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Journal of Virology Nov 2022Herpes simplex virus 1 (HSV-1) utilizes cellular RNA polymerase II (Pol) to transcribe its genes in one of two phases. In the latent phase, viral transcription is highly...
Herpes simplex virus 1 (HSV-1) utilizes cellular RNA polymerase II (Pol) to transcribe its genes in one of two phases. In the latent phase, viral transcription is highly restricted, but during the productive lytic phase, more than 80 genes are expressed in a temporally coordinated cascade. In this study, we used Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to characterize early viral transcriptional events using HSV-1 immediate early (IE) gene mutants, corresponding genetically repaired viruses, and wild-type virus. Unexpectedly, in the absence of the IE genes ICP4, ICP22, and ICP0 at 1.5 hours postinfection (hpi), we observed high levels of aberrant transcriptional activity across the mutant viral genomes but substantially less on either wild-type or the congenic repaired virus genomes. This feature was particularly prominent in the absence of ICP4 expression. Cycloheximide treatment during infection with both the ICP4 and ICP22 mutants and their respective genetic repairs did not alter the relative distribution of Pol activity, but it increased overall activity across both viral genomes, indicating that both virion components and at least some protein synthesis were required for full repression. Overall, these data reveal that prior to their role in transcriptional activation, IE gene products and virion components first repress transcription and that the HSV-1 lytic transcriptional cascade is mediated through subsequent derepression steps. HSV-1 transcription during productive replication is believed to comprise a series of activation steps leading to a specific sequence of gene expression. Here, we show that virion components and IE gene products ICP0, ICP4, and ICP22 first repress viral gene transcription to various degrees before subsequently activating specific gene subsets. It follows that the entire HSV transcriptional program involves a series of steps to sequentially reverse this repression. This previously uncharacterized repressive activity of IE genes very early in infection may represent an important checkpoint allowing HSV-1 to orchestrate either the robust lytic transcriptional cascade or the more restricted transcriptional program during latency.
Topics: Animals; Humans; Chlorocebus aethiops; Gene Expression Regulation, Viral; Herpes Simplex; Herpesvirus 1, Human; Immediate-Early Proteins; Ubiquitin-Protein Ligases; Vero Cells; Viral Transcription; Virus Replication
PubMed: 36300939
DOI: 10.1128/jvi.01416-22 -
Frontiers in Cell and Developmental... 2020Mammalian Mediator (Med) is a key regulator of gene expression by linking transcription factors to RNA polymerase II (Pol II) transcription machineries. The Mediator...
Mammalian Mediator (Med) is a key regulator of gene expression by linking transcription factors to RNA polymerase II (Pol II) transcription machineries. The Mediator subunit 23 (Med23) is a member of the conserved Med protein complex and plays essential roles in diverse biological processes including adipogenesis, carcinogenesis, osteoblast differentiation, and T-cell activation. However, its potential functions in the nervous system remain unknown. We report here that Med23 is required for adult hippocampal neurogenesis in mouse. Deletion of Med23 in adult hippocampal neural stem cells (NSCs) was achieved in Nestin-Cre:Med23 mice by oral administration of tamoxifen. We found an increased number of proliferating NSCs shown by pulse BrdU-labeling and immunostaining of MCM2 and Ki67, which is possibly due to a reduction in cell cycle length, with unchanged GFAP/Sox2 NSCs and Tbr2 progenitors. On the other hand, neuroblasts and immature neurons indicated by NeuroD and DCX were decreased in number in the dentate gyrus (DG) of Med23-deficient mice. In addition, these mice also displayed defective dendritic morphogenesis, as well as a deficiency in spatial and contextual fear memory. Gene ontology (GO) analysis of hippocampal NSCs revealed an enrichment in genes involved in cell proliferation, Pol II-associated transcription, Notch signaling pathway and apoptosis. These results demonstrate that Med23 plays roles in regulating adult brain neurogenesis and functions.
