Changes In Cellular Gene Expression Research Articles

Abstract PO1-06-13: Plasma Exosomes in Obesity-driven Diabetes Exacerbate Progression of Triple Negative Breast Cancer: Insights from Animal Models

Abstract Objective: Our objective is to examine how obesity and type 2 diabetes (T2D) drive systemic and local metabolic changes in adipose tissue to promote progression of triple negative breast cancer (TNBC). We investigate the role of plasma exosomes as significant mediators of gene expression and functional changes, increasing TNBC progression and metastasis. We hypothesize that T2D and obesity alter the payload of plasma exosomes. These modified exosomes, specifically differentially expressed microRNAs in T2D, critically reshape the tumor microenvironment, ultimately promoting epithelial to mesenchymal transition (EMT) and distant metastasis of TNBC cells. Our goal is to gain a deeper understanding of the underlying mechanisms connecting T2D, obesity and TNBC metastasis, through the novel factor of exosome crosstalk. Our findings shed light on novel therapeutic targets and strategies to mitigate the impact of metabolic disorders on cancer outcomes. Methods: We subjected C57BL/6J female mice to high-fat diet (HFD; 60% Kcal fat) for 12 weeks with weight monitoring, whereupon oral glucose tolerance test confirmed glucose intolerance and insulin resistance. Plasma exosomes were then isolated from peripheral blood and used to treat E0771 cells, as a TNBC model, for 72 hours for in vitro and in vivo analysis. To assess gene expression changes, we employed a commercial EMT array kit and performed qPCR. Gene expression data were uploaded to Qiagen software, specifically the Ingenuity Pathway Analysis tool, for analysis. To evaluate cell migration as a functional test of aggressiveness, a migration assay was conducted using 24-hour FBS deprivation, followed by 24-hour migration, utilizing transwell plates with 8μm pores. For in vivo analysis, mice were injected with 50k E0771-GFP cells in the 4th mammary fat pad. Tumors were allowed to grow for 28 days, at which time histology and flow cytometry analyses were performed with harvested brain, lung, and spleen. Results: In-vitro analysis showed that HFD-derived exosomes reprogram gene expression in E0771 cells, driving a strong EMT signature, compared to LFD control or exosome-free negative control. This reprogramming elicited a pro-metastatic phenotype and upregulated signaling pathways associated with metastasis. Furthermore, HFD exosomes upregulated PD-L1 receptor expression compared to negative controls, linking EMT to immune tolerance. In-vivo analysis confirmed these findings, demonstrating increased metastatic burden in both lung and brain in the HFD context. Conclusions: Plasma exosomes derived from HFD fed mice dramatically reprogram EMT gene networks in E0771 cells, exerting genomic alterations that persist for at least 28 days. These induced changes in cellular gene expression likely promote the development of brain metastases and immune evasion. The model suggests that TNBC patients with obesity-driven diabetes should be evaluated for increased risk of distant metastases. Plasma exosomes miRNA profiling may be a valuable biomarker in cancer disparities populations where the prevalence of obesity and diabetes are high. Citation Format: Pablo Llevenes. Plasma Exosomes in Obesity-driven Diabetes Exacerbate Progression of Triple Negative Breast Cancer: Insights from Animal Models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-13.

Joint inference of clonal structure using single-cell genome and transcriptome sequencing data.

Latest advancements in the high-throughput single-cell genome (scDNA) and transcriptome (scRNA) sequencing technologies enabled cell-resolved investigation of tissue clones. However, it remains challenging to cluster and couple single cells for heterogeneous scRNA and scDNA data generated from the same specimen. In this study, we present a computational framework called CCNMF, which employs a novel Coupled-Clone Non-negative Matrix Factorization technique to jointly infer clonal structure for matched scDNA and scRNA data. CCNMF couples multi-omics single cells by linking copy number and gene expression profiles through their general concordance. It successfully resolved the underlying coexisting clones with high correlations between the clonal genome and transcriptome from the same specimen. We validated that CCNMF can achieve high accuracy and robustness using both simulated benchmarks and real-world applications, including an ovarian cancer cell lines mixture, a gastric cancer cell line, and a primary gastric cancer. In summary, CCNMF provides a powerful tool for integrating multi-omics single-cell data, enabling simultaneous resolution of genomic and transcriptomic clonal architecture. This computational framework facilitates the understanding of how cellular gene expression changes in conjunction with clonal genome alternations, shedding light on the cellular genomic difference of subclones that contributes to tumor evolution.

