Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Monocytes
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Latent Kaposi’s Sarcoma-Associated Herpesvirus Infection of Monocytes Downregulates Expression of Adaptive Immune Response Costimulatory Receptors and Proinflammatory Cytokines Sean M. Gregory,a,b Ling Wang,a John A. West,a Dirk P. Dittmer,a,b and Blossom Damaniaa,b Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA,a and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USAb Kaposi’s sarcoma-associated herpesvirus (KSHV) infection is associated with the development of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. We report the establishment of a monocytic cell line latently infected with KSHV (KSHV-THP-1). We profiled viral and cytokine gene expression in the KSHV-THP-1 cells compared to that in unin- fected THP-1 cells and found that several genes involved in the host immune response were downregulated during latent infec- tion, including genes for CD80, CD86, and the cytokines tumor necrosis factor alpha (TNF-␣) and interleukin-1 (IL-1). Thus, KSHV minimizes its immunological signature by suppressing key immune response factors, enabling persistent infection and evasion from host detection. aposi’s sarcoma-associated herpesvirus (KSHV), also known cells, monocytes, and other lineages to replenish depleted pools of Kas human herpesvirus 8 (HHV8), is a member of the gamma- infected, differentiated cells (45). herpesvirus subfamily. KSHV is the etiological agent of Kaposi’s Immunological detection of KSHV infection by T cells requires sarcoma (KS) (8), primary effusion lymphoma (PEL), and multi- T cell receptor (TCR) cognate interactions with the antigen- centric variant of Castleman’s disease (MCD) (1, 8, 36). KS is a presenting cell (APC) major histocompatibility complex (MHC) highly inflammatory and angiogenic vascular tumor defined by surface molecules displaying KSHV peptide. On inactivated T characteristic spindle cells, which are believed to originate from cells, CD28 is expressed at low levels. Upon TCR-MHC contact endothelial cells. PEL and MCD are both B cell lymphoprolifera- and APC surface receptor CD40 ligation by CD40 ligand tive diseases. (CD40L), CD28 expression is upregulated. Concomitantly, the Like other herpesviruses, KSHV establishes latent infection in costimulatory molecules CD80 and CD86 on the APCs, which its host. A number of PEL cell lines have been established where interact with CD28 to increase the immune response, are upregu- most of the cells are latently infected, with only a small population lated. Despite the existence of KSHV-specific T cells and immune of cells undergoing spontaneous lytic reactivation (28, 38). Dur- control in healthy individuals, latent virus is unable to be elimi- ing latency, a limited number of viral proteins are expressed, in- nated from the host (41, 42). It is known that KSHV lytic proteins cluding the latency-associated nuclear antigen (LANA), vFLIP, K3 and K5 actively downregulate CD80 and MHC class I (MHC I) vCyclin, kaposin, vIRF3, K1, and vIL-6 (7, 12, 35, 37). Mainte- surface expression; however, the suppression of adaptive immune nance of the viral genome is absolutely dependent on the LANA molecules during latency also contributes to evasion of the host protein, which tethers the latent viral episome to the host cell response (22). Cells involved in immunity that are tropic for chromosome, ensuring that the viral genome is replicated with the host genome and is not diluted out of the expanding population of KSHV may facilitate suppression of host immune responses. latently infected cells (10, 13). The LANA protein has been shown Given that KSHV infects monocytes in vivo, we established a to be expressed in latently infected B cells and endothelial cells, as latently infected monocytic cell line by using the monocytic leu- well as in the KSHV-positive tumors associated with these cell kemia cell line THP-1 to characterize viral gene expression in la- types (3, 10, 13). tently infected monocytes (2, 39). THP-1 cells are susceptible to KSHV can successfully infect human monocytes and macro- human cytomegalovirus (HCMV) infection and support viral la- phages in vitro and in vivo (4–6, 24). Rappocciolo et al. demon- tency (43). Importantly, although we have previously shown that strated that KSHV uses the receptor DC-SIGN to enter macro- KSHV can infect primary human monocytes (44), we could not phages and dendritic cells (DCs) (31, 32). Kerur et al. showed that establish a long-term latent culture in these cells because of the in the THP-1 acute monocytic leukemia cell line, KSHV primary primary nature of the monocytes. In contrast, THP-1 cells enable infection was dependent on ␣31, ␣v3, ␣v5, and ␣51 integ- rins and that it was preceded by endosomal entry, which activated FAK, Src, PI3K, NF-B, and