PubMed: 32850819
DOI: 10.3389/fcell.2020.00699 -
Journal, Genetic Engineering &... Dec 2022The spread of HIV is on the rise and has become a global issue, especially for underdeveloped and developing countries. This is due to the fact that HIV majorly occurs... (Review)
Review
The spread of HIV is on the rise and has become a global issue, especially for underdeveloped and developing countries. This is due to the fact that HIV majorly occurs asymptomatically and is implausible for early diagnosis. Recent advances in research and science have enabled the investigation of a new potential treatment involving gene-based therapy, known as RNA interference (RNAi) that will direct gene silencing and further compensate for natural variants and viral mutants. Several types of small regulatory RNA are discussed in this present study, including microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA).This paper examines the mechanism of RNAi as a viable HIV therapy, using a minimum of four shRNAs to target both dispensable host components (CCR5) and viral genes (Gag, Env, Tat, Pol I, Pol II and Vif). Moreover, a multiplexed mechanism of shRNAs and miRNA is known to be effective in preventing viral escape due to mutation as the miRNA develops a general polycistronic platform for the expression of a large amount of shRNA-miRs. Several administration methods as well as the advantages of this RNAi treatment are also discussed in this study. The administration methods include (1) ex vivo delivery with the help of viral vectors, nanoparticles, and electroporation, (2) nonspecific in vivo delivery using non-viral carriers including liposomes, dendrimers and aptamers, as well as (3) targeted delivery that uses antibodies, modified nanoparticles, nucleic acid aptamers, and tissue-specific serotypes of AAV. Moreover, the advantages of this treatment are related to the effectiveness in silencing the HIV gene, which is more compatible compared to other gene therapy treatments, such as ZFN, TALEN, and CRISPR/Cas9.
PubMed: 36576612
DOI: 10.1186/s43141-022-00451-z -
Medicine Jul 2021In recent years, with the development of molecular epidemiology, molecular transmission networks based on evolutionary theory and sequence analysis have been widely used...
INTRODUCTION
In recent years, with the development of molecular epidemiology, molecular transmission networks based on evolutionary theory and sequence analysis have been widely used in research on human immunodeficiency virus (HIV)-1 transmission dynamics and precise intervention for high-risk populations. The HIV-1 molecular transmission network is a new method to study the population's access to the network, the characteristics of clustering, and the characteristics of interconnection in the network. Here, we analyzed the characteristics of the HIV-1 molecular transmission network of sexually transmitted people in Liaoning Province.
METHODS
A study of HIV-infected persons who were sexually transmitted in Liaoning Province from 2003 to 2019. HIV-1 RNA was extracted, amplified and sequenced, and a phylogenetic tree was constructed to determine the subtype using the well matched pol gene region sequence. The gene distance between sequences was calculated, the threshold was determined, and the molecular transmission network was constructed.
RESULTS
109 samples of pol gene region were obtained. The main subtype of HIV-1 was CRF01_AE, followed by B, CRF07_BC, etc. 12.8% of them were resistant to HIV. At the threshold of 0.55 gene distance, 60.6% of them entered the HIV-1 molecular transmission network. Workers, sample source voluntary counseling and testing, other testing, subtype B and drug resistance are the factors influencing the access to HIV-1 molecular transmission network. The subtype of CRF01_AE formed 6 clusters in the molecular transmission network. In the network, the difference of connection degree between different subtypes was statistically significant.
DISCUSSION
The three subtypes CRF01_AE, CRF07_BC and B that enter the molecular transmission network do not have interconnections, and they form clusters with each other. It shows that the risk of transmission among the three subtypes is less than the risk of transmission within each subtype. The factors affecting HIV-1 entry into the molecular transmission network were occupation, sample source, genotype and drug resistance. The L33F mutation at the HIV-1 resistance mutation site constitutes the interconnection in the largest transmission cluster in the network. The epidemiological characteristics of HIV-infected persons in each molecular transmission cluster show that 97% of the study subjects come from the same area and have a certain spatial aggregation.
CONCLUSION
Constructing a molecular transmission network and conducting long-term monitoring, while taking targeted measures to block the spread of HIV can achieve precise prevention and control.
Topics: Adult; China; Female; Genotype; HIV Infections; HIV-1; Health Behavior; Humans; Male; Middle Aged; Molecular Epidemiology; RNA, Viral; Sequence Analysis, DNA; Sexually Transmitted Diseases, Viral; Socioeconomic Factors
PubMed: 34260561
DOI: 10.1097/MD.0000000000026640 -
Scientific Reports Mar 2020To investigate the genetic diversity, spatiotemporal dynamics, and transmission networks of HIV-1 CRF55_01B epidemic in China. A total of 209 partial pol gene sequences...
