The project was funded by the National Institutes of Health (AI128406 www.nih.gov) to ZY and in part by Johnson Cancer Research Center (http://cancer.k-state.edu) in the form of Graduate Student Summer Stipend to PD.

1. Preparation of DNA Template by PCR for In Vitro Transcription
<ol>
	<li>To prepare the DNA template by PCR, design primers. When designing primers consider crucial characteristics like primer length, annealing temperature (Tm), GC content, 3&#8217; end with G or C etc. 
	NOTE: Discussed in detail in these literature25,26,27.</li>
	<li>Design primers to generate PCR amplicon containing the following elements in 5&#8217; to 3&#8217; direction: T7-Promoter, poly(A) leader, firefly luciferase ORF and a poly(A) tail referred hereafter to as T7_12A-Fluc. Design primers (Forward and Reverse) to encompass all the additional elements not present in the template DNA (Figure 2A). 
	NOTE: The sequence of all elements can be found in Table 1.</li>
	<li>Include several extra nucleotides in forward primer (5&#8217;-3&#8217;)28, followed by T7-promoter, poly(A) leader or desired 5&#8217;-UTR sequence and approximately 20 nucleotides, adjust based on Tm, corresponding to the 5&#8217; end of the reporter gene&#8217;s ORF. Ensure the corresponding region in the primer is identical to the sense strand (+ strand) of the gene. 
	NOTE: For long 5&#8217;-UTR, synthesize two DNA fragments: one with T7 promoter followed by long 5&#8217;-UTR and second with reporter gene&#8217;s ORF. Join these two fragments using overlap extension PCR29.</li>
	<li>Design reverse primer (5&#8217;-3&#8217;) to include poly(A) tail and approximately 20 nucleotides, adjust based on Tm, corresponding to the 3&#8217; end of the reporter gene&#8217;s ORF. Ensure the corresponding region in the primer is identical to the anti-sense strand (- strand) of the gene and an in-frame stop codon is present before the poly(A) tail. 
	NOTE: The desired length of A&#8217;s in a poly(A) leader or poly(A) tail can be customized in the primers. For example, to add 50 A&#8217;s in the poly(A) tail, the reverse primer should entail 50 T&#8217;s. Similarly, to add 20 A&#8217;s in the poly(A) leader, the forward primer should entail 20 A&#8217;s.</li>
	<li>For internal control, design another set of primers containing the following elements in 5&#8217; to 3&#8217; direction: T7 Promoter, a random 5&#8217;-UTR coding sequence containing Kozak sequence, Renilla luciferase ORF and poly(A) tail referred hereafter to as T7_Kozak-Rluc.</li>
	<li>In a PCR tube, add the reagents in the following order: DNase free water, 2x high-fidelity DNA polymerase, primers, and sequence confirmed luciferase template DNA (Table 2). 
	NOTE: Amounts of individual components in the mixture should be adjusted according to the reaction volume.</li>
	<li>Use a standard 3-step (Denaturation, Annealing, Extension) PCR cycle to generate a DNA template as shown in Table 3. 
	NOTE: Annealing temperature X &#176;C depends on the primer set being used and extension time T min depend on the PCR amplicon size and DNA polymerase used.</li>
	<li>Detect the PCR product by running 5-10% of PCR reaction in 1% agarose Tris-acetate-EDTA (TAE) gel electrophoresis (containing 0.1&#181;g/ml ethidium bromide) along with commercially available molecular weight standard. Visualize the gel under a UV illuminator to determine the size of the PCR product.</li>
	<li>After determining the correct size of the PCR product, ~1.7 kb for T7_12A-Fluc and ~1.0 kb for T7_Kozak-Rluc, purify it using a commercially available PCR purification kit. Elute the DNA using 100 &#181;L nuclease free water (Figure 2B).</li>
	<li>Once purified, check the concentration of the DNA using a spectrophotometer and determine the A260/A280 ratio (~1.8-2.0 is acceptable).</li>
	<li>Store purified DNA at -20 &#176;C or use for in vitro transcription immediately.</li>
</ol>
2. Generate mRNA by In Vitro Transcription
<ol>
	<li>Synthesize RNA from the PCR product in vitro, using an in vitro transcription kit (Figure 3A). 
	NOTE: T7_12A-Fluc and T7_Kozak-Rluc DNA templates are used to synthesize 12A-Fluc and Kozak-Rluc mRNAs, respectively.
	<ol>
