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Nature Communications Jun 2024Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and...
Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and transposons. Eukaryotic Argonautes are subdivided into two clades: AGOs generally facilitate miRNA- or siRNA-mediated silencing, while PIWIs generally facilitate piRNA-mediated silencing. It is currently unclear when and how Argonaute-based RNA silencing mechanisms arose and diverged during the emergence and early evolution of eukaryotes. Here, we show that in Asgard archaea, the closest prokaryotic relatives of eukaryotes, an evolutionary expansion of Argonaute proteins took place. In particular, a deep-branching PIWI protein (HrAgo1) encoded by the genome of the Lokiarchaeon 'Candidatus Harpocratesius repetitus' shares a common origin with eukaryotic PIWI proteins. Contrasting known prokaryotic Argonautes that use single-stranded DNA as guides and/or targets, HrAgo1 mediates RNA-guided RNA cleavage, and facilitates gene silencing when expressed in human cells and supplied with miRNA precursors. A cryo-EM structure of HrAgo1, combined with quantitative single-molecule experiments, reveals that the protein displays structural features and target-binding modes that are a mix of those of eukaryotic AGO and PIWI proteins. Thus, this deep-branching archaeal PIWI may have retained an ancestral molecular architecture that preceded the functional and mechanistic divergence of eukaryotic AGOs and PIWIs.
Topics: Argonaute Proteins; Humans; RNA Interference; Archaea; RNA, Small Interfering; Archaeal Proteins; Cryoelectron Microscopy; MicroRNAs; Evolution, Molecular; Phylogeny
PubMed: 38951509
DOI: 10.1038/s41467-024-49452-1 -
Nature Communications Jun 2024Like many other viruses, KSHV has two life cycle modes: the latent phase and the lytic phase. The RTA protein from KSHV is essential for lytic reactivation, but how this...
Like many other viruses, KSHV has two life cycle modes: the latent phase and the lytic phase. The RTA protein from KSHV is essential for lytic reactivation, but how this protein's activity is regulated is not fully understood. Here, we report that linear ubiquitination regulates the activity of RTA during KSHV lytic reactivation and de novo infection. Overexpressing OTULIN inhibits KSHV lytic reactivation, whereas knocking down OTULIN or overexpressing HOIP enhances it. Intriguingly, we found that RTA is linearly polyubiquitinated by HOIP at K516 and K518, and these modifications control the RTA's nuclear localization. OTULIN removes linear polyubiquitin chains from cytoplasmic RTA, preventing its nuclear import. The RTA orthologs encoded by the EB and MHV68 viruses are also linearly polyubiquitinated and regulated by OTULIN. Our study establishes that linear polyubiquitination plays a critically regulatory role in herpesvirus infection, adding virus infection to the list of biological processes known to be controlled by linear polyubiquitination.
Topics: Herpesvirus 8, Human; Ubiquitination; Humans; Virus Replication; Immediate-Early Proteins; HEK293 Cells; Trans-Activators; Ubiquitin-Protein Ligases; Virus Activation; Herpesviridae Infections; Cell Nucleus
PubMed: 38951495
DOI: 10.1038/s41467-024-49887-6 -
Molecular Metabolism Jun 2024Aberrant glucolipid metabolism in the heart is a characteristic factor in diabetic cardiomyopathy (DbCM). Super-enhancers-driven noncoding RNAs (seRNAs) are emerging as...
OBJECTIVE
Aberrant glucolipid metabolism in the heart is a characteristic factor in diabetic cardiomyopathy (DbCM). Super-enhancers-driven noncoding RNAs (seRNAs) are emerging as powerful regulators in the progression of cardiac diseases. However, the functions of seRNAs in DbCM have not been fully elucidated.
METHODS
Super enhancers and their associated seRNAs were screened and identified by H3K27ac ChIP-seq data in the Encyclopedia of DNA Elements (ENCODE) dataset. A dual-luciferase reporter assay was performed to analyze the function of super-enhancers on the transcription of peroxisome proliferator-activated receptor α-related seRNA (PPARα-seRNA). A DbCM mouse model was established using db/db leptin receptor-deficient mice. Adeno-associated virus serotype 9-seRNA (AAV9-seRNA) was injected via the tail vein to evaluate the role of seRNA in DbCM. The underlying mechanism was explored through RNA pull-down, RNA and chromatin immunoprecipitation, and chromatin isolation by RNA purification.
RESULTS
PPARα-seRNA was regulated by super-enhancers and its levels were increased in response to high glucose and palmitic acid stimulation in cardiomyocytes. Functionally, PPARα-seRNA overexpression aggravated lipid deposition, reduced glucose uptake, and repressed energy production. In contrast, PPARα-seRNA knockdown ameliorated metabolic disorder in vitro. In vivo, overexpression of PPARα-seRNA exacerbated cardiac metabolic disorder and deteriorated cardiac dysfunction, myocardial fibrosis, and hypertrophy in DbCM. Mechanistically, PPARα-seRNA bound to the histone demethylase KDM4B (Lysine-specific demethylase 4B) and decreased H3K9me3 levels in the promoter region of PPARα, ultimately enhancing its transcription.
