Name: Establishment and Quantification of De Novo Lytic Infection by Cell-free Kaposis Sarcoma-Associated Herpesvirus (Video) | JoVE Journal
Uploaded: 2025-08-15T14:40:00.000+00:00
Duration: 7 min 10 s
Description: Learn to establish a robust in vitro platform for KSHV de novo lytic infection using HCT116 cells and BAC16-derived virions. This method enables quantification of viral replication and study of early herpesvirus events.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors would like to thank Dr. Jae U. Jung (Cleveland Clinic) for sharing the iSLK-BAC16 cell line producing recombinant KSHV. Graphic design was created using BioRender.com, for which the authors possess a license. This work was supported by NIH awards R21 DE028256, R01 CA140964, and R01 CA262631 to C. Liang (PI), the Wistar Science Accelerator Postdoctoral Award to Q. Zhu.

KSHV is classified as a Biosafety Level 2 (BSL-2) human pathogen. Perform all procedures involving infectious material in a certified BSL-2 biosafety cabinet. Decontaminate work surfaces with 70% ethanol before and after each procedure. Dispose of all waste in compliance with institutional biosafety regulations and WHO BSL-2 guidelines. A schematic overview of the full workflow is provided in Figure 1. The procedure includes the generation of cell-free BAC16-derived KSHV from reactivated iSLK cells, de novo infection of HCT 116 cells, quantification of virus titers by infectious unit (IU) assay, and qPCR analysis of virion-associated viral DNA. The reagents and the equipment used are listed in the Table of Materials.
1. Production of cell-free BAC16-derived KSHV
<ol>
	<li>Seed five T75 flasks of Dox-inducible iSLK cells harboring wide-type (WT) KSHV BAC 16 (iSLK-BAC16) at approximately 80% confluency. Incubate at 37 &#176;C in a humidified 5% CO 2 incubator for 24 h.</li>
	<li>Induce lytic reactivation by adding 1 &#181;g/mL Dox and 1 mM sodium butyrate to each flask. Continue incubation for 72-96 h.</li>
	<li>Monitor cells daily by phase-contrast microscopy. Collect supernatant once &#62;90% of cells exhibit rounding, detachment from the flask surface, and/or visible lysis, hallmarks of cytopathic effect (CPE) indicative of robust lytic reaction. Following supernatant collection, decontaminate and discard the lytically induced iSLK-BAC16 cells using 10% bleach.</li>
	<li>Centrifuge the collected supernatants at 1,500 x g for 10 min at 4 &#176;C to remove debris. Filter the clarified supernatant through a 0.45 &#181;m polyethersulfone (PES) membrane filter without disturbing the pellet.</li>
	<li>Transfer the filtered supernatants to ultracentrifuge tubes. Pellet the viral particles by centrifugation at 25,000 x g for 3 h at 4 &#176;C using a SW28 rotor.</li>
	<li>Carefully discard the supernatant into 10% bleach. Resuspend the viral pellet in 1 mL serum-free Dulbecco&#39;s Modified Eagle Medium (DMEM).</li>
	<li>Aliquot the resuspended virus into sterile, screwcap 1.5 mL microcentrifuge tubes. Store aliquots at -80 &#176;C until use. Determine infectious titers of the virus stock using GFP-based infectious unit (IU) assays in SLK cells (see step 3).</li>
</ol>
2. De novo infection of HCT116 cells
NOTE: A high multiplicity of infection (MOI) is critical to efficiently initiate lytic replication in HCT116 cells following de novo KSHV infection. This condition enables robust viral gene expression and productive replication without the need for exogenous stimuli. A representative result demonstrating the MOI-dependent effect on virion production is provided in Supplementary Figure 1A.
<ol>
	<li>Seed 1 x 105 HCT116 cells per well in a 12-well tissue culture plate. Incubate for 24 h at 37 &#176;C in a humidified 5% CO 2 incubator.</li>
	<li>Dilute the concentrated virus stock in pre-warmed, serum-free DMEM to a final volume of 250 &#181;L to achieve a multiplicity of infection (MOI) of 10.</li>
	<li>Aspirate culture medium from each well. Add 250 &#181;L of diluted virus directly to the cells.</li>
	<li>Centrifuge the plate at 1,500 x g for 1 h at 30 &#176;C to synchronize infection (spinoculation). Immediately transfer the plate to a 37 &#176;C incubator and continue incubation for an additional 1 h.</li>
	<li>Aspirate the inoculum and wash the cells three times with pre-warmed PBS to remove residual virus. Add 1 mL of complete DMEM to each well.</li>
