Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection

Background Human cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection. Methods Viral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay. Results Abundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33. Conclusions Viral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency

Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte

Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease

PPAR Gamma and Viral Infections of the Brain

Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a master regulator of metabolism, adipogenesis, inflammation and cell cycle, and it has been extensively studied in the brain in relation to inflammation or neurodegeneration. Little is known however about its role in viral infections of the brain parenchyma, although they represent the most frequent cause of encephalitis and are a major threat for the developing brain. Specific to viral infections is the ability to subvert signaling pathways of the host cell to ensure virus replication and spreading, as deleterious as the consequences may be for the host. In this respect, the pleiotropic role of PPARγ makes it a critical target of infection. This review aims to provide an update on the role of PPARγ in viral infections of the brain. Recent studies have highlighted the involvement of PPARγ in brain or neural cells infected by immunodeficiency virus 1, Zika virus, or human cytomegalovirus. They have provided a better understanding on PPARγ functions in the infected brain, and revealed that it can be a double-edged sword with respect to inflammation, viral replication, or neuronogenesis. They unraveled new roles of PPARγ in health and disease and could possibly help designing new therapeutic strategies

Message in a Scaffold: Natural Biomaterials for Three-Dimensional (3D) Bioprinting of Human Brain Organoids

Brain organoids are invaluable tools for pathophysiological studies or drug screening, but there are still challenges to overcome in making them more reproducible and relevant. Recent advances in three-dimensional (3D) bioprinting of human neural organoids is an emerging approach that may overcome the limitations of self-organized organoids. It requires the development of optimal hydrogels, and a wealth of research has improved our knowledge about biomaterials both in terms of their intrinsic properties and their relevance on 3D culture of brain cells and tissue. Although biomaterials are rarely biologically neutral, few articles have reviewed their roles on neural cells. We here review the current knowledge on unmodified biomaterials amenable to support 3D bioprinting of neural organoids with a particular interest in their impact on cell homeostasis. Alginate is a particularly suitable bioink base for cell encapsulation. Gelatine is a valuable helper agent for 3D bioprinting due to its viscosity. Collagen, fibrin, hyaluronic acid and laminin provide biological support to adhesion, motility, differentiation or synaptogenesis and optimize the 3D culture of neural cells. Optimization of specialized hydrogels to direct differentiation of stem cells together with an increased resolution in phenotype analysis will further extend the spectrum of possible bioprinted brain disease models

Correction: Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection.

[This corrects the article DOI: 10.1371/journal.pone.0088531.]

Comparison of miRNA expression in HCMV infected HEL and THP-1 cells.

<p>Expression kinetics for four HCMV miRNAs representing the three expression pattern groups are shown side-by-side to compare expression patterns in HELs vs. THP-1s (data are from experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088531#pone-0088531-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088531#pone.0088531.s001" target="_blank">S1</a>). The compared representatives of group 1 (miR-112 and miR-70-3p), group 2 (miR-22A-5p), and group 3 (miR-US33-3p) are shown. Note that HELs and THP-1 cells were infected with HCMV strain Towne at MOIs of 5 and 10, respectively. *P<0.05, **P<0.01.</p

Impact of ectopic expression of miRNAs on HCMV replication.

<p>(A) HCMV miRNA levels were quantitated by stem-loop RT-PCR in HEL cells 48 h after transduction with empty vector lentivirus (control) or with lentiviruses engineered to express miR-UL22A, miR-UL70, miR-US33, or 48 hpi of HELs with HCMV strain Towne (MOI = 5). Results represent fold-differences for each miRNA relative to control. (B) Levels of IE1, IE2, UL44, or gB were determined by western blotting. Actin serves as a loading control. Control lentivirus (−) or lentiviruses expressing according miRNAs (+) are indicated. (C) HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33. At the times indicated post infection HCMV genome copy numbers were determined by qPCR. (D) HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33 Infectious virus titers in supernatants at 96 and 144 hpi were determined. Inhibition efficiencies are % changes in virus titers in medium from HCMV-infected miR-US33-transduced cells vs. control-transduced cells. **P<0.01, *P<0.05.</p

Down-regulation of HCMV US29 by miR-US33.

<p>(A) Complementarity of miR-US33 with its putative target sequence in <i>US29</i>; the miR-US33 “seed” sequence is underlined. (B) <i>US29</i> sequences are a target of miR-US33. A plasmid expressing luciferase from a transcript containing the putative miR-US33 target sequence from <i>US29</i> was co-transfected into 293 cells with a plasmid expressing miR-US33 or an empty vector control plasmid. Each transfection also included a β-gal-expressing plasmid to normalize transfection efficiencies. Luciferase and β-gal activities were measured 48 h post transfection and β-gal activities were used to normalize the luciferase activities. (C) Regulation of <i>US29</i> mRNA by miR-US33 during HCMV infection. HELs were infected with HCMV strain Towne at an MOI of 0.01 48 h after transduction with control lentivirus or lentivirus expressing miR-US33 and at the indicated hpi <i>US29</i> mRNA levels were quantitated by RT-PCR. **P<0.01.</p