Cis and Trans Acting Factors Involved in Human Cytomegalovirus Experimental and Natural Latent Infection of CD14 (+) Monocytes and CD34 (+) Cells

AbstractLatency enables human cytomegalovirus (HCMV) to persist in the hematopoietic cells of infected individuals indefinitely and prevents clearance of the pathogen. Despite its critical importance to the viral infectious cycle, viral mechanisms that contribute to latency have not been identified. We compared the ability of low-passage clinical and laboratory-adapted strains of HCMV to establish a latent infection in primary human CD34+ cells. The low-passage strains, Toledo and FIX, established an infection with the hallmarks of latency, whereas the laboratory strains, AD169 and Towne, replicated producing progeny virus. We hypothesized that ULb′ region of the genome, which is unique to low-passage strains, may encode a latency-promoting activity. We created and analyzed recombinant viruses lacking segments or individual open reading frames (ORFs) in the ULb′ region. One 5-kb segment, and more specifically the UL138 ORF, was required for HCMV to establish and/or maintain a latent infection in hematopoietic progenitor cells infected in vitro. This is the first functional demonstration of a virus-coded sequence required for HCMV latency. Importantly, UL138 RNA was expressed in CD34+ cells and monocytes from HCMV-seropositive, healthy individuals. UL138 might be a target for antivirals against latent virus.

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Advances in Model Systems for Human Cytomegalovirus Latency and Reactivation

mBio ◽

10.1128/mbio.01724-21 ◽

2022 ◽

Author(s):

Lindsey B. Crawford ◽

Nicole L. Diggins ◽

Patrizia Caposio ◽

Meaghan H. Hancock

Keyword(s):

Human Cytomegalovirus ◽

Congenital Abnormalities ◽

Latent Infection ◽

Organ Transplant ◽

Solid Organ Transplant ◽

Model Systems ◽

Morbidity And Mortality ◽

Solid Organ ◽

Hematopoietic Progenitor ◽

Myeloid Lineage

Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells.

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.BETA.-herpesvirus(human cytomegalovirus and human herpesvirus 6) latent infection.

Uirusu ◽

10.2222/jsv.46.161 ◽

1996 ◽

Vol 46 (2) ◽

pp. 161-168

Author(s):

Kazuhiro Kondo

Keyword(s):

Human Cytomegalovirus ◽

Latent Infection ◽

Human Herpesvirus ◽

Human Herpesvirus 6 ◽

Herpesvirus 6

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Interleukin-2 from Adaptive T Cells Enhances Natural Killer Cell Activity against Human Cytomegalovirus-Infected Macrophages

Journal of Virology ◽

10.1128/jvi.00435-15 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6435-6441 ◽

Cited By ~ 9

Author(s):

Zeguang Wu ◽

Giada Frascaroli ◽

Carina Bayer ◽

Tatjana Schmal ◽

Thomas Mertens

Keyword(s):

Innate Immunity ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Natural Killer ◽

Adaptive Immunity ◽

Human Cytomegalovirus ◽

Latent Infection ◽

Immune Surveillance ◽

Interleukin 2

ABSTRACTControl of human cytomegalovirus (HCMV) requires a continuous immune surveillance, thus HCMV is the most important viral pathogen in severely immunocompromised individuals. Both innate and adaptive immunity contribute to the control of HCMV. Here, we report that peripheral blood natural killer cells (PBNKs) from HCMV-seropositive donors showed an enhanced activity toward HCMV-infected autologous macrophages. However, this enhanced response was abolished when purified NK cells were applied as effectors. We demonstrate that this enhanced PBNK activity was dependent on the interleukin-2 (IL-2) secretion of CD4+T cells when reexposed to the virus. Purified T cells enhanced the activity of purified NK cells in response to HCMV-infected macrophages. This effect could be suppressed by IL-2 blocking. Our findings not only extend the knowledge on the immune surveillance in HCMV—namely, that NK cell-mediated innate immunity can be enhanced by a preexisting T cell antiviral immunity—but also indicate a potential clinical implication for patients at risk for severe HCMV manifestations due to immunosuppressive drugs, which mainly suppress IL-2 production and T cell responsiveness.IMPORTANCEHuman cytomegalovirus (HCMV) is never cleared by the host after primary infection but instead establishes a lifelong latent infection with possible reactivations when the host′s immunity becomes suppressed. Both innate immunity and adaptive immunity are important for the control of viral infections. Natural killer (NK) cells are main innate effectors providing a rapid response to virus-infected cells. Virus-specific T cells are the main adaptive effectors that are critical for the control of the latent infection and limitation of reinfection. In this study, we found that IL-2 secreted by adaptive CD4+T cells after reexposure to HCMV enhances the activity of NK cells in response to HCMV-infected target cells. This is the first direct evidence that the adaptive T cells can help NK cells to act against HCMV infection.

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In vitro infection of megakaryocytes and their precursors by human cytomegalovirus

Blood ◽

10.1182/blood.v95.2.487 ◽

2000 ◽

Vol 95 (2) ◽

pp. 487-493 ◽

Cited By ~ 55

Author(s):

Kirsten Crapnell ◽

Esmail D. Zanjani ◽

Aniruddho Chaudhuri ◽

Joao L. Ascensao ◽

Stephen St. Jeor ◽

...