	<li>Monitor GFP expression in infected cells by fluorescence microscopy at 36 h, 60 h, and 96 h post-infection. At each time point, collect 1 mL of culture supernatant from each well. 
	NOTE: After the supernatant collection, KSHV-infected HCT116 cells can&#160;be retained for downstream applications such as RNA extraction, protein analysis, immunofluorescence, or flow cytometry, based on experimental objectives.</li>
	<li>Centrifuge the collected supernatants at 1,500 x g for 10 min at 4 &#176;C to remove cell debris. Transfer clarified supernatants to fresh tubes. Snap-freeze in liquid nitrogen and store at -80 &#176;C for further analysis.</li>
</ol>
3. Infectious units assay (IU assay)
NOTE: SLK cells are permissive to KSHV entry and support early lytic gene expression. This assay quantifies infectious units based on GFP expression in infected SLK cells following exposure to serially diluted virus-containing supernatants.
<ol>
	<li>Seed 1 x 104 SLK cells per well in a 96-well plate 24 h prior to infection. Ensure cells are evenly distributed and reach ~60%-70% confluence on the day of infection.</li>
	<li>Label nine 1.5 mL microcentrifuge tubes for serial two-fold dilutions: Tube 1 (undiluted, 1:1), Tube 2 (1:2), Tube 3 (1:4), Tube 4 (1:8), Tube 5 (1:16), Tube 6 (1:32), Tube 7 (1:64), Tube 8 (1:128), and Tube 9 (1:256).</li>
	<li>Add 200 &#181;L of virus supernatant to Tube 1. Add 100 &#181;L of pre-warmed, serum-free DMEM to each of Tubes 2 through 9. To begin the dilution series, transfer 100 &#181;L from Tube 1 to Tube 2 and mix thoroughly by pipetting up and down. Then transfer 100 &#181;L from Tube 2 to Tube 3 and repeat this process sequentially through Tube 9, mixing thoroughly at each step. 
	NOTE: Each tube should contain a final volume of 100 &#181;L. Perform all dilutions in triplicate.</li>
	<li>Aspirate the culture medium from SLK cells. Add 100 &#181;L of each virus dilution to the designated wells. Use 27 wells in total (3 wells per dilution from 1:1 to 1:256).</li>
	<li>Centrifuge the 96-well plate at 1,500 x g for 1 h at 30 &#176;C to promote vial adsorption. Transfer the plate to a 37 &#176;C CO 2 incubator for an additional 1 h.</li>
	<li>Remove the inoculum and gently add 100 &#181;L of complete DMEM to each well. Incubate the plate for 24 h at 37 &#176;C.</li>
	<li>Harvest the cells and analyze GFP expression by flow cytometry. Record the number of GFP-positive cells per well.</li>
	<li>Calculate infectious units per mL (IU/mL) using the following formula13: 
	IU/mL = [(number of GFP-positive cells per well)/&#160;inoculum volume (mL)] x dilution factor. Determine the final virus titer using the last dilution with detectable GFP-positive cells</li>
</ol>
4. Quantification of encapsidated viral genomes by qPCR
<ol>
	<li>Add DNase I to a final concentration of 100 U/mL to 400 &#181;L of clarified supernatant (see step 1 for supernatant clarification and debris removal). Incubate for 15 min at 37 &#176;C to digest non-encapsidated viral and cellular DNAs. A representative result demonstrating the specificity and effectiveness of DNase treatment is shown in Supplementary Figure 1B.</li>
	<li>Add EDTA to a final concentration of 50 mM to stop the DNase reaction. Incubate at 75 &#176;C for 15 min to inactivate DNase I.</li>
	<li>Extract viral DNA from the DNase-treated supernatant using the DNA Mini Kit according to the manufacturer&#39;s protocol.</li>
	<li>Elute the viral DNA in 30 &#181;L of elution buffer. Use 5 &#181;L of eluted DNA for each qPCR reaction.</li>
	<li>Amplify the KSHV open reading frame 73 (ORF 73) using the following primers: Forward: 5&#39;-CCGAGGACGAAATGGAAGTG-3&#39;; Reverse: 5&#39;-GGTGATGTTCTGAGTACATAGCGG-3.</li>
	<li>Prepare qPCR reactions in triplicate using SYBR Green Master SuperMix. Set up the cycling conditions as follows: Initial denaturation: 95 &#176;C for 3 min; Amplification: 40 cycles of 95 &#176;C for 10 s, 52 &#176;C for 30 s, and 72 &#176;C for 30 s; Melting curve analysis: standard ramp for amplicon specificity.</li>
	<li>Generate a standard curve using 10-fold serial dilutions of BAC-16-derived viral DNA, ranging from 109 to 101 copies per reaction. Plot Ct values against the log10&#160;of input copy number to assess assay linearity. A representative standard curve is shown in Supplementary Figure 1C.</li>
	<li>Determine viral genome copy number by comparing Ct values to the standard curve. Normalize and report results as viral DNA copies per mL of supernatant.</li>
</ol>

