J Virol 88:3861C3873. US28 during latency in the Kasumi-3 latency model system and MBP146-78 in primary or HCMV model systems. HCMV latency culture systems that utilize primary hematopoietic cells (3,C14) and model systems (15,C27) are gaining momentum and being used more widely, and thus we are learning more about these stages of contamination. Repressive chromatin marks are critical in HCMV genomic silencing during latency, and both histone deacetylases and methyltransferases function to aid in this repression (reviewed in reference 28). The major immediate early promoter (MIEP) contains multiple transcription factor binding sites, and these are also modulated by chromatinization and associated with repressive marks during latency (reviewed in reference 28). Although chromatinization plays a critical role in latency and reactivation, it is clear that other viral and cellular factors are involved. For example, viral proteins including UL138 (29, 30), pp71 (13), LUNA (31), UL144 (32), and viral interleukin-10 (latency-associated HCMV homolog of IL-10 [LAcmvIL-10]) (33,C36) contribute to successful latency and reactivation in culture. HCMV has co-opted cellular factors as well, such as cellular microRNAs (miRNAs) (36,C38), transcription factors (32, 38), and cell signaling (38, 39). It is MBP146-78 clear that HCMV latency and reactivation are multifaceted processes and thus likely that our full understanding of these stages of infection remains incomplete. HCMV is usually a large virus, made up of over 200 open reading frames (ORFs) (40,C43). However, during latency only a small subset of genes is usually expressed (5, 44). US28 is usually one of four HCMV-encoded G-protein coupled receptor (GPCR) homologs and is expressed during both the latent (5, 32, 44, 45) and lytic (46, 47) cycles. Although many studies have focused on understanding US28’s functions during lytic replication (reviewed in reference 48), there is little known about the role MBP146-78 US28 plays during latency although it is one of only a few genes associated with latent transcription. US28 transcripts have been detected both during natural latency (32, 45) and during latent contamination studies (4,C6, 44, 49). To begin to elucidate the role of US28 during latency, we have utilized the Kasumi-3 model for HCMV latency and reactivation (23). The Kasumi-3 cell line is a CD34+ hematopoietic progenitor cell (HPC) line that shares many of the same cell surface markers described for the systems utilizing primary CD34+ HPCs isolated from either bone marrow or cord blood (50). We have previously shown that this Kasumi-3 cell line supports all of the hallmarks of HCMV latency, including reactivation resulting in the production of infectious virus (23). Using this model for HCMV latency and a panel of viral recombinants, we show that US28 aids in promoting successful latent contamination. Additionally, we found that this phenotype also occurs during contamination of primary CD34+ HPCs. Together, our PTPSTEP findings reveal that US28 plays a role in successful latent contamination of HPCs. MATERIALS AND METHODS Cells and viruses. Kasumi-3 cells (ATCC CRL-2725) were cultured in RPMI 1640 medium (ATCC 30-2001) supplemented with 20% fetal bovine serum (FBS), 100 U/ml each of penicillin and streptomycin, and 100 g/ml gentamicin at a density of 3 105 to 3 106 cells/ml. Primary newborn human foreskin fibroblasts (NuFF-1 cells; GlobalStem) were maintained in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% FBS, 2 mM l-glutamine, 0.1 mM nonessential amino acids, 10 mM HEPES, and 100 U/ml each of penicillin and streptomycin. Irradiated stromal cells (1:1 mixture of S1/S1 and MG3 cells) were a kind gift from Felicia D. Goodrum (University of Arizona) and were thawed directly into human CD34+ long-term culture medium (hLTCM) consisting of MyeloCult H5100 (Stem Cell Technologies) supplemented with 1 M hydrocortisone and 100 U/ml each of penicillin and streptomycin. Primary CD34+ hematopoietic progenitor cells (HPCs) were isolated from deidentified cord blood samples by magnetic separation, as described MBP146-78 elsewhere (4, 5, 51). Cells were immediately infected after isolation (see below). All cells were maintained at 37C with 5% CO2. Isolation and culture conditions for primary CD34+ cells are described in the next section. HCMV bacterial MBP146-78 artificial chromosome (BAC)-derived strain TB40/E (clone 4) (52) was used in this study. We previously engineered this strain to express mCherry (TB40/E-mCherry) (53). TB40/E-mCherry-US28 (US28), in.