CONCLUSIONS
Our study revealed the pivotal function of a super-enhancer-driven long noncoding RNA (lncRNA), PPARα-seRNA, in the deterioration of cardiac function and the exacerbation of metabolic abnormalities in diabetic cardiomyopathy, which recruited KDM4B to the promoter region of PPARα and repression of its transcription. This suggests a promising therapeutic strategy for the treatment of DbCM.
PubMed: 38950776
DOI: 10.1016/j.molmet.2024.101978 -
The Journal of Clinical Investigation May 2024In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated...
In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.
Topics: Animals; Female; Dependovirus; Mice; Pregnancy; Gene Editing; Humans; Immunoglobulin G; CRISPR-Associated Protein 9; Genetic Vectors; Maternal-Fetal Exchange; Antibodies, Viral; CRISPR-Cas Systems; Fetus; Immunity, Maternally-Acquired
PubMed: 38950310
DOI: 10.1172/JCI179848 -
BioRxiv : the Preprint Server For... Jun 2024Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a...
Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a variant of the type 1F Cascade known as type 1F-3. This Cascade variant complexes with a homodimer of the transposition protein TniQ and leverages the sequence specificity of Cascade to direct the integration activity of the heteromeric transposase tnsA/B, resulting in site-specific transposition of Tn6677. We desire to uncover the molecular details behind R Loop formation of 'Cascade-TniQ.' Due to the lack of a complete model of Cascade-TniQ available at atom-level resolution, we first build a complete model using AlphaFold V2.1. We then simulate this model via classical molecular dynamics and umbrella sampling to study an important regulatory component within Cascade-TniQ, known as the Cas8 'bundle.' Particularly, we show that this alpha helical bundle experiences a free energy barrier to its large-scale translatory motions and relative free energies of its states primarily dependent on a loop within a Cas7 subunit in Cascade-TniQ. Further, we comment on additional structural and dynamical regulatory points of Cascade-TniQ during R Loop formation, such as Cascade-TniQ backbone rigidity, and the potential role TniQ plays in regulating bundle dynamics. In summary, our outcomes provide the first all-atom dynamic representation of one of the largest CRISPR systems, with information that can contribute to understanding the mechanism of nucleic acid binding and, eventually, to transposase recruitment itself. Such information may prove informative to advance genome engineering efforts.
PubMed: 38948825
DOI: 10.1101/2024.06.21.600075 -
BioRxiv : the Preprint Server For... Jun 2024Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These...
UNLABELLED
Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA ) and CypA-depleted (CypA ) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA cells but not in CypA cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA cells but not in CypA cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA cells had lower activity compared to those from CypA cells. PICs from cells depleted for CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC activity is independent of TRIM5α. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA and CypA cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor.
IMPORTANCE
HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.
PubMed: 38948800
DOI: 10.1101/2024.06.15.599180 -
Journal of Family Medicine and Primary... May 2024The Sunderban area of West Bengal is home to tribal and religious minorities inhabiting various islands. There is a high prevalence of thalassemia among poverty-stricken...
INTRODUCTION
The Sunderban area of West Bengal is home to tribal and religious minorities inhabiting various islands. There is a high prevalence of thalassemia among poverty-stricken residents of this region living with meagre health care facilities. This work was planned to determine the proportion of four viral transfusion-transmitted infections (TTIs): HIV-1, HIV-2, hepatitis B virus (HBV) and hepatitis C virus (HCV) among thalassemia patients attending the sole rural medical college in the region.
MATERIALS AND METHODS
Thalassemia patients ( = 359, age ranging from 1 year to 60 years) attending the thalassemia clinic or being admitted to the indoor facilities for better management were included in the study. Only patients diagnosed with high-performance liquid chromatography (HPLC) and with classical clinical features were included in the study. Blood samples of these patients were tested for HIV as per NACO protocol. For HBV and HCV, samples were first tested serologically; reactive samples were collected and sent in the cold chain to a higher centre for nucleic acid amplification testing (NAAT) for qualitative and quantitative estimation. Clinical and laboratory data was collected, patients were followed up for complications and hospitalisation during the study period, and statistical analysis was performed.
RESULTS
Majority of our patients had E-beta-thalassemia (245, 59.81%), followed by beta-thalassemia major (102, 28.30%). NAAT-confirmed HCV infection (14.21%) infection was the most common, followed by HBV (2.51%), and lastly by HIV-1 (0.58%) infection. Among infected thalassemia patients, the mean HCV RNA was 741063 ± 438514.67 IU/ml while the mean HBV DNA level was 4082863 ± 7298514 IU/ml. Co-infections of HIV-1 and HCV and that of HBV and HCV were noted in one patient each (0.28%). HCV-related liver disease (14.21%) and growth retardation (10.31%) were the most typical complication noted, and death occurred in five patients (1.39%) during the study period.
CONCLUSION
Primary care physicians should know HCV infection is the most common TTI among thalassemia patients in rural eastern India.