ERK1/2 signaling (20). Coinfection Received 28 September 2011 Accepted 17 January 2012 of monocytes with KSHV and HIV increased the replication of Published ahead of print 25 January 2012 HIV in the presence of KSHV (6). Monocytes present in KS lesions Address correspondence to Blossom Damania, [email protected]. have been shown to support viral replication (5). Additionally, S. M. Gregory and L. Wang contributed equally to the work. KSHV has been found to infect CD34ϩ stem cell precursors in Copyright © 2012, American Society for Microbiology. All Rights Reserved. vitro, suggesting that stem cells or later-stage committed progen- doi:10.1128/JVI.06437-11 itor cells may be infected and subsequently differentiate into B 3916 jvi.asm.org 0022-538X/12/$12.00 Journal of Virology p. 3916–3923 Effect of Latent KSHV Infection in Monocytes the establishment of a monocytic latent cell line that harbors described previously (11, 12). The primer name is as published before or KSHV and which can be passaged over a long period. indicates the orf name in KSHV followed by position of the forward primer. In addition, the original primer set was replaced with improved primers for orf 11 (KS1008-1), orf20 (kshv10021-1), orf33 (kshv10034-1), MATERIALS AND METHODS orf50 (kshv10052-1), orf53 (kshv10055-1), orf63 (kshv10069-1), orf65 Production of recombinant rKSHV.219 virus. Vero cells containing la- (kshv10070-1), orf71 (kshv10076-1), orf K5 (kshv10014-1), and orf K15 tent rKSHV.219 (KSHV-Vero) and a recombinant baculovirus KSHV (kshv10082-1). Primer sequences are available from the UNC Vironomics Orf50 (Bac50) were kindly provided by Jeffrey Vieira (40). rKSHV.219 core. Cycle threshold (CT) values were determined by automated analysis. expresses green fluorescent protein (GFP) and also contains a puromycin The threshold was set to five times the standard deviation (SD) of the resistance gene as a selectable marker. Insect SF9 cells were grown in nontemplate control (NTC). For each qPCR run, dissociation curves were SF900-II serum-free medium at 28°C. SF9 cells were infected with bacu- analyzed to verify that identical primer-specific, single reaction products lovirus expressing KSHV Orf50 (Bac50) for 3 days, after which time the were generated in each run. Genes with multiple primer pairs indicate two baculovirus-containing supernatant was clarified by centrifugation (1,500 independent primer sets. Samples were normalized to GAPDH levels, rpm for 10 min). KSHV-Vero cells were then infected with Bac50 and centered by median of gene, and ordered by hierarchical clustering using treated with 2 mM sodium butyrate for 3 days. Supernatant was harvested, ArrayMiner (Optimal Design, Inc., Brussels, Belgium) software with stan- and cells were removed by centrifugation (1,500 rpm for 10 min). Super- dard correlation metrics (11, 12). Further statistical analysis was con- natants were subsequently passed through a 0.45- m filter. ducted using the R programming environment (v 2.5.1). Establishment of the KSHV-THP-1 cell line. THP-1 cells were cul- Flow cytometry. To assess the expression of cell surface receptors, tured in RPMI with 10% fetal bovine serum (FBS) and were maintained at THP-1 and KSHV-THP-1 monocytes were suspended at 1 ϫ 107 cells/ml 37°C in a 5% CO2 environment. The rKSHV.219 produced from KSHV- in staining buffer (1ϫ PBS, 2% BSA) and labeled with 20 l of mouse Vero cells was used to infect THP-1 cells. We first made an infection allophycocyanin-conjugated anti-human CD86 (BD Pharmingen), cocktail containing complete RPMI medium and rKSHV.219 superna- mouse allophycocyanin-conjugated anti-human CD83 (BD Pharmin- tants (volume ratio of 1:2) with 4 g/ml Polybrene. THP-1 cells were gen), or isotype control allophycocyanin-conjugated IgG1 kappa (BD centrifuged at 1,500 rpm for 5 min, and then 2 ϫ 106 THP-1 cells were Pharmingen) antibody for 30 min at 4°C, protected from light. Cells were resuspended in 3 ml infection cocktail and added to one well of a 6-well then thoroughly washed twice with staining buffer by centrifugation at plate. The cells in the 6-well plate were spun for 90 min at 30°C at 2,500 1,500 rpm at 4°C. Next, labeled cells were fixed using 1% formaldehyde for rpm; then, the supernatants were removed and the cells were resuspended 30 min at 4°C, followed by washing with staining buffer. Samples were in 3 ml complete RPMI medium. The cells were incubated at 37°C for 72 resuspended in 250 l of staining buffer and analyzed using a BD FACs h and then selected in complete RPMI medium containing 1.0 g/ml Calibur (BD Biosciences) flow cytometer with data analysis using Summit puromycin for 3 to 5 weeks to establish stable KSHV-THP-1 cells. Once version 4.3 (Dako). Data were acquired for a minimum of 25,000 total THP-1 cells were 100% KSHV positive, KSHV-THP-1 cells were main- events.