Keyword(s):

Life Cycle ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Allogeneic Bone Marrow Transplantation ◽

Laboratory Strain ◽

Allogeneic Bone ◽

Cd34 Cells ◽

Hcmv Disease

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34+ cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42+ MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the β-galactosidase (β-gal) gene. Using chromogenic β-gal substrates, HCMV was detected during MK differentiation of infected CD34+ cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV.

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Human cytomegalovirus latent infection of granulocyte-macrophage progenitors.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.25.11879 ◽

1994 ◽

Vol 91 (25) ◽

pp. 11879-11883 ◽

Cited By ~ 252

Author(s):

K. Kondo ◽

H. Kaneshima ◽

E. S. Mocarski

Keyword(s):

Human Cytomegalovirus ◽

Latent Infection

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Latent infection of myeloid progenitors by human cytomegalovirus protects cells from FAS-mediated apoptosis through the cellular IL-10/PEA-15 pathway

Journal of General Virology ◽

10.1099/vir.0.000180 ◽

2015 ◽

Vol 96 (8) ◽

pp. 2355-2359 ◽

Cited By ~ 14

Author(s):

Emma Poole ◽

Jonathan C. H. Lau ◽

John Sinclair

Keyword(s):

Human Cytomegalovirus ◽

Latent Infection ◽

Myeloid Progenitors

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Human Cytomegalovirus miR-US33as-5p Targets IFNAR1 to Achieve Immune Evasion During Both Lytic and Latent Infection

Frontiers in Immunology ◽

10.3389/fimmu.2021.628364 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qian Zhang ◽

Xin Song ◽

Ping Ma ◽

Liping Lv ◽

Yangyang Zhang ◽

...

Keyword(s):

Viral Load ◽

Human Cytomegalovirus ◽

Immune Evasion ◽

Latent Infection ◽

Signal Transduction Pathway ◽

Luciferase Reporter ◽

Type I ◽

Immune Evasion Mechanism ◽

Reporter Assays

As the first line of antiviral defense, type I interferon (IFN) binds IFN receptor 1 (IFNAR1) and IFNAR2 to activate the Jak-STAT signal transduction pathway, producing IFN-stimulated genes (ISGs) to control viral infection. The mechanisms by which human cytomegalovirus (HCMV) counteracts the IFN pathway are only partially defined. We show that miR-US33as-5p encoded by HCMV is expressed in both lytic and latent infection. By analysis with RNA hybrid and screening with luciferase reporter assays, we identified IFNAR1 as a target of hcmv-miR-US33as-5p, which was further verified by examining the expression of two IFNAR1 mutants and the binding of IFNAR1 to miR-US33as-5p/miR-US33as-5p-M1/miR-US33as-5p-M2. We found that after the transfection of miR-US33as-5p mimics into different cell lines, the phosphorylation of downstream proteins and ISG expression were downregulated. Immunofluorescence showed that the miR-US33as-5p mimics also inhibited STAT1 translocation into the nucleus. Furthermore, we constructed HCMV with mutant miR-US33as-5p and determined that the mutation did not affect HCMV replication. We found that MRC-5/human foreskin fibroblast (HFF) cells infected with ΔmiRNA HCMV exhibited higher IFNAR1 and ISG expression and a reduced viral load in the presence of exogenous IFN than cells infected with WT HCMV did, confirming that the knockout of miR-US33as-5p impaired viral resistance to IFN. Finally, we tested the effect of ΔmiRNA HCMV on THP-1 and d-THP-1 cells, common in vitro models of latent infection and reactivation, respectively. Again, we found that cells infected with ΔmiRNA HCMV showed a reduced viral load in the presence of IFN than the control cells did, confirming that miR-US33as-5p also affects IFN resistance during both latency and reactivation. These results indicate a new microRNA (miRNA)-based immune evasion mechanism employed by HCMV to achieve lifelong infection.

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Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Blood ◽

10.1182/blood.v88.7.2510.bloodjournal8872510 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2510-2516 ◽

Cited By ~ 11

Author(s):

M Holberg-Petersen ◽

H Rollag ◽

S Beck ◽

I Overli ◽

G Tjonnfjord ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Clinical Isolate ◽

Human Cytomegalovirus ◽

Mononuclear Cells ◽

Colony Growth ◽

Structural Protein ◽

Hematopoietic Stem ◽

Cd34 Cells

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

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Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo

Blood ◽

10.1182/blood.2020010020 ◽

2021 ◽

Author(s):

Nikoletta Psatha ◽

Aphrodite Georgakopoulou ◽

Chang Li ◽

Vivek Nandakumar ◽

Grigorios Georgolopoulos ◽

...

Keyword(s):

Fetal Hemoglobin ◽

Therapeutic Window ◽

Pronounced Increase ◽

Human Red Blood Cells ◽

Transfusion Independence ◽

Cd34 Cells ◽

Proliferation And Differentiation ◽

Cis And Trans

Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) have an ameliorated clinical phenotype and in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites have been shown to significantly increase expression of endogenous fetal hemoglobin (HbF). To broaden the therapeutic window beyond a single editing approach, we have explored combinations of cis and trans editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of one trans and one cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0 thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe beta globin phenotype.

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