<ol><li>Mesri, E. A., Cesarman, E., Boshoff, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kaposi%27s+sarcoma+herpesvirus%2Fhuman+herpesvirus-8+%28KSHV%2FHHV8%29%2C+and+the+oncogenesis+of+Kaposi%27s+sarcoma%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Kaposi's sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV8), and the oncogenesis of Kaposi's sarcoma.</a> Nat Rev Cancer. 10 (10), 707(2010).</li>
<li>Cesarman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gammaherpesvirus+and+lymphoproliferative+disorders+in+immunocompromised+patients%5BTitle%5D)+AND+%22Cancer+Lett%22%5BJournal%5D)">Gammaherpesvirus and lymphoproliferative disorders in immunocompromised patients.</a> Cancer Lett. 305 (2), 163-174 (2011).</li>
<li>Golas, G., Alonso, J. D., Toth, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+de+novo+lytic+infection+of+dermal+lymphatic+microvascular+endothelial+cells+by+Kaposi%27s+sarcoma-associated+herpesvirus%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Characterization of de novo lytic infection of dermal lymphatic microvascular endothelial cells by Kaposi's sarcoma-associated herpesvirus.</a> Virology. 536, 27-31 (2019).</li>
<li>Dittmer, D. P., Damania, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kaposi+sarcoma-associated+herpesvirus%3A+immunobiology%2C+oncogenesis%2C+and+therapy%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Kaposi sarcoma-associated herpesvirus: immunobiology, oncogenesis, and therapy.</a> J Clin Invest. 126 (9), 3165-3175 (2016).</li>
<li>Chang, H. H., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+unique+herpesviral+transcriptional+program+in+KSHV-infected+lymphatic+endothelial+cells+leads+to+mTORC1+activation+and+rapamycin+sensitivity%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">A unique herpesviral transcriptional program in KSHV-infected lymphatic endothelial cells leads to mTORC1 activation and rapamycin sensitivity.</a> Cell Host Microbe. 13 (4), 429-440 (2013).</li>
<li>Brulois, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+and+manipulation+of+a+new+Kaposi%27s+sarcoma-associated+herpesvirus+bacterial+artificial+chromosome+clone%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Construction and manipulation of a new Kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome clone.</a> J Virol. 86 (18), 9708-9720 (2012).</li>
<li>Myoung, J., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+a+doxycycline-inducible+KSHV+producer+cell+line+of+endothelial+origin%3A+Maintenance+of+tight+latency+with+efficient+reactivation+upon+induction%5BTitle%5D)+AND+%22J+Virol+Methods%22%5BJournal%5D)">Generation of a doxycycline-inducible KSHV producer cell line of endothelial origin: Maintenance of tight latency with efficient reactivation upon induction.</a> J Virol Methods. 174 (1-2), 12-21 (2011).</li>
<li>Grundhoff, A., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inefficient+establishment+of+KSHV+latency+suggests+an+additional+role+for+continued+lytic+replication+in+Kaposi+sarcoma+pathogenesis%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Inefficient establishment of KSHV latency suggests an additional role for continued lytic replication in Kaposi sarcoma pathogenesis.</a> J Clin Invest. 113 (1), 124-136 (2004).</li>
<li>Gao, S. J., Deng, J. H., Zhou, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Productive+lytic+replication+of+a+recombinant+Kaposi%27s+sarcoma-associated+herpesvirus+in+efficient+primary+infection+of+primary+human+endothelial+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Productive lytic replication of a recombinant Kaposi's sarcoma-associated herpesvirus in efficient primary infection of primary human endothelial cells.</a> J Virol. 77 (18), 9738-9749 (2003).</li>