To investigate the genetic diversity, spatiotemporal dynamics, and transmission networks of HIV-1 CRF55_01B epidemic in China. A total of 209 partial pol gene sequences of HIV-1 CRF55_01B were sampled during 2007-2015 from 7 provinces of China. Phylogenetic analyses and trait diffusion process of these sequences were performed using Bayesian methods. Distance-based molecular network analyses were performed to infer putative relationships. Characteristics of genetically linked individuals were analyzed. Our study identified that HIV-1 CRF55_01B likely originated among men who have sex with men (MSM) in Guangdong province in January 2003 (April 2000-April 2005), and that Guangdong province and MSM are major hubs for the spread of the HIV-1 CRF55_01B epidemic in China. A Bayesian Skygrid plot revealed that the effective population size of HIV-1 CRF55_01B experienced increased phase followed by a plateau. All sequences from persons of unknown risk clustered within groups who reported MSM risk. This could be because Chinese MSM may not report such risk due to HIV/AIDS-related stigmatization and discrimination. This study inferred the transmission dynamics of the HIV-1 CRF55_01B epidemic in China at high resolution. The methods developed in this study may be critical for designing effective HIV prevention strategies in China and beyond.
Topics: China; Female; Genetic Linkage; Genome, Viral; HIV Infections; HIV-1; Homosexuality, Male; Humans; Male; Phylogeny; pol Gene Products, Human Immunodeficiency Virus
PubMed: 32198405
DOI: 10.1038/s41598-020-61870-x -
Viruses May 2022Interferon is a marker of host antiviral immunity, which is disordered in COVID-19 patients. ERV can affect the secretion of interferon through the cGAS-STING pathway....
Interferon is a marker of host antiviral immunity, which is disordered in COVID-19 patients. ERV can affect the secretion of interferon through the cGAS-STING pathway. In this study, we explored whether IFN-I and HERV-K (HML-2) were activated in COVID-19 patients and whether there was an interaction between them. We collected blood samples from COVID-19 patients and healthy controls. We first detected the expression of HERV-K (HML-2) , , and genes and IFN-I-related genes between patients and healthy people by qPCR, synchronously detected VERO cells infected with SARS-CoV-2. Then, the chromosome distributions of highly expressed HERV-K (HML-2) , , and genes were mapped by the next-generation sequencing results, and GO analysis was performed on the related genes. We found that the HERV-K (HML-2) , , and genes were highly expressed in COVID-19 patients and VERO cells infected with SARS-CoV-2. The interferon-related genes , and were also activated in COVID-19 patients, and GO analysis showed that HERV-K (HML-2) can regulate the secretion of interferon. The high expression of HERV-K (HML-2) might activate the increase of interferon in COVID-19 patients, proving that HERV-K does not only play a negative role in the human body.
Topics: Animals; Antiviral Agents; COVID-19; Chlorocebus aethiops; Endogenous Retroviruses; Genes, Viral; Humans; Interferons; SARS-CoV-2; Vero Cells
PubMed: 35632738
DOI: 10.3390/v14050996 -
Cell & Bioscience Feb 2023Loss of the transcription factor GLI-Similar 3 (GLIS3) function causes congenital hypothyroidism (CH) in both humans and mice due to decreased expression of several...
BACKGROUND
Loss of the transcription factor GLI-Similar 3 (GLIS3) function causes congenital hypothyroidism (CH) in both humans and mice due to decreased expression of several thyroid hormone (TH) biosynthetic genes in thyroid follicular cells. Whether and to what extent, GLIS3 regulates thyroid gene transcription in coordination with other thyroid transcriptional factors (TFs), such as PAX8, NKX2.1 and FOXE1, is poorly understood.
METHODS
PAX8, NKX2.1, and FOXE1 ChIP-Seq analysis with mouse thyroid glands and rat thyrocyte PCCl3 cells was performed and compared to that of GLIS3 to analyze the co-regulation of gene transcription in thyroid follicular cells by these TFs.
RESULTS
Analysis of the PAX8, NKX2.1, and FOXE1 cistromes identified extensive overlaps between these TF binding loci and those of GLIS3 indicating that GLIS3 shares many of the same regulatory regions with PAX8, NKX2.1, and FOXE1, particularly in genes associated with TH biosynthesis, induced by thyroid stimulating hormone (TSH), and suppressed in Glis3KO thyroid glands, including Slc5a5 (Nis), Slc26a4, Cdh16, and Adm2. ChIP-QPCR analysis showed that loss of GLIS3 did not significantly affect PAX8 or NKX2.1 binding and did not cause major alterations in H3K4me3 and H3K27me3 epigenetic signals.