		<li>To do this, take a microcentrifuge tube and add the reagents in the following order: DNase-RNase free water, NTP Buffer Mix, Cap Analog, Template PCR Product, T7-RNA polymerase Mix (Table 4). 
		NOTE: Other capping systems can also be used to cap RNA sequentially after in vitro transcription following the manufacturer&#8217;s instruction.</li>
		<li>Mix thoroughly and incubate at 37 &#176;C for 2 h.</li>
		<li>Proceed to the purification of the synthesized RNA using an RNA purification kit.</li>
	</ol>
	</li>
	<li>Run purified RNA in 1.5% agarose Tris-borate-EDTA (TBE) gel (containing 0.5 &#181;g/mL ethidium bromide) to check the RNA. Visualize the gel under a UV illuminator (Figure 3B).</li>
	<li>Check the concentration of the RNA using a spectrophotometer and determine the A260/A280 ratio (~1.8-2.0 is acceptable).</li>
	<li>Aliquot the purified RNA and store at -80 &#176;C.</li>
</ol>
3. Transfect mRNA to Cells
<ol>
	<li>Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent next day) and incubate overnight in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Infect HeLa cells with vaccinia virus (VACV) at a Multiplicity of Infection (MOI) of 5 or keep uninfected HeLa cells for comparison. 
	NOTE: MOI is the number of infectious viral particles per cell.</li>
	<li>MOI of X= {[(Number of cells * X) / Virus Titer] * 1000} &#181;l of virus per 1 mL medium.</li>
	<li>After desired h post infection (hpi) (in this experiment at 10-12 hpi), transfect mRNA (500 ng of total mRNA per well of 24-well plates) using a cationic lipid transfection reagent as shown in Figure 3C.
	<ol>
		<li>For one well of a 24-well plate, mix 480 ng of 12A sequence bearing firefly luciferase (12A-Fluc) mRNA and 20 ng of Kozak sequence bearing Renilla luciferase (Kozak-Rluc) mRNA in one microcentrifuge tube. In another microcentrifuge tube add 1.1 &#181;L of cationic lipid transfection reagent.</li>
		<li>Add 55 &#181;L of reduced serum medium in both tubes. Mix and incubate at room temperature for 5 min.</li>
		<li>After 5 min of incubation, add 55 &#181;L cationic lipid transfection reagent containing reduced serum medium in mRNA containing tube.</li>
		<li>Mix gently but thoroughly, and incubate at room temperature for 15 min.</li>
		<li>During the incubation, remove the cell culture medium and add 400 &#181;L of reduced serum medium per well of 24-well plates.</li>
		<li>After incubation, add 100 &#181;L of the mixture dropwise and evenly to one well of 24-well plates.</li>
	</ol>
	</li>
</ol>
4. Measure Luciferase Activities
<ol>
	<li>Five-hours post-co-transfection of 12A-Fluc and Kozak-Rluc mRNA, measure luciferase activity using a luciferase assay system capable of performing two reporter assays (e.g., Dual Luciferase Reporter assay kit).</li>
	<li>Remove the reduced serum medium and lyse the cells by adding 150 &#181;L 1x lysis buffer, a component of the luciferase assay kit.</li>
	<li>After 10 min incubation at room temperature, collect the lysate by scrapping the cells and transfer to a microcentrifuge tube.</li>
	<li>Centrifuge the lysate at 12,000 x g for 10 min at 4 &#176;C to pellet cell debris.</li>
	<li>Add 30 &#181;L of supernatant in opaque-walled 96 well white assay plate with a solid bottom.</li>
	<li>Measure the dual luminescence using the luciferase assay kit and a multimode plate reader luminometer.</li>
	<li>Perform the measurement using kinetics function (on a per-well basis) using the settings described in Table 5. 
	NOTE: The reading can also be taken using manual luminometer. Add an equal volume of lysate and substrate for Fluc in a cuvette. Wait for 2 s and measure for 10 s using luminometer. Following Fluc measurement, quickly take out cuvette from luminometer and add an equal volume of the substrate for Rluc manually. Again, wait for 2 s and measure for 10 s using luminometer.</li>
	<li>Export the luminescence reading data into a desirable file format.</li>
	<li>Determine relative translation rate from 12A-Fluc mRNA in uninfected and VACV infected HeLa cells by dividing Fluc value by internal control Rluc value. 
	NOTE: Supplementary Figure 1 shows the step-by-step analysis of raw data to get relative Fluc activity.</li>
</ol>