<li>Atyeo, N., Chae, M. Y., Toth, Z., Sharma, A., Papp, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kaposi%27s+sarcoma-associated+herpesvirus+immediate+early+proteins+trigger+FOXQ1+expression+in+oral+epithelial+cells%2C+engaging+in+a+novel+lytic+cycle-sustaining+positive+feedback+loop%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Kaposi's sarcoma-associated herpesvirus immediate early proteins trigger FOXQ1 expression in oral epithelial cells, engaging in a novel lytic cycle-sustaining positive feedback loop.</a> J Virol. 97 (3), e0169622(2023).</li>
<li>Lagunoff, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+infection+and+serial+transmission+of+Kaposi%27s+sarcoma-associated+herpesvirus+in+cultured+endothelial+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells.</a> J Virol. 76 (5), 2440-2448 (2002).</li>
<li>ViejoBorbolla, A., Schulz, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kaposi%27s+sarcoma-associated+herpesvirus+and+innate+immunity%5BTitle%5D)+AND+%22J+Gen+Virol%22%5BJournal%5D)">Kaposi's sarcoma-associated herpesvirus and innate immunity.</a> J Gen Virol. 98 (2), 271-284 (2017).</li>
<li>Chandran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+events+in+Kaposi%27s+sarcoma-associated+herpesvirus+infection+of+target+cells%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Early events in Kaposi's sarcoma-associated herpesvirus infection of target cells.</a> J Virol. 84 (5), 2188-2199 (2010).</li>
<li>Zhu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kaposi%27s+sarcoma-associated+herpesvirus+induces+mitochondrial+fission+to+evade+host+immune+responses+and+promote+viral+production%5BTitle%5D)+AND+%22Nat+Microbiol%22%5BJournal%5D)">Kaposi's sarcoma-associated herpesvirus induces mitochondrial fission to evade host immune responses and promote viral production.</a> Nat Microbiol. 10 (6), 1501-1520 (2025).</li>
<li>Lee, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+and+HIF-1alpha+promote+lytic+de+novo+KSHV+infection%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Hypoxia and HIF-1alpha promote lytic de novo KSHV infection.</a> J Virol. 97 (11), e0097223(2023).</li>
<li>White, I. E., Campbell, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+of+cell-free+and+cell-associated+Kaposi%27s+sarcoma-associated+herpesvirus+DNA+by+real-time+PCR%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Quantitation of cell-free and cell-associated Kaposi's sarcoma-associated herpesvirus DNA by real-time PCR.</a> J Clin Microbiol. 38 (5), 1992-1995 (2000).</li>
<li>Bechtel, J., Grundhoff, A., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNAs+in+the+virion+of+Kaposi%27s+sarcoma-associated+herpesvirus%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">RNAs in the virion of Kaposi's sarcoma-associated herpesvirus.</a> J Virol. 79 (16), 10138-10146 (2005).</li>
<li>Lin, X., Li, X., Liang, D., Lan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNAs+and+unusual+small+RNAs+discovered+in+Kaposi%27s+sarcoma-associated+herpesvirus+virions%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">MicroRNAs and unusual small RNAs discovered in Kaposi's sarcoma-associated herpesvirus virions.</a> J Virol. 86 (23), 12717-12730 (2012).</li>
<li>Watzinger, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+quantitative+PCR+assays+for+detection+and+monitoring+of+pathogenic+human+viruses+in+immunosuppressed+pediatric+patients%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.</a> J Clin Microbiol. 42 (11), 5189-5198 (2004).</li>
<li>Cho, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antiviral+activity+of+angelicin+against+gammaherpesviruses%5BTitle%5D)+AND+%22Antiviral+Res%22%5BJournal%5D)">Antiviral activity of angelicin against gammaherpesviruses.</a> Antiviral Res. 100 (1), 75-83 (2013).</li>
<li>Coen, N., Duraffour, S., Snoeck, R., Andrei, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((KSHV+targeted+therapy%3A+An+update+on+inhibitors+of+viral+lytic+replication%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">KSHV targeted therapy: An update on inhibitors of viral lytic replication.</a> Viruses. 6 (11), 4731-4759 (2014).</li>
</ol>

