cofactor2B (approximately 23KDa) will be separated from the NS3 protease and TAD which together is approximately 33KDa.Once a stable cell population has been raised expressing all of the necessary elements of the assay, clonalselection will begin. The cells expressing the non-cleavable control fusion can be directly sorted based on GFPexpression. The cells expressing the wild type fusion will involve more rounds of sorting. These cells will besorted for no GFP expression with Dox but no inhibitor eliminating background and then sorted for GFPexpression when Dox and an inhibitor are present. Selection will be performed to obtain the best clones, lowestbackground and highest GFP expression, in the assay. After a yet to be determined number of rounds of sorting,cells will be individually plated in a 96-well plate and amplified to obtain clonal cell lines. These cell lines willthen be tested in order to find a cell line that will show robust GFP expression when Dox and inhibitor arepresent and little to no background when Dox is present without inhibitor.After generating stable cell lines expressing all of the components of the assay, optimization of enzyme kineticscan be performed. This will involve titration experiments with Doxto determine the optimal conditions for themaximum induction of Gal4.Preliminary Data:Gal4 WNV protease fusions, wild type and mutated, were constructed in pcDNA vectors with a CMV promoterand confirmed by sequencing. Transfection experiments were performed with these constructs in both naïveHEK293T cells and Huh 7.5.1 cells, a human hepatocytic cell line proving a second model, and in both Huh 7.5.1and HEK293T cells that expressed 5xUAS GFP. In all these experiments,Gal4alone positive controlinducted GFPexpression in approximately 10-15% of cells. Similar level of GFP induction was seen with transfection byGal4/NS2B/3m,which serves as a positive control for Gal4 fusion protein (and negative for protease activity).Little to no GFP expression was observed with naïve cells as with cells transfected with Gal4/NS2B/3wt, asexpected (data not shown).A population of HEK293T cells engineered to stably express rtTA and 5xUAS GFP has already been established.Preliminary transient expression experiments with these cells have shown that the Gal4/NS2B/3 fusion with amutated cleavage site (Gal4/PRm) activate GFP expression, proving that the fusion is indeed a single polyprotein
and that the approximately 1500bp-long protease sequence does not disrupt the ability of the fusion to bind to5xUAS and induce the downstream gene (GFP).Transfection of HEK293T cells that express both 5xUAS GFP and rtTA also yielded similar results whentransfected with pH-7xTRE constructs in the presence of Dox. The Gal4 control without protease resulted in1.9% GFP expression in the absence of Doxand 10.3% with Dox. The Gal4/NS2B/3m construct resulted in 9%GFP in the presence of Dox, while the Gal4/NS2B/3wt construct showed no GFP expression (Figure 7). While thepercentage of positive green cells is not very high, it is very close in both control and mutated fusion,demonstrating the expected trend (10.3% and 9% respectively). The cell samples analyzed by flow cytometrywere transfected in these preliminary experiments for the purpose of determining the best vectors of choice forvirus production and Gal4 fusion constructs. In addition, the non-specific effect of Dox will be determined infuture experiments.Figure 7. Gal4/NS2B/3 Fusion Transfection in HEK293T Cells Expressing 5xUAS GFP and rtTA.The Gal4 fusion constructs with a mutated cleavage site (Gal4/WNVm)showed similar GFP expression as the Gal4 with Doxpositive control. The wild type Gal4 fusion (Gal4/WNVwt) showed almost no GFP expression indicating cleavage of theprotease within the Gal4 domains. The Gal4 control without Dox showed 1.9% GFP as background.
Timeline:The next step will be aimed at obtaining stably-expressing cell lines, where expression of protease will beverified based on GFP expression in the presence of Dox. In order to corroborate expression of the wild typeconstruct western blotting will be performed. In this case, as wild type protease does not lead to GFP expressionunless inhibited, selection will be performed in the presence of a putative protease inhibitor. Upon selection oftwo cell populations stably expressing rtTA and harboring the 5xUAS GFP construct, one expressing the mutatedversion and the second, the wild type Gal4/NS2B/3 fusion, clonal selection will be utilized to obtain a cell linewith optimal sensitivity. We expect to obtain a cell population expressing rtTA, 5xUAS GFP, andGal4/NS2B/NS3m by the end of Spring 2012. Next, we intend to obtain the Gal4/NS2B/NS3wt. Upon successfulinhibition of Gal4/NS2B/3wt, a stable population of cells can be selected and clonal selection can begin. Theclonal cell lines should obtained by the summer of 2012, allowing to optimize the assayFall 2012. The assay willultimately be used to screen libraries in search for novel inhibitors as well as for the study of protease andcofactor activity.1. Hilton BJ, Wolkowicz R. An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitorsin T-Cells.PLoS ONE. 2010;5(6):e10940.2. Johnston SA, Zavortink MJ, Debouck C, Hopper JE. Functional domains of the yeast regulatory proteinGAL4.ProcNatlAcadSci U S A. 1986;83(17):6553–6557.3. Murray MG, Hung W, Sadowski I, Das Mahapatra B. Inactivation of a yeast transactivator by the fused HIV-1proteinase: a simple assay for inhibitors of the viral enzyme activity. Gene. 1993;134(1):123–128.4. Robin G, Chappell K, Stoermer MJ, et al. Structure of West Nile virus NS3 protease: ligand stabilization of thecatalytic conformation. J. Mol. Biol. 2009;385(5):1568–1577.5. Ebel GD, Fitzpatrick KA, Lim P-Y, et al. Nonconsensus West Nile Virus Genomes Arising During MosquitoInfection Suppress Pathogenesis and Modulate Virus Fitness In Vivo. J. Virol. 2011;85(23):12605–12613.6. Stiasny K, Fritz R, Pangerl K, Heinz F. Molecular mechanisms of flavivirus membrane fusion. Amino Acids.2011;41(5):1159–1163.7. Murray KO, Mertens E, Desprès P. West Nile virus and its emergence in the United States of America.Veterinary Research. 2010;41(6):67.8. Sejvar JJ, Haddad MB, Tierney BC, et al. Neurologic Manifestations and Outcome of West Nile Virus Infection.JAMA. 2003;290(4):511–515.9. Farfán-Ale JA, Blitvich BJ, Marlenee NL, et al. Antibodies to West Nile virus in asymptomatic mammals, birds,and reptiles in the Yucatan Peninsula of Mexico. Am. J. Trop. Med. Hyg. 2006;74(5):908–914.10. Campbell GL, Marfin AA, Lanciotti RS, Gubler DJ. West Nile virus.The Lancet Infectious Diseases.2002;2(9):519–529.11. Chappell KJ, Stoermer MJ, Fairlie DP, Young PR. Insights to substrate binding and processing by West NileVirus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies. J. Biol. Chem.2006;281(50):38448–38458.
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