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

Summary

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.

Abstract

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.

Introduction

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus etiologically linked to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KSHV inflammatory cytokine syndrome^1,2. 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 progression^4,5. 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 butyrate^7,8. 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 infection^3,9,10,11. 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'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.

Protocol

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

1. 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 °C in a humidified 5% CO ₂ incubator for 24 h.
2. Induce lytic reactivation by adding 1 µg/mL Dox and 1 mM sodium butyrate to each flask. Continue incubation for 72-96 h.
3. Monitor cells daily by phase-contrast microscopy. Collect supernatant once >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.
4. Centrifuge the collected supernatants at 1,500 x g for 10 min at 4 °C to remove debris. Filter the clarified supernatant through a 0.45 µm polyethersulfone (PES) membrane filter without disturbing the pellet.
5. Transfer the filtered supernatants to ultracentrifuge tubes. Pellet the viral particles by centrifugation at 25,000 x g for 3 h at 4 °C using a SW28 rotor.
6. Carefully discard the supernatant into 10% bleach. Resuspend the viral pellet in 1 mL serum-free Dulbecco's Modified Eagle Medium (DMEM).
7. Aliquot the resuspended virus into sterile, screwcap 1.5 mL microcentrifuge tubes. Store aliquots at -80 °C until use. Determine infectious titers of the virus stock using GFP-based infectious unit (IU) assays in SLK cells (see step 3).

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.

1. Seed 1 x 10⁵ HCT116 cells per well in a 12-well tissue culture plate. Incubate for 24 h at 37 °C in a humidified 5% CO ₂ incubator.
2. Dilute the concentrated virus stock in pre-warmed, serum-free DMEM to a final volume of 250 µL to achieve a multiplicity of infection (MOI) of 10.
3. Aspirate culture medium from each well. Add 250 µL of diluted virus directly to the cells.
4. Centrifuge the plate at 1,500 x g for 1 h at 30 °C to synchronize infection (spinoculation). Immediately transfer the plate to a 37 °C incubator and continue incubation for an additional 1 h.
5. 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.
6. 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 be retained for downstream applications such as RNA extraction, protein analysis, immunofluorescence, or flow cytometry, based on experimental objectives.
7. Centrifuge the collected supernatants at 1,500 x g for 10 min at 4 °C to remove cell debris. Transfer clarified supernatants to fresh tubes. Snap-freeze in liquid nitrogen and store at -80 °C for further analysis.

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.

1. 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.
2. 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).
3. Add 200 µL of virus supernatant to Tube 1. Add 100 µL of pre-warmed, serum-free DMEM to each of Tubes 2 through 9. To begin the dilution series, transfer 100 µL from Tube 1 to Tube 2 and mix thoroughly by pipetting up and down. Then transfer 100 µL 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 µL. Perform all dilutions in triplicate.
4. Aspirate the culture medium from SLK cells. Add 100 µL of each virus dilution to the designated wells. Use 27 wells in total (3 wells per dilution from 1:1 to 1:256).
5. Centrifuge the 96-well plate at 1,500 x g for 1 h at 30 °C to promote vial adsorption. Transfer the plate to a 37 °C CO ₂ incubator for an additional 1 h.
6. Remove the inoculum and gently add 100 µL of complete DMEM to each well. Incubate the plate for 24 h at 37 °C.
7. Harvest the cells and analyze GFP expression by flow cytometry. Record the number of GFP-positive cells per well.
8. Calculate infectious units per mL (IU/mL) using the following formula¹³:
   IU/mL = [(number of GFP-positive cells per well)/ inoculum volume (mL)] x dilution factor. Determine the final virus titer using the last dilution with detectable GFP-positive cells

4. Quantification of encapsidated viral genomes by qPCR

1. Add DNase I to a final concentration of 100 U/mL to 400 µL of clarified supernatant (see step 1 for supernatant clarification and debris removal). Incubate for 15 min at 37 °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.
2. Add EDTA to a final concentration of 50 mM to stop the DNase reaction. Incubate at 75 °C for 15 min to inactivate DNase I.
3. Extract viral DNA from the DNase-treated supernatant using the DNA Mini Kit according to the manufacturer's protocol.
4. Elute the viral DNA in 30 µL of elution buffer. Use 5 µL of eluted DNA for each qPCR reaction.
5. Amplify the KSHV open reading frame 73 (ORF 73) using the following primers: Forward: 5'-CCGAGGACGAAATGGAAGTG-3'; Reverse: 5'-GGTGATGTTCTGAGTACATAGCGG-3.
6. Prepare qPCR reactions in triplicate using SYBR Green Master SuperMix. Set up the cycling conditions as follows: Initial denaturation: 95 °C for 3 min; Amplification: 40 cycles of 95 °C for 10 s, 52 °C for 30 s, and 72 °C for 30 s; Melting curve analysis: standard ramp for amplicon specificity.
7. 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 of input copy number to assess assay linearity. A representative standard curve is shown in Supplementary Figure 1C.
8. 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.

Results

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...

Discussion

Modeling de novo lytic infection of KSHV has long been hindered by the virus'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 infection^9,10, their limited lifespan, high cost, and technical difficulty in culture maintenance reduce reproducibility and scalability. Recent studies h...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45 µm polyethersulfome (PES) filter	VWR	76479-020
Biorender	Biorender	N/A	Graphic design was created with BioRender.com, for which the authors possess a licence
CFX Opus 96 Real-time PCR System	Bio-Rad	184-5072
Complete DMEM			Dulbecco's modified Eagle'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).
DNase I	New England Biolabs	M0303S
doxycycline	Sigma-Aldrich	D9891
LSR II 18 flow cytometer	BD Bioscience	N/A
PerfeCTa SYBR Green Master SuperMix	Quantabio	95054-02K
QIAamp DNA Mini Kit	Qiagen	56304
sodium butyrate (NaB)	Sigma-Aldrich	B5887
UltraPure 0.5M EDTA, pH 8.0	Thermo Fisher	15575020