Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell ...quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.
Peatland restoration has become a common land-use management practice in recent years, with the water table depth (WTD) being one of the key monitoring elements, where it is used as a proxy for ...various ecosystem functions. Regular, uninterrupted, and spatially representative WTD data in situ can be difficult to collect, and therefore, remotely sensed data offer an attractive alternative for landscape-scale monitoring. In this study, we illustrate the application of Sentinel-1 SAR backscatter for water table depth monitoring in near-natural and restored blanket bogs in the Flow Country of northern Scotland. Among the study sites, the near-natural peatlands presented the smallest fluctuations in the WTD (with depths typically between 0 and 15 cm) and had the most stable radar signal throughout the year (~3 to 4 dB amplitude). Previously drained and afforested peatlands undergoing restoration management were found to have higher WTD fluctuations (depths up to 35 cm), which were also reflected in higher shifts in the radar backscatter (up to a ~6 dB difference within a year). Sites where more advanced restoration methods have been applied, however, were associated with shallower water table depths and smoother surfaces. Three models—simple linear regression, multiple linear regression, and the random forest model—were evaluated for their potential to predict water table dynamics in peatlands using Sentinel-1 SAR backscatter. The random forest model was found to be the most suited, with the highest correlation scores, lowest RMSE values, and overall good temporal fit (R2 = 0.66, RMSE = 2.1 cm), and multiple linear regression came in a close second (R2 = 0.59, RMSE = 4.5 cm). The impact of standing water, terrain ruggedness, and the ridge and furrow aspect on the model correlation scores was tested but found not to have a statistically significant influence. We propose that this approach, using Sentinel-1 and random forest models to predict the WTD, has strong potential and should be tested in a wider range of peatland sites.
Small mammals, such as small rodents (Rodentia: Muroidea) and shrews (Insectivora: Soricidae), present particular challenges in camera trap surveys. Their size is often insufficient to trigger ...infra-red sensors, whilst resultant images may be of inadequate quality for species identification. The conventional survey method for small mammals, live-trapping, can be both labour-intensive and detrimental to animal welfare. Here, we describe a method for using camera traps for monitoring small mammals. We show that by attaching the camera trap to a baited tunnel, fixing a close-focus lens over the camera trap lens, and reducing the flash intensity, pictures or videos can be obtained of sufficient quality for identifying species. We demonstrate the use of the method by comparing occurrences of small mammals in a peatland landscape containing (i) plantation forestry (planted on drained former blanket bog), (ii) ex-forestry areas undergoing bog restoration, and (iii) unmodified blanket bog habitat. Rodents were detected only in forestry and restoration areas, whilst shrews were detected across all habitat. The odds of detecting small mammals were 7.6 times higher on camera traps set in plantation forestry than in unmodified bog, and 3.7 times higher on camera traps in restoration areas than in bog. When absolute abundance estimates are not required, and camera traps are available, this technique provides a low-cost survey method that is labour-efficient and has minimal animal welfare implications.
Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that ...myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.
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. The study of HCMV latency has been hampered by difficulties in obtaining and culturing primary cells, as well as an inability to quantitatively measure reactivating virus, but recent advances in both
and
models of HCMV latency and reactivation have led to a greater understanding of the interplay between host and virus. Key differences in established model systems have also led to controversy surrounding the role of viral gene products in latency establishment, maintenance, and reactivation. This review will discuss the details and challenges of various models including hematopoietic progenitor cells, monocytes, cell lines, and humanized mice. We highlight the utility and functional differences between these models and the necessary experimental design required to define latency and reactivation, which will help to generate a more complete picture of HCMV infection of myeloid-lineage cells.
Human cytomegalovirus (HCMV) microRNAs play essential roles in latency and reactivation in CD34
hematopoietic progenitor cells (HPCs) via regulation of viral and cellular gene expression. In the ...present study, we show that HCMV miR-US25-1 targets RhoA, a small GTPase required for CD34
HPC self-renewal, proliferation, and hematopoiesis. Expression of miR-US25-1 impairs signaling through the nonmuscle myosin II light chain, which leads to a block in cytokinesis and an inhibition of proliferation. Moreover, infection with an HCMV mutant lacking miR-US25-1 resulted in increased proliferation of CD34
HPCs and a decrease in the proportion of genome-containing cells at the end of latency culture. These observations provide a mechanism by which HCMV limits proliferation to maintain latent viral genomes in CD34
HPCs.
Each herpesvirus family establishes latency in a unique cell type. Since herpesvirus genomes are maintained as episomes, the virus needs to devise mechanisms to retain the latent genome during cell division. Alphaherpesviruses overcome this obstacle by infecting nondividing neurons, while gammaherpesviruses tether their genome to the host chromosome in dividing B cells. The betaherpesvirus human cytomegalovirus (HCMV) establishes latency in CD34
hematopoietic progenitor cells (HPCs), but the mechanism used to maintain the viral genome is unknown. In this report, we demonstrate that HCMV miR-US25-1 downregulates expression of RhoA, a key cell cycle regulator, which results in inhibition of CD34
HPC proliferation by blocking mitosis. Mutation of miR-US25-1 during viral infection results in enhanced cellular proliferation and a decreased frequency of genome-containing CD34
HPCs. These results reveal a novel mechanism through which HCMV is able to regulate cell division to prevent viral genome loss during proliferation.
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.
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
and
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
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.
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 CX
CR1, 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.
Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is ...regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFβ interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.
Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. Several studies have shown that HCMV induces NF-κB ...signaling early in infection, resulting in the induction of antiviral proinflammatory cytokines with a subsequent reduction of these cytokines late in infection. The mechanism for late cytokine reduction is unknown. In this study, we show that HCMV microRNAs (miRNAs) miR-US5-1 and miR-UL112-3p target the IκB kinase (IKK) complex components IKKα and IKKβ to limit production of proinflammatory cytokines in response to interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α). Transfection of miR-UL112-3p and miR-US5-1 mimics reduced endogenous IKKα and IKKβ protein levels, and site-directed mutagenesis of the 3' untranslated regions (UTRs) identified the binding sites for each miRNA. Infection with mutant viruses lacking these miRNAs resulted in increased levels of IKKα and IKKβ proteins, an impaired ability to control NF-κB signaling at late times of lytic infection, and increased production of proinflammatory cytokines compared to wild-type virus in cell types relevant to HCMV infection
These phenotypes were rescued by preexpression of miR-US5-1 and miR-UL112-3p in infected cells or by a miR-US5-1/miR-UL112-3p double mutant virus that expresses short hairpin RNAs (shRNAs) targeting IKKα and IKKβ, demonstrating the gene specificity of the miRNAs. These observations describe a mechanism through which HCMV miRNAs expressed late in the infectious cycle downregulate proinflammatory cytokine production to create a cellular proviral environment.
Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients and causes hearing loss and mental retardation when acquired congenitally. Initial events during HCMV infection result in the activation of NF-κB signaling, which culminates in the production of IL-6, CCL5, and TNF-α. Several viruses have developed mechanisms to block the antiviral effects of these cytokines. We show here that two HCMV miRNAs, miR-US5-1 and miR-UL112-3p, specifically downregulate IKKα and IKKβ signaling factors necessary to propagate NF-κB signaling and subsequent IL-6, CCL5, and TNF-α production. Regulation of these proinflammatory cytokines during lytic infection and during latency is critical to viral survival in the host.
Introduction: Fashion and American Culture Sauro, Clare; Hancock, Joseph H.
Journal of American culture (Malden, Mass.),
March 2023, 2023-03-00, 20230301, Letnik:
46, Številka:
1
Journal Article
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