No conflicts of interest declared.

Modeling de novo lytic infection of KSHV has long been hindered by the virus&#39;s strong propensity to establish latency in most permissive cell types. Although certain primary cells, such as endothelial and oral epithelial cells, have been shown to support lytic replication following de novo infection9,10, their limited lifespan, high cost, and technical difficulty in culture maintenance reduce reproducibility and scalability. Recent studies h...

Kaposi&#39;s sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus etiologically linked to Kaposi&#39;s sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KSHV inflammatory cytokine syndrome1,2. Like all herpesviruses, KSHV establishes lifelong persistence in the host, alternating between latent and lytic phases. While latency predominates in most infected cells, a subset can enter the lytic cycle either during primary (de novo) infection or through reactivation from latency3. Lytic replication involves an orderly cascade of viral gene expression, culminating in genome replication, virion assembly, and progeny release. This process is critical for viral dissemination and disease progression4,5. Early studies of KSHV lytic replication often relied on ectopic expression of individual viral genes in heterologous systems. Though informative, these approaches did not capture the coordinated gene regulation and virus-host interaction present during authentic infection. The development of BAC-16, a recombinant bacterial artificial chromosome (BAC) carrying the full-length KSHV genome, enabled the production of genetically manipulable viruses that retain competency for replication and infectious virion production6. This system facilitates targeted mutagenesis and functional analysis of viral genes within the context of the complete genome. Reactivation models employing latently infected iSLK or BCBL-1 cells with BAC-16 or KSHV have since become standard for studying KSHV lytic replication. These systems permit synchronized induction of the lytic cycle using agents such as doxycycline (Dox) and sodium butyrate7,8. However, because reactivation models circumvents early infection steps -- including viral entry, intracellular trafficking, and the initial decision between latency and lytic replication -- it cannot fully recapitulate primary infection dynamics.
De novo infection models offer a complementary framework for addressing these gaps. Several primary cell models, such as human endothelial and gingival epithelial cells, have been shown to support lytic replication following KSHV de novo infection3,9,10,11. However, their limited lifespan, high cost, and poor amenability to genetic manipulation restrict their reproducibility and scalability for mechanistic studies. In this protocol, we describe a tractable and scalable model of KSHV de novo lytic infection using the HCT 116 human colorectal carcinoma cell line. While not a natural target of KSHV, HCT116 cells exhibit robust permissiveness to infection and provide a stable and cost-effective platform suitable for mechanistic studies and high-throughput applications.Given KSHV&#39;s limited plaque-forming ability in culture, we utilize the BAC16 system incorporating a GFP reporter to generate cell-free virus for infection. This enables direct visualization of infected cells and quantification of infectious titers via GFP-based infectious unit (IU) assays12. To complement this, virion-associated DNA is measured by quantitative real-time PCR (qPCR) following DNase treatment, offering a sensitive method for detecting encapsidated viral genomes. Together, these assays permit temporally resolved, quantitative analysis of lytic replication dynamics during primary infection.
This protocol provides a reliable and flexible platform for modeling KSHV de novo lytic infection in vitro, facilitating dissection of early events in the viral lifecycle, enabling evaluation of gene function and antiviral activity.