Transcriptomic forecasting with neural ordinary differential equations

Single-cell transcriptomics technologies can uncover changes in the molecular states that underlie cellular phenotypes. However, understanding the dynamic cellular processes requires extending from inferring trajectories from snapshots of cellular states to estimating temporal changes in cellular gene expression. To address this challenge, we have developed a neural ordinary differential-equation-based method, RNAForecaster, for predicting gene expression states in single cells for multiple future time steps in an embedding-independent manner. We demonstrate that RNAForecaster can accurately predict future expression states in simulated single-cell transcriptomic data with cellular tracking over time. We then show that by using metabolic labeling single-cell RNA sequencing (scRNA-seq) data from constitutively dividing cells, RNAForecaster accurately recapitulates many of the expected changes in gene expression during progression through the cell cycle over a 3-day period. Thus, RNAForecaster enables short-term estimation of future expression states in biological systems from high-throughput datasets with temporal information.

A6A-seq: N6-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing

The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics. The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs. Here we developed an a6A-seq method, which takes advantage of N6-allyladenosine (a6A) metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner. a6A plays a role as a chemical sequencing tag in that the iodination of a6A in mRNAs results in 1,N6-cyclized adenosine (cyc-A), which induces base misincorporation during RNA reverse transcription, thus making a6A-labelled mRNAs detectable by sequencing. A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine. a6A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition. Compared with regular RNA-seq, a6A-seq could more sensitively detect the change of mRNA production over a time scale. The experiment of a6A-containing mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a6A-seq data. Together, our results show a6A-seq is an effective tool to study RNA dynamics.

Subviral Dense Bodies of Human Cytomegalovirus Induce an Antiviral Type I Interferon Response.

(1) Background: Cells infected with the human cytomegalovirus (HCMV) produce subviral particles, termed dense bodies (DBs), both in-vitro and in-vivo. They are released from cells, comparable to infectious virions, and are enclosed by a membrane that resembles the viral envelope and mediates the entry into cells. To date, little is known about how the DB uptake influences the gene expression in target cells. The purpose of this study was to investigate the impact of DBs on cells, in the absence of a viral infection. (2) Methods: Mass spectrometry, immunoblot analyses, siRNA knockdown, and a CRISPR-CAS9 knockout, were used to investigate the changes in cellular gene expression following a DB exposure; (3) Results: A number of interferon-regulated genes (IRGs) were upregulated after the fibroblasts and endothelial cells were exposed to DBs. This upregulation was dependent on the DB entry and mediated by the type I interferon signaling through the JAK-STAT pathway. The induction of IRGs was mediated by the sensing of the DB-introduced DNA by the pattern recognition receptor cGAS. (4) Conclusions: The induction of a strong type I IFN response by DBs is a unique feature of the HCMV infection. The release of DBs may serve as a danger signal and concomitantly contribute to the induction of a strong, antiviral immune response.

Cat8 Response to Nutritional Changes and Interaction With Ehrlich Pathway Related Factors.

