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. 2019 Aug 20;10(4):e01889-19.
doi: 10.1128/mBio.01889-19.

Human Cytomegalovirus US28 Ligand Binding Activity Is Required for Latency in CD34+ Hematopoietic Progenitor Cells and Humanized NSG Mice

Lindsey B Crawford  1 , Patrizia Caposio  1 , Craig Kreklywich  1 , Andrew H Pham  1 , Meaghan H Hancock  1 , Taylor A Jones  1 , Patricia P Smith  1 , Andrew D Yurochko  2 , Jay A Nelson  1 , Daniel N Streblow  3
Affiliations

Human Cytomegalovirus US28 Ligand Binding Activity Is Required for Latency in CD34+ Hematopoietic Progenitor Cells and Humanized NSG Mice

Lindsey B Crawford et al. mBio. .

Abstract

Human cytomegalovirus (HCMV) infection of CD34+ hematopoietic progenitor cells (CD34+ HPCs) provides a critical reservoir of virus in stem cell transplant patients, and viral reactivation remains a significant cause of morbidity and mortality. The HCMV chemokine receptor US28 is implicated in the regulation of viral latency and reactivation. To explore the role of US28 signaling in latency and reactivation, we analyzed protein tyrosine kinase signaling in CD34+ HPCs expressing US28. US28-ligand signaling in CD34+ HPCs induced changes in key regulators of cellular activation and differentiation. In vitro latency and reactivation assays utilizing CD34+ HPCs indicated that US28 was required for viral reactivation but not latency establishment or maintenance. Similarly, humanized NSG mice (huNSG) infected with TB40E-GFP-US28stop failed to reactivate upon treatment with granulocyte-colony-stimulating factor, but viral genome levels were maintained. Interestingly, HCMV-mediated changes in hematopoiesis during latency in vivo and in vitro was also dependent upon US28, as US28 directly promoted differentiation toward the myeloid lineage. To determine whether US28 constitutive activity and/or ligand-binding activity were required for latency and reactivation, we infected both huNSG mice and CD34+ HPCs in vitro with HCMV TB40E-GFP containing the US28-R129A mutation (no CA) or Y16F mutation (no ligand binding). TB40E-GFP-US28-R129A was maintained during latency and exhibited normal reactivation kinetics. In contrast, TB40E-GFP-US28-Y16F exhibited high levels of viral genome during latency and reactivation, indicating that the virus did not establish latency. These data indicate that US28 is necessary for viral reactivation and ligand binding activity is required for viral latency, highlighting the complex role of US28 during HCMV latency and reactivation.IMPORTANCE Human cytomegalovirus (HCMV) can establish latency following infection of CD34+ hematopoietic progenitor cells (HPCs), and reactivation from latency is a significant cause of viral disease and accelerated graft failure in bone marrow and solid-organ transplant patients. The precise molecular mechanisms of HCMV infection in HPCs are not well defined; however, select viral gene products are known to regulate aspects of latency and reactivation. The HCMV-encoded chemokine receptor US28, which binds multiple CC chemokines as well as CX3CR1, is expressed both during latent and lytic phases of the virus life cycle and plays a role in latency and reactivation. However, the specific timing of US28 expression and the role of ligand binding in these processes are not well defined. In this report, we determined that US28 is required for reactivation but not for maintaining latency. However, when present during latency, US28 ligand binding activity is critical to maintaining the virus in a quiescent state. We attribute the regulation of both latency and reactivation to the role of US28 in promoting myeloid lineage cell differentiation. These data highlight the dynamic and multifunctional nature of US28 during HCMV latency and reactivation.

Keywords: US28; hematopoiesis; human cytomegalovirus; latency; reactivation.