<table><tbody><tr><td>0.45 &amp;micro;m polyethersulfome (PES) filter&amp;nbsp;</td><td>VWR</td><td>76479-020</td><td></td></tr><tr><td>Biorender</td><td>Biorender</td><td>N/A</td><td>Graphic design was&lt;br/&gt; created with BioRender.com, for which the authors possess a licence</td></tr><tr><td>CFX Opus 96 Real-time PCR System</td><td>Bio-Rad</td><td>184-5072</td><td></td></tr><tr><td>Complete DMEM</td><td></td><td></td><td>Dulbecco&#039;s modified Eagle&#039;s medium (DMEM; Sigma-Aldrich) supplemented with 10%(v/v) fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Gibco), and 100U/ml penicillin-streptomycin (Pen-Strep; GenClone).</td></tr><tr><td>DNase I</td><td>New England Biolabs</td><td>M0303S</td><td></td></tr><tr><td>doxycycline</td><td>Sigma-Aldrich</td><td>D9891</td><td></td></tr><tr><td>LSR II 18 flow cytometer</td><td>BD Bioscience</td><td>N/A</td><td></td></tr><tr><td>PerfeCTa SYBR Green Master SuperMix</td><td>Quantabio</td><td>95054-02K</td><td></td></tr><tr><td>QIAamp DNA Mini Kit</td><td>Qiagen</td><td>56304</td><td></td></tr><tr><td>sodium butyrate (NaB)</td><td>Sigma-Aldrich</td><td>B5887</td><td></td></tr><tr><td>UltraPure 0.5M EDTA, pH 8.0</td><td>Thermo Fisher</td><td>15575020</td><td></td></tr></tbody></table>

establishment and quantification of de novo lytic infection by cell-free kaposi's sarcoma-associated herpesvirus

Establishment and Quantification of De Novo Lytic Infection by Cell-free Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus implicated in multiple human malignancies, can undergo lytic replication during primary infection, a process that contributes to viral dissemination, immune evasion, and disease pathogenesis. However, the lack of robust in vitro systems for de novo lytic infection has limited insights into early infection events. Here, we present a tractable protocol that employs human colorectal cancer HCT 116 cells as targets for infection with cell-free virions derived from KSHV bacterial artificial chromosome 16 (BAC16)-reactivated iSLK producer cells. This model recapitulates key steps in primary infection, including viral entry, genome delivery, lytic gene expression, and progeny production. Infection is synchronized via spinoculation and monitored temporally. Virus production is quantified using a dual readout system comprising green fluorescence protein (GFP)-based infectious unit (IU) assays and qPCR of encapsidated viral DNA. These approaches enable detailed analysis of viral replication kinetics. While the current protocol focuses on the detection of late-stage events, the system is adaptable for studying early phases of infection through modified sampling time points. By offering a reproducible, scalable platform for productive de novo infection, this system addresses a methodological gap in KSHV research and supports mechanistic studies of herpesvirus-host interaction and antiviral strategies.

To quantify the titer of infectious virus, supernatants harvested 72 h after lytic reactivation of iSLK-BAC16 cells were subjected to two-fold serial dilution and used to infect SLK cells. The number of GFP-positive SLK cells at each dilution is shown in Figure 2A, and corresponding infectious units (IU) per mL were calculated and presented in Figure 2B. To evaluate the efficiency of de novo lytic replication, HCT 116 ce...

This protocol describes an in vitro system to model de novo lytic infection of Kaposi's sarcoma-associated herpesvirus using BAC16-derived virions. The method enables investigation of early viral replication and dissemination. Infectious virus production is quantified by GFP-based infectious units assays and real-time PCR of encapsidated viral genomes.

Kaposi's sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus implicated in multiple human malignancies, can undergo lytic replication during primary ...