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The authors would like to declare no competing financial interest.

All four-core steps are critical to the success of the in vitro transcribed RNA-based luciferase reporter assay. Special attention should be given to primer design, especially for the T7 promoter sequence. T7 RNA polymerase starts transcription from the underlined first G (GGG-5&#39;-UTR-AUG-) in T7 promoter added before the 5&#39;-UTR sequence. Although the transcription start site (TSS) starts from the first G at the 5&#39; end, decreasing the number of G&#39;s less than three in T7 promoter region decreased the...

According to the central dogma, genetic information flows from DNA to RNA and then finally to protein1,2. This flow of genetic information is highly regulated at many levels including mRNA translation3,4. Development of reporter assays to measure regulation of gene expression will facilitate understanding of regulatory mechanisms involved in this process. Here we describe a protocol to study mRNA translation using an in vitro transcribed RNA-based luciferase reporter assay in poxvirus-infected cells.
Poxviruses comprise many highly dangerous human and animal pathogens5. Like all other viruses, poxviruses exclusively rely on host cell machinery for protein synthesis6,7,8. To efficiently synthesize viral proteins, viruses evolved many strategies to hijack cellular translational machinery to redirect it for translation of viral mRNAs7,8. One commonly employed mechanism by viruses is to use cis-acting elements in their transcripts. Notable examples include the Internal Ribosome Entry Site (IRES) and cap-independent translation enhancer (CITE)9,10,11. These cis-elements render the viral transcripts a translational advantage by attracting translational machinery via diverse mechanisms12,13,14. Over 100 poxvirus mRNAs have an evolutionarily conserved cis-acting element in the 5&#8217;-untranslated region (5&#8217;-UTR): a 5&#8217;-poly(A) leader at the very 5&#8217; ends of these mRNAs15,16. The lengths of these 5&#8217;-poly(A) leaders are heterogeneous and are generated by slippage of the poxvirus-encoded RNA polymerase during transcription17,18. We, and others, recently discovered that the 5&#8217;-poly(A) leader confers a translation advantage to an mRNA in cells infected with vaccinia virus (VACV), the prototypic member of poxviruses19,20.
The in vitro transcribed RNA-based luciferase reporter assay was initially developed to understand the role of 5&#8217;-poly(A) leader in mRNA translation during poxvirus infection19,21. Although plasmid DNA-based luciferase reporter assays have been widely used, there are several drawbacks that will complicate the result interpretation in poxvirus-infected cells. First, plasmids are able to replicate in VACV-infected cells22. Second, cryptic transcription often occurs from plasmid DNA18,23,24. Third, VACV promoter-driven transcription generates poly(A)-leader of heterogeneous lengths consequently making it difficult to control the poly(A)-leader length in some experiments18. An in vitro transcribed RNA-based luciferase reporter assay circumvents these issues and the data interpretation is straightforward.
There are four key steps in this method: (1) polymerase chain reaction (PCR) to generate the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA; (3) transfection to deliver mRNA into cells; and (4) detection of luciferase activity as indicator of translation (Figure 1). The resulting PCR amplicon contains the following elements in 5&#8217; to 3&#8217; direction: T7-Promoter, poly(A) leader or desired 5&#8217;-UTR sequence, firefly luciferase open reading frame (ORF) followed by a poly(A) tail. PCR amplicon is used as the template to synthesize mRNA by in vitro transcription using T7 polymerase. During in vitro transcription, m7G cap or other cap analog is incorporated in newly synthesized mRNA. The capped transcripts are transfected into uninfected or VACV-infected cells. The cell lysate is collected at the desired time after transfection to measure luciferase activities that indicate protein production from transfected mRNA. This reporter assay can be used to study translation regulation by cis-element present in 5&#8217;-UTR, 3&#8217;-UTR or other regions of an mRNA. Furthermore, the in vitro transcribed RNA-based assay can be used to study different mechanisms of translation initiation including cap-dependent initiation, cap-independent initiation, re-initiation and internal initiation like IRES.