Cat8 is an important transcription factor regulating the utilization of non-fermentative carbon sources in Saccharomyces cerevisiae. However, our previous studies found that Cat8 may play a critical role in nitrogen metabolism, but the regulatory mechanism has not been elucidated. In this study, the nuclear localization and analysis of regulatory activity showed that the Cat8 function relies on Snf1 kinase. In the fermentation with glucose or glycerol as carbon sources under phenylalanine (Phe) induction, by comparing the changes of cellular gene expression and Cat8 target gene binding profiles after Cat8 overexpression, enhanced transcription was shown among key genes involved in the Ehrlich pathway (e.g., ARO9, ARO10, and ADH2) and its upstream and downstream related factors (e.g., GAP1, AGP1, GAT1, PDR12, and ESPB6), indicating that Cat8 participated in the regulation of nitrogen metabolism. Moreover, highly active Cat8 interacts with transcriptional activator Aro80 and GATA activator Gat1 coordinately to regulate the transcription of ARO10. Altogether, our results showed that Cat8 may act as a global transcription factor in response to nutritional changes, regulating both carbon and nitrogen utilization. This provides a new insight for us to explore the regulation of cell nutrient metabolism networks in yeast.

Network inference with Granger causality ensembles on single-cell transcriptomics.

Cellular gene expression changes throughout a dynamic biological process, such as differentiation. Pseudotimes estimate cells' progress along a dynamic process based on their individual gene expression states. Ordering the expression data by pseudotime provides information about the underlying regulator-gene interactions. Because the pseudotime distribution is not uniform, many standard mathematical methods are inapplicable for analyzing the ordered gene expression states. Here we present single-cell inference of networks using Granger ensembles (SINGE), an algorithm for gene regulatory network inference from ordered single-cell gene expression data. SINGE uses kernel-based Granger causality regression to smooth irregular pseudotimes and missing expression values. It aggregates predictions from an ensemble of regression analyses to compile a ranked list of candidate interactions between transcriptional regulators and target genes. In two mouse embryonic stem cell differentiation datasets, SINGE outperforms other contemporary algorithms. However, a more detailed examination reveals caveats about poor performance for individual regulators and uninformative pseudotimes.

Multi-omic evaluation of metabolic alterations in multiple sclerosis identifies shifts in aromatic amino acid metabolism

SummaryThe circulating metabolome provides unique insights into multiple sclerosis (MS) pathophysiology, but existing studies are relatively small or characterized limited metabolites. We test for differences in the metabolome between people with MS (PwMS; n = 637 samples) and healthy controls (HC; n = 317 samples) and assess the association between metabolomic profiles and disability in PwMS. We then assess whether metabolic differences correlate with changes in cellular gene expression using publicly available scRNA-seq data and whether identified metabolites affect human immune cell function. In PwMS, we identify striking abnormalities in aromatic amino acid (AAA) metabolites (p = 2.77E−18) that are also strongly associated with disability (p = 1.01E−4). Analysis of scRNA-seq data demonstrates altered AAA metabolism in CSF and blood-derived monocyte cell populations in PwMS. Treatment with AAA-derived metabolites in vitro alters monocytic endocytosis and pro-inflammatory cytokine production. We identify shifts in AAA metabolism resulting in the reduced production of immunomodulatory metabolites and increased production of metabotoxins in PwMS.

Influence of the MUC1 Cell Surface Mucin on Gastric Mucosal Gene Expression Profiles in Response to Helicobacter pylori Infection in Mice.