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Figures

FIG 1
FIG 1
US28 promotes cellular kinase phosphorylation in CD34+ HPCs. CD34+ HPCs infected with Ad-US28 were left untreated or were treated with PBS, RANTES (CCL5), or Fractalkine (CX3CL1). HPCs infected with Ad-Empty that were treated with PBS were used as a background control for phosphorylation status. At 30 min posttreatment, cellular lysates were collected and analyzed using the PathScan RTK chip to quantify protein phosphorylation. The data are represented as fold change versus values for Ad-Empty and are representative of two independent experiments.
FIG 2
FIG 2
HCMV-TB40E-GFP constructs. A bacterial artificial chromosome containing the HCMV TB40E-GFP genome was used as the genetic backbone for recombineering of US28 mutants using the 2-step galK-Kan method. US28 mutations and ddFKBP C-terminal fusion are depicted.
FIG 3
FIG 3
HCMV US28 is required for viral reactivation. CD34+ HPCs were infected with HCMV TB40E-GFP-WT or -US28stop at an MOI of 3. At 2 days postinfection (dpi), the cells were sorted by FACS for viable GFP+ CD34+ HPCs. HPCs were cultured for an additional 12 days in transwells over stromal cells (14 dpi) to establish latency. (A) Equal numbers of latently infected HPCs were either directly cocultured with NHDFs in cytokine-enriched media to induce viral reactivation (reactivation) or lysed and plated onto NHDFs (prereactivation) to assess the amount of virus present prior to reactivation. At 14 days postplating, the number of GFP-positive wells was determined by fluorescence microscopy, and the frequency of infectious centers was determined by ELDA software. (B) Total genomic DNA from latent HPCs was isolated, and quantitative real-time PCR was used to quantify the ratio of viral genomes (copies of HCMV UL141) to cellular genomes (per two copies of human [Hu] β-globin). Data are representative of three independent experiments; additional experiments are shown in Fig. S1.
FIG 4
FIG 4
FKBP protein destabilization domain validates that HCMV US28 is required for viral reactivation. CD34+ HPCs were infected with HCMV TB40E-GFP-WT or -US28-ddFKBP and cultured to establish latency as described for Fig. 3. Following the establishment of latency, equal numbers of cells were either cocultured with NHDFs with or without 1 μM Shield-1 (reactivation conditions) or lysed and plated onto NHDFs (prereactivation). At 14 days postplating, the number of GFP-positive wells was determined by fluorescence microscopy, and the frequency of infectious centers was determined by ELDA software.
FIG 5
FIG 5
US28 ligand binding is required for maintenance of latency. CD34+ HPCs were infected with HCMV TB40E-GFP-WT, -US28stop, US28-R129A, or -US28-Y16F and cultured to establish latency as described for Fig. 3. Following the establishment of latency, equal numbers of cells were either cocultured with NHDFs in cytokine-enriched media (reactivation conditions) or lysed and plated onto NHDFs (prereactivation). At 21 days postplating, the number of GFP-positive wells was determined by fluorescence microscopy, and the frequency of infectious centers was determined by ELDA software.
FIG 6
FIG 6
HCMV US28 is required for reactivation in vivo. Humanized NSG mice were injected with fibroblasts infected with either HCMV TB40E-GFP-WT or -US28stop (n = 8 to 10 per group). At 8 weeks postinfection, half of the mice were treated with G-CSF and AMD-3100 to induce cellular mobilization and promote HCMV reactivation. Control mice were left untreated. At 1 week postmobilization, mice were euthanized and tissues were collected. Total DNA was extracted using DNAzol, and HCMV viral load was determined by qPCR on 1 μg of total DNA prepared from spleen (A) or liver (B) tissue. Error bars represent standard deviations between average DNA copies from two (A) or four (B) tissue sections for individual animals. All samples were compared by one-way analysis of variance (ANOVA) within experimental groups (nonmobilized versus mobilized [+G-CSF] for each virus and between all virus groups for both nonmobilized and mobilized conditions). P values are listed for significant comparisons where P < 0.05.
FIG 7
FIG 7
HCMV US28 ligand binding is required to maintain latency in vivo. Humanized NSG mice were injected with fibroblasts infected with either HCMV TB40E-GFP-WT, -US28stop, -US28-R129A, or -US28-Y16F (n = 10 per group). At 8 weeks postinfection, half of the mice were treated with G-CSF and AMD-3100 to induce cellular mobilization and promote HCMV reactivation. Control mice were left untreated. At 1 week postmobilization, mice were euthanized and tissues were collected. Total DNA was extracted using DNAzol, and HCMV viral load was determined by qPCR on 1 μg of total DNA prepared from spleen (A) or liver (B) tissue. Error bars represent standard deviations between average DNA copies from two (A) or four (B) tissue sections for individual animals. All samples were compared by one-way ANOVA within experimental groups (nonmobilized versus mobilized [+G-CSF] for each virus and between all virus groups for both nonmobilized and mobilized conditions). P values are listed for significant comparisons where P < 0.05.
FIG 8
FIG 8
US28 alters hematopoiesis in vivo in huNSG mice. Humanized NSG mice were injected with mock-, HCMV TB40E-GFP-WT-, or HCMV TB40E-GFP-ΔUS28-infected fibroblasts (n = 4 to 5 per group). At 8 weeks postinfection, phenotypic analysis of human CD45+ blood leukocytes or splenocytes was performed by flow cytometry for T cell (CD3), B cell (CD19), and monocyte/macrophage (CD14) populations. Data from mock- and WT-infected huNSG mice shown here were previously published (39). Error bars represent standard deviations between individual animals. All samples were compared by one-way ANOVA between all virus groups, and P values are listed for significant comparisons where P < 0.05.
FIG 9
FIG 9
US28 induces myeloid colony formation in CD34+ HPCs. (A) CD34+ HPCs were mock infected or infected with HCMV TB40E-GFP-WT or TB40E-GFP-ΔUS28 for 2 days. FACS-isolated viable GFP+ CD34+ HPCs were plated in Methocult H4434 at 500 cells/well and counted at 7 days. Data shown are average numbers of myeloid colonies per well for triplicate wells. Data are representative of three independent experiments. (B) CD34+ HPCs were infected with Ad-US28 or Ad-Empty (control). At 24 hpi, cells were plated in Methocult H4434 for 7 days. Data are representative of two independent experiments. Error bars represent standard deviations between three replicate wells per experiment. P values were determined one-way ANOVA (A) or by t test (B) and are listed as exact values. Replicate experiments are shown in Fig. S2.
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References