<table><tbody><tr><td>2X-Q5 Master mix</td><td>New England Biolabs</td><td>M0492</td><td>High-Fidelity DNA Polymerase used in PCR</td></tr><tr><td>3&amp;acute;-O-Me-m7G(5&#039;)ppp(5&#039;)G RNA Cap Structure Analog</td><td>New England Biolabs</td><td>S1411L</td><td>Anti reverse Cap analog or ARCA</td></tr><tr><td>Corning 96 Well Half-Area white flat bottom polystyrene microplate</td><td>Corning</td><td>3693</td><td>Opaque walled 96 well white plate with solid bottom</td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega</td><td>E1960</td><td>Dual-Luciferase Assay Kit (DLAK)</td></tr><tr><td>E.Z.N.A. Cycle Pure Kit</td><td>OMEGA BIO-TEK</td><td>D6492</td><td>PCR purification kit</td></tr><tr><td>GloMax Navigator Microplate Luminometer</td><td>Promega</td><td>GM2010</td><td>Referred as multimode plate reader luminometer</td></tr><tr><td>HiScribe T7 Quick High Yield RNA synthesis Kit</td><td>New England Biolabs</td><td>E2050S</td><td>In Vitro transcription kit</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668019</td><td>Cationic lipid transfection reagent</td></tr><tr><td>NanoDrop2000</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td>Used to measure DNA and RNA concentration</td></tr><tr><td>Opti-MEM</td><td>Thermo Fisher Scientific</td><td>31985070</td><td>Reduced serum media</td></tr><tr><td>Purelink RNA Mini Kit</td><td>Thermo Fisher Scientific</td><td>12183018A</td><td>RNA purification kit</td></tr><tr><td>Vaccinia Capping System</td><td>New England Biolabs</td><td>M2080</td><td>Capping system</td></tr></tbody></table>

in vitro transcribed rna-based luciferase reporter assay to study translation regulation in poxvirus-infected cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

The four steps of in vitro transcribed RNA-based luciferase reporter assay: PCR to generate DNA template for In vitro transcription, in vitro transcription to generate mRNA, mRNA transfection, and luciferase measurement, can be seen in the schematic diagram (Figure 1). Designing of primers for both DNA templates (Fluc and Rluc) and the general scheme of overhang extension PCR is illustrated in the schematic (Figure 2A). After PCR...

Watch this Scientific Journal Video about In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells at JoVE.com

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

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14.9K Views.  Kansas State University. We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5’-untranslated region (UTR) and 3’-UTR. Different translation initiation modes can also be examined using this method.

Video: In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Genetics

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Cancer Research

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Biology

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections

Source: Chiem, K., et al. <a href="https://app.jove.com/v/59890">A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection.</a> J. Vis. Exp. (150), (2019).
This video demonstrates a method to track viral infections in mouse models using a recombinant luciferase and fluorescence protein-expressing bi-reporter Influenza A virus. This method enables both in vivo and ex vivo studies, enabling researchers to gain insights into viral dynamics and the effects of interventions.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Summary

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