The cell surface mucin MUC1 is an important host factor limiting Helicobacter pylori (H. pylori) pathogenesis in both humans and mice by providing a protective barrier and modulating mucosal epithelial and leukocyte responses. The aim of this study was to establish the time-course of molecular events in MUC1-modulated gene expression profiles in response to H. pylori infection in wild type (WT) and MUC1-deficient mice using microarray-determined mRNA expression, gene network analysis and Ingenuity Pathway Analysis (IPA). A time-course over the first 72 h of infection showed significantly higher mucosal loads of bacteria at 8 h of infection in Muc1−/− mice compared with WT, confirming its importance in the early stages of infection (P = 0.0003). Microarray analysis revealed 266 differentially expressed genes at one or more time-points over 72 h in the gastric mucosa of Muc1−/− mice compared with WT control using a threshold of 2-fold change. The SPINK1 pancreatic cancer canonical pathway was strongly inhibited in Muc1−/− mice compared with WT at sham and 8 h infection (P = 6.08E-14 and P = 2.25 E-19, respectively) but potently activated at 24 and 72 h post-infection (P = 1.38E-22 and P = 5.87E-13, respectively). The changes in this pathway are reflective of higher expression of genes mediating digestion and absorption of lipids, carbohydrates, and proteins at sham and 8 h infection in the absence of MUC1, but that this transcriptional signature is highly down regulated as infection progresses in the absence of MUC1. Uninfected Muc1−/− gastric tissue was highly enriched for expression of factors involved in lipid metabolism and 8 h infection further activated this network compared with WT. As infection progressed, a network of antimicrobial and anti-inflammatory response genes was more highly activated in Muc1−/− than WT mice. Key target genes identified by time-course microarrays were independently validated using RT-qPCR. These results highlight the dynamic interplay between the host and H. pylori, and the role of MUC1 in host defense, and provide a general picture of changes in cellular gene expression modulated by MUC1 in a time-dependent manner in response to H. pylori infection.

Aristotle: A single blood test for pan-cancer screening.

e15037 Background: Blood participates in physiological and pathological changes throughout the body which are reflected as changes in cellular gene expression. The involvement of the immune system in cancer suppression and promotion through immunoediting exemplifies this. These characteristics make blood a promising non-invasive surrogate tissue for cancer diagnosis and monitoring. Methods: A multi-class cancer classification model, Aristotle, was developed with logistic regression using Affymetrix gene expression profiles generated from 2,845 unique human whole peripheral blood samples. The model was evaluated through 100 iterations of 2-fold cross-validation. Results: The 1,013 cancer patient samples (age 61±13, 40.6% ♀), include cancer of the bladder (n = 87, age 65±11, 23.3% ♀), breast (n = 94, age 54±10, 98.4% ♀), colon (n = 205, age 64±13, 39.5% ♀)/colon polyps (n = 226, age 61±11, 36.0% ♀), cervix (n = 36, age 55±13), endometrium (n = 25, age 49±12), liver (n = 16, age 57±11, 12.5% ♀), nasopharynx (n = 78, age 51±13, 26.9% ♀), ovaries (n = 64, age 55±12), prostate (n = 160, age 68±9) or stomach (n = 22, age 59±11, 28.6% ♀); and 1,832 control samples (age 53±16, 38.9% ♀), including healthy subjects (n = 1,042, age 55±16, 37.0 ♀) and subjects with ankylosing spondylitis (n = 59, age 39±12, 18.6% ♀), heart failure (n = 193, age 67±12, 40.0% ♀), inflammatory bowel disease (n = 51, age 37±9, 40.0% ♀), osteoarthritis (n = 248, age 53±15, 45.6% ♀), rheumatoid arthritis (n = 40, age 57±17, 84.6% ♀), schizophrenia (n = 110, age 36±13, 43.6% ♀) or other (n = 89, age 50±14, 41.1% ♀). Aristotle classified cancer profiles with sensitivities from 55.6-100% and positive predictive values from 5.6-77.7% at a specificity of 99.0% (Table). The mean false positive rate for the 11 cancer classes ranged from 0.3- 6.8%. Conclusions: Our study demonstrates the clinical viability of using whole peripheral blood for multi-class cancer classification. Aristotle was capable of classifying patients with different types of cancer with clinically relevant accuracy. We are confident that Aristotle holds great promise to help address the need for practical and non-invasive early-stage cancer diagnostics. [Table: see text]

White spot syndrome viral protein VP9 alters the cellular higher-order chromatin structure.