    1. Cannon MJ, Schmid DS, Hyde TB. 2010. Review of cytomegalovirus seroprevalence and demographic characteristics associated with infection. Rev Med Virol 20:202–213. doi:10.1002/rmv.655. - DOI - PubMed
    1. Ljungman P, Hakki M, Boeckh M. 2011. Cytomegalovirus in hematopoietic stem cell transplant recipients. Hematol Oncol Clin North Am 25:151–169. doi:10.1016/j.hoc.2010.11.011. - DOI - PMC - PubMed
    1. Ramanan P, Razonable RR. 2013. Cytomegalovirus infections in solid organ transplantation: a review. Infect Chemother 45:260–271. doi:10.3947/ic.2013.45.3.260. - DOI - PMC - PubMed
    1. Nichols WG, Corey L, Gooley T, Davis C, Boeckh M. 2002. High risk of death due to bacterial and fungal infection among cytomegalovirus (CMV)-seronegative recipients of stem cell transplants from seropositive donors: evidence for indirect effects of primary CMV infection. J Infect Dis 185:273–282. doi:10.1086/338624. - DOI - PubMed
    1. Jenkins C, Garcia W, Abendroth A, Slobedman B. 2008. Expression of a human cytomegalovirus latency-associated homolog of interleukin-10 during the productive phase of infection. Virology 370:285–294. doi:10.1016/j.virol.2007.09.002. - DOI - PubMed

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