Tractable and reproducible systems to model KSHV primary lytic infection are limited. We established a reliable and robust in vitro platform for modeling KSHV de novo lytic infection. It's challenging to model KSHV Lytic infection because of its strong latency bias, and the few primary cells that support it are delicate and have a limited lifespan, which severely limits reproducibility and scalability. We developed a tractable and reproducible model in HCT116 cells that capture efficient KSHV lytic replication during primary infection, offering a unique platform to study early events in herpes virus replication. To begin, seed five T75 flasks with dox-inducible iSLK cells containing wild-type KSHV BAC16 at approximately 80% confluency. Incubate the flasks at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 24 hours. Add one microgram per milliliter of doxycycline and one millimolar sodium butyrate to each flask to induce lytic reactivation. Using phase-contrast microscopy, monitor the cells daily.Collect the supernatant once more than 90% of cells show rounding, detachment from the flask surface, or visible lysis. Then, centrifuge the collected supernatants at 1, 500g for 10 minutes at four degrees Celsius to remove cellular debris. Filter the resulting supernatant through a 0.45 micrometer polyether-sulfone membrane filter without disturbing the pellet.Transfer the filtered supernatants to ultracentrifuge tubes. Centrifuge the tubes at 25, 000g for three hours at four degrees Celsius using an SW28 rotor to pellet the viral particles. Carefully discard the supernatant into a container with 10%bleach. Resuspend the viral pellet in one-milliliter of serum-free DMEM. Seed one times 10 to the power of five HCT116 cells per well in a 12-well tissue culture plate. Incubate the plate at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 24 hours.Dilute the concentrated virus stock in prewarmed, serum-free DMEM to a final volume of 250 microliters to achieve a multiplicity of infection, or MOI of 10. Aspirate the culture medium from each well and add 250 microliters of the diluted virus directly onto the cells. Centrifuge the plate at 1, 500g for one hour at 30 degrees Celsius to synchronize infection.Immediately transfer the plate to a 37 degrees Celsius incubator and continue incubation for one hour. Aspirate the viral inoculum from each well. Wash the cells three times with prewarmed PBS to remove residual virus.Add one milliliter of complete DMEM to each well. Then, collect one milliliter of culture supernatant from each well. Seed one times 10 to the power of four SLK cells per well in a 96-well plate 24 hours before infection. Label nine 1.5 milliliter microcentrifuge tubes for a serial two-fold dilution series. Add 200 microliters of virus supernatant to tube one. Add 100 microliters of prewarmed, serum-free DMEM to each of tubes two through nine.Begin the serial dilution by transferring 100 microliters from tube one to tube two and mixing thoroughly by pipetting. Now, aspirate the culture medium from SLK cells. Add 100 microliters of each virus dilution to the designated wells, using 27 wells in total.Centrifuge the 96-well plate at 1, 500g for one hour at 30 degrees Celsius to promote viral absorption. Transfer the plate to a 37 degrees Celsius incubator with 5%carbon dioxide and incubate for an additional hour. Remove the inoculum from each well.Then, gently add 100 microliters of complete DMEM to each well. Incubate the plate for 24 hours at 37 degrees Celsius. Then, analyze the GFP-positive cells.A two-fold serial dilution of BAC16-derived virus stock showed a progressive decrease in the number of GFP-positive SLK cells, confirming a measurable and dilution-sensitive infectious titer. The calculated infectious titer of the BAC16-derived virus stock was approximately 1.3 million infectious units per milliliter. GFP expression in HCT116 cells increased over time, following de novo infection, with the most intense signal observed at 60 hours post-infection, indicating robust viral replication at that time point.Flow cytometry analysis confirmed a peak in GFP-positive SLK cells at 60 hours post-infection, reaching 74.4% positivity, followed by a decrease to 62.7% at 96 hours. Quantitative PCR of viral DNA in supernatants showed a sharp increase in viral genome copies, peaking at 60 hours post-infection and declining thereafter.

establishment-quantification-de-novo-lytic-infection-cell-free-kaposi

Research

JoVE Journal

Immunology and Infection

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS).   We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue.  Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (γHV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for γHV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

γ-Herpesviruses (γ-HVs) establish life-long persistency in their host. Infection of mice with γ-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of γHVs. This protocol describes the detection and quantitation of γHV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

Measurement of &#947;HV68 Infection in Mice

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

This video describes the virus plaque assay of the infectious herpes simplex virus or HSV. The diluted viral particles are inoculated to the monolayer of epithelial cells. Upon incubation, the virus forms plaques and is counted visually to determine the infectious virus titer.

Virus Plaque Assay: A Technique to Measure Infectious Herpes Simplex Virus via Quantification of Plaques Formed in Monolayer of Virus-Infected Cells

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (γHV68) that is closely-related to human Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. “Reconstituting” the expression of innate immune components in knockout embryonic...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

This protocol permits the isolation of Epstein-Barr virus particles from the human P3HR1 cell line upon inducing the viral lytic cycle with phorbol 12-myristate 13-acetate. DNA is subsequently extracted from the viral preparation and subjected to real-time PCR to quantify the viral particle concentration.

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line

Learn to establish a robust in vitro platform for KSHV de novo lytic infection using HCT116 cells and BAC16-derived virions. This method enables quantification of viral replication and study of early herpesvirus events.

Establishment and Quantification of De Novo Lytic Infection by Cell-free Kaposi's Sarcoma-Associated Herpesvirus

Explore More Videos