Viral protein 9 (VP9) is a non‐structural protein of white spot syndrome virus (WSSV) highly expressed during the early stage of infection. The crystal structure of VP9 suggests that the polymers of VP9 dimers resemble a DNA mimic, but its function remains elusive. In this study, we demonstrated that VP9 impedes histones binding to DNA via single‐molecule manipulation. We established VP9 expression in HeLa cells due to the lack of a WSSV‐susceptible cell line, and observed abundant VP9 in the nucleus, which mirrors its distribution in the hemocytes of WSSV‐infected shrimp. VP9 expression increased the dynamics and rotational mobility of histones in stable H3‐GFP HeLa cells as revealed by fluorescent recovery after photobleaching and fluorescence anisotropy imaging, which suggested a loosened compaction of chromatin structure. Successive salt fractionation showed that a prominent population of histones was solubilized in high salt concentrations, which implies alterations of bulk chromatin structure. Southern blotting identified that VP9 alters juxtacentromeric chromatin structures to be more accessible to micrococcal nuclease digestion. RNA microarray revealed that VP9 expression also leads to significant changes of cellular gene expression. Our findings provide evidence that VP9 alters the cellular higher‐order chromatin structure, uncovering a potential strategy adopted by WSSV to facilitate its replication.

NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

BackgroundAdenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear.MethodsThe dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding.ResultsPMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells.ConclusionsOur data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

Cellular Genes Involved in Redox Regulation Are Altered by Inhibitors of Epstein‐Barr Virus Lytic Gene Expression

Epstein‐Barr virus (EBV), a member of the herpesvirus family, causes infectious mononucleosis and is associated with human cancers, such as Burkitt and Hodgkin lymphomas, gastric carcinoma, and nasopharyngeal carcinoma. EBV establishes a latent infection in B cells, but must be reactivated into the viral lytic cycle to produce more virions and spread the infection among cells and hosts. The viral lytic cascade is initiated by the expression of two viral genes BZLF1 and BRLF1. The transcription of these viral immediate‐early genes is highly regulated. Reactivation of the viral lytic cycle is triggered by a variety of chemical and environmental stimuli. Chemical lytic inducing agents must alter cellular signaling pathways and transcription factors that control viral lytic gene expression. To discover cellular genes involved in viral reactivation, EBV‐positive cells were treated with activators or inhibitors of the viral lytic cycle. Changes in cellular gene expression were measured by next‐generation RNA sequencing (RNA‐seq). Transcriptome analysis identified large changes in expression of cellular genes involved in the redox status of the cell. Future work will connect these cellular pathways to the control of viral lytic gene expression.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling.

Background: Hepatitis C virus (HCV) infects human liver hepatocytes, often leading to liver cirrhosis and hepatocellular carcinoma (HCC). It is believed that chronic infection alters host gene expression and favors HCC development. In particular, HCV replication in Endoplasmic Reticulum (ER) derived membranes induces chronic ER stress. How HCV replication affects host mRNA translation and transcription at a genome wide level is not yet known. Methods: We used Riboseq (Ribosome Profiling) to analyze transcriptome and translatome changes in the Huh-7.5 hepatocarcinoma cell line replicating HCV for 6 days. Results: Established viral replication does not cause global changes in host gene expression—only around 30 genes are significantly differentially expressed. Upregulated genes are related to ER stress and HCV replication, and several regulated genes are known to be involved in HCC development. Some mRNAs (PPP1R15A/GADD34, DDIT3/CHOP, and TRIB3) may be subject to upstream open reading frame (uORF) mediated translation control. Transcriptional downregulation mainly affects mitochondrial respiratory chain complex core subunit genes. Conclusion: After establishing HCV replication, the lack of global changes in cellular gene expression indicates an adaptation to chronic infection, while the downregulation of mitochondrial respiratory chain genes indicates how a virus may further contribute to cancer cell-like metabolic reprogramming (“Warburg effect”) even in the hepatocellular carcinoma cells used here.

Single cell sequencing to identify temporal and cellular changes in gene expression post ischemic injury

Single cell sequencing to identify temporal and cellular changes in gene expression post ischemic injury

Proteome analysis of differential protein expression in porcine alveolar macrophages regulated by porcine reproductive and respiratory syndrome virus nsp1β protein.