PubMed: 38948618
DOI: 10.4103/jfmpc.jfmpc_1751_23 -
Journal of Extracellular Biology Jul 2024multiple nucleopolyhedrovirus (AcMNPV) is an enveloped DNA virus of the family. This baculovirus is widely exploited for the biological control of insect pest species...
multiple nucleopolyhedrovirus (AcMNPV) is an enveloped DNA virus of the family. This baculovirus is widely exploited for the biological control of insect pest species and as an expression platform to produce recombinant proteins in insect cells. Extracellular vesicles (EVs) are secreted by all cells and are involved in key roles in many biological processes through their cargo consisting of proteins, RNA or DNA. In viral infections, EVs have been found to transfer both viral and cellular cargo that can elicit either a pro- or antiviral response in recipient cells. Here, small EVs (sEVs) released by (Sf) insect cells were characterised for the first time. Using (SfC1B5) cells stably expressing the baculovirus , the viral envelope protein GP64 was shown to be incorporated into sEVs. Sf9 cells were also transfected with a bacmid AcMNPV genome lacking (AcΔP6.9) to prevent budded virus production. The protein content of sEVs from both mock- and AcΔP6.9-transfected cells were analysed by mass spectrometry. In addition to GP64, viral proteins Ac-F, ME-53 and viral ubiquitin were identified, as well as many host proteins including TSG101-which may be useful as a protein marker for sEVs.
PubMed: 38947876
DOI: 10.1002/jex2.163 -
International Journal of General... 2024The aim of this study was to describe the demographic and clinical characteristics of hepatitis B virus (HBV) associated hepatocellular carcinoma (HCC), analyse the risk...
BACKGROUND
The aim of this study was to describe the demographic and clinical characteristics of hepatitis B virus (HBV) associated hepatocellular carcinoma (HCC), analyse the risk factors associated with HBV-associated HCC, and to provide some references to the diagnosis and treatment of HCC.
METHODS
This study retrospectively enrolled 730 patients, including 390 patients with chronic hepatitis B (CHB) as controls, and 340 patients with CHB complicated with HCC as patients. Relevant information and medical records of these participants were collected, including age, sex, cigarette smoking, alcoholism, diabetes mellitus (DM), hypertension, coronary heart disease (CHD), cirrhosis, occupation, ascites, HBV-DNA load, the qualitative analysis of HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb serological markers, and levels of alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), gamma-glutamyltransferase (GGT), TNM stage, tumor size and tumor number. The test, Chi-square test, non-parametric rank-sum test, logistic regression analyses were used to explore the influencing factors and their degree of association with HCC in patients with HBV.
RESULTS
The proportion of smoking, alcoholism, married status, DM, hypertension, and the rate of HBV-DNA with a viral load of ≥500 copies/mL were significantly higher in the HCC group than in the controls (all <0.05). Cirrhosis was more common among patients with CHB+HCC than in controls (=0.013). The proportion of patients with HBsAg, HBeAb, and HBcAb positive was greater in CHB+HCC group than that in CHB group. Logistic regression analysis indicated that age ≥60 years (OR: 1.835, 95% CI: 1.020-3.302, =0.043), HBeAb positive (OR: 9.105, 95% CI: 4.796-17.288, <0.001), antiviral treatment with entecavir (OR: 2.209, 95% CI: 1.106-4.409, =0.025), and GGT (OR: 1.004, 95% CI: 1.001-1.007, =0.002) were risk factors for HCC in patients with CHB.
CONCLUSION
Advanced age, HBeAb positive, antiviral treatment with entecavir, and GGT were independent risk factors for HCC in HBV patients.
PubMed: 38947567
DOI: 10.2147/IJGM.S464083 -
Journal of Veterinary Research Jun 2024Exosomes are nanosized lipid bilayer membranous microvesicles, extracellularly released from a variety of mammalian cells. They mediate intercellular signalling by...
INTRODUCTION
Exosomes are nanosized lipid bilayer membranous microvesicles, extracellularly released from a variety of mammalian cells. They mediate intercellular signalling by transporting several types of RNA, lipids and proteins and participate in the intercellular exchange of DNA, RNA, micro RNA, proteins and other components. These microvesicles are present in all body fluids in physiological and pathological conditions and reflect the state of the host organism. The aim of the study was the isolation and molecular determination of exosomes in blood and supernatant fluids of bovine dendritic cell cultures infected with bovine leukaemia virus (BLV).
MATERIAL AND METHODS
Exosomes were isolated by ultracentrifugation from the blood sera, plasma and supernatant of bovine BLV-infected and uninfected control dendritic cell cultures and their presence was confirmed with scanning electron and transmission electron microscopy. Western blot analysis of the structural BLV glycoprotein 51 (Env) and protein 24 (Gag) and of the tetraspanin exosomal markers CD9, CD63 and flotillin-1 was undertaken in BLV+ and control BLV- cattle.
RESULTS
In exosomes of leukaemic cattle both BLV proteins and exosomal markers were detected. In healthy control animals only exosomal markers were determined.
CONCLUSION
Proteins of BLV were released with exosomes and could be transferred into recipient cells as an alternative propagation route not requiring virus infection.
PubMed: 38947160
DOI: 10.2478/jvetres-2024-0031