CONCLUSIONS
Our study indicates that GLIS3 regulates transcription of TH biosynthetic and TSH-inducible genes in thyroid follicular cells in coordination with PAX8, NKX2.1, and FOXE1 by binding within the same regulatory hub. GLIS3 does not cause major changes in chromatin structure at these common regulatory regions. GLIS3 may induce transcriptional activation by enhancing the interaction of these regulatory regions with other enhancers and/or RNA Polymerase II (Pol II) complexes.
PubMed: 36793061
DOI: 10.1186/s13578-023-00979-8 -
RNA (New York, N.Y.) Feb 2022Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to...
Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing embryo.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Introns; Polyadenylation; RNA Polymerase II; RNA Splicing; Transcription Termination, Genetic
PubMed: 34667107
DOI: 10.1261/rna.078933.121 -
AIDS Research and Human Retroviruses Jun 2022HIV-1 gene sequences were analyzed from 77 HIV-1 positive children infected perinatally and exhibiting virological failure (VF). Viral subtyping, phylogenetic analysis,...
HIV-1 gene sequences were analyzed from 77 HIV-1 positive children infected perinatally and exhibiting virological failure (VF). Viral subtyping, phylogenetic analysis, and genotypic drug resistance analysis were carried out on samples collected before start of anti retroviral treatment (ART) (baseline, BL), and at 12 months post-ART initiation (M12). Subtype C was found to be most predominant, seen in 75 of the 77 (97.4%) children. The level of pretreatment drug resistance (PDR) was 14% among these children. At BL, K103N (5), E138A/G (4), and M184V (3) were the most common mutations. At M12 the prevalence of any resistance-associated mutation (RAM) (acquired drug resistance/ADR) was 81.8% (63/77). Dual class resistance mutations were seen in 64% (49/77) of children. M184V/I, K103N/S, and Y181C were the most commonly occurring mutations, seen in 76%, 51%, and 36% children. RAMs to the second-generation non-nucleoside reverse transcriptase inhibitors (NNRTI), etravirine (ETR) and rilpivirine (RPV), were seen in 40.2% (31/77) and 48.05% (37/77) of the children, respectively. Our findings reveal similar prevalence rates of PDR and ADR in children with VF as reported in other studies. Occurrence of ETR and RPV resistance associated mutations (RAMs) is of concern and highlights the need for timely switch of regimens guided by genotypic resistance testing in perinatally infected children from India.
Topics: Anti-HIV Agents; Child; Drug Resistance, Viral; Genes, pol; Genotype; HIV Infections; HIV Seropositivity; HIV-1; Humans; Infectious Disease Transmission, Vertical; Mutation; Phylogeny; Pyridazines; Reverse Transcriptase Inhibitors; Rilpivirine
PubMed: 35302390
DOI: 10.1089/AID.2021.0227 -
Nucleic Acids Research Jun 2022ARMC5 is implicated in several pathological conditions, but its function remains unknown. We have previously identified CUL3 and RPB1 (the largest subunit of RNA...
ARMC5 is implicated in several pathological conditions, but its function remains unknown. We have previously identified CUL3 and RPB1 (the largest subunit of RNA polymerase II (Pol II) as potential ARMC5-interacting proteins. Here, we show that ARMC5, CUL3 and RBX1 form an active E3 ligase complex specific for RPB1. ARMC5, CUL3, and RBX1 formed an active E3 specific for RPB1. Armc5 deletion caused a significant reduction in RPB1 ubiquitination and an increase in an accumulation of RPB1, and hence an enlarged Pol II pool in normal tissues and organs. The compromised RPB1 degradation did not cause generalized Pol II stalling nor depressed transcription in the adrenal glands but did result in dysregulation of a subset of genes, with most upregulated. We found RPB1 to be highly expressed in the adrenal nodules from patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) harboring germline ARMC5 mutations. Mutant ARMC5 had altered binding with RPB1. In summary, we discovered that wildtype ARMC5 was part of a novel RPB1-specific E3. ARMC5 mutations resulted in an enlarged Pol II pool, which dysregulated a subset of effector genes. Such an enlarged Pol II pool and gene dysregulation was correlated to adrenal hyperplasia in humans and KO mice.
Topics: Adrenal Hyperplasia, Congenital; Animals; Armadillo Domain Proteins; DNA-Directed RNA Polymerases; Humans; Ligases; Mice; Mice, Knockout; RNA Polymerase II; Ubiquitin-Protein Ligases
PubMed: 35687106
DOI: 10.1093/nar/gkac483