Porcine reproductive and respiratory syndrome virus (PRRSV), an acute infectious disease agent in swine, causes enormous economic losses to the global swine industry. PRRSV nonstructural protein 1β (nsp1β) plays a critical role in viral subgenomic mRNA synthesis and host immune regulation. However, the global changes of cellular gene expression in natural target cells regulated by the nsp1β have not yet been identified. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry were used to quantitatively identify cellular proteins in porcine alveolar macrophage (PAM) 3D4/21 cells transduced with recombinant lentivirus expressing PRRSV nsp1β that are differentially expressed compared with PAM 3D4/21 cells transduced with recombinant lentivirus expressing GFP. Of the 425 cellular proteins detected as differentially expressed, 186 were upregulated and 239 were downregulated. Based on the identities of the differentially expressed cellular proteins and the essential role of nsp1β in interferon (IFN) activation and inflammatory factor antagonism during PRRSV infection, we propose a potential mechanism in which nsp1β inhibits IFN induction and nuclear factor κB (NF-κB) signaling pathways. Our results suggest that mitochondrial antiviral signaling (MAVS) protein and translocases of outer membrane complex 70 (TOM70), involved in type I IFN induction, were downregulated, while protein phosphatase 1A (PPM1A), related to the inhibition of NF-κB pathway activation, was upregulated in nsp1β-overexpressed PAM 3D4/21 cells. These data provide valuable information for better understanding the potential biological function of nsp1β during PRRSV infection and the mechanism of virus escape from host immune surveillance of viral replication.

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.

Expression and replication of virus-like circular DNA in human cells

The consumption of bovine milk and meat is considered a risk factor for colon- and breast cancer formation, and milk consumption has also been implicated in an increased risk for developing Multiple Sclerosis (MS). A number of highly related virus-like DNAs have been recently isolated from bovine milk and sera and from a brain sample of a MS patient. As a genetic activity of these Acinetobacter-related bovine milk and meat factors (BMMFs) is unknown in eukaryotes, we analyzed their expression and replication potential in human HEK293TT cells. While all analyzed BMMFs show transcriptional activity, the MS brain isolate MSBI1.176, sharing homology with a transmissible spongiform encephalopathy-associated DNA molecule, is transcribed at highest levels. We show expression of a replication-associated protein (Rep), which is highly conserved among all BMMFs, and serological tests indicate a human anti-Rep immune response. While the cow milk isolate CMI1.252 is replication-competent in HEK293TT cells, replication of MSBI1.176 is complemented by CMI1.252, pointing at an interplay during the establishment of persistence in human cells. Transcriptome profiling upon BMMF expression identified host cellular gene expression changes related to cell cycle progression and cell viability control, indicating potential pathways for a pathogenic involvement of BMMFs.

Stage-specific follicular extracellular vesicle uptake and regulation of bovine granulosa cell proliferation.

Follicular fluid within ovarian antral follicles contains numerous factors, which influence the development of a healthy oocyte including nucleic acids, steroids, proteins, and extracellular vesicles (EVs). Current evidence indicates that follicular EVs promote changes in cellular gene expression and support cumulus-oocyte complex expansion in vitro. In this study, we found EVs from different sized follicles differentially stimulate granulosa cell proliferation and this could be explained by both the differential contents associated, on or within the vesicles and by the preferential uptake of EVs dependent on follicle size from which they were isolated. Antibody array and inhibitor studies indicated that the Src, PI3K/Akt, and MAPK signaling pathways mediate the stimulatory effects of EVs on granulosa cell proliferation. This study demonstrates for the first time that EVs isolated from follicular fluid are capable of stimulating granulosa cell proliferation and that this stimulatory response is associated with the size of antral follicle from which the EVs originated. The study further also provides the first evidence that vesicles released by small antral follicles are preferentially taken up when compared to those isolated from large follicles, suggesting that vesicular surface proteins change during follicular maturation.

Viral Ubiquitin Ligase Stimulates Selective Host MicroRNA Expression by Targeting ZEB Transcriptional Repressors.

Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/δ-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.