Mechanism Of Biogenesis Research Articles

The double-membrane vesicle (DMV): a virus-induced organelle dedicated to the replication of SARS-CoV-2 and other positive-sense single-stranded RNA viruses.

Positive single-strand RNA (+ RNA) viruses can remodel host cell membranes to induce a replication organelle (RO) isolating the replication of their genome from innate immunity mechanisms. Some of these viruses, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMVs) for this purpose. Viral non-structural proteins are essential for DMV biogenesis, but they cannot form without an original membrane from a host cell organelle and a significant supply of lipids. The endoplasmic reticulum (ER) and the initial mechanisms of autophagic processes have been shown to be essential for the biogenesis of SARS-CoV-2 DMVs. However, by analogy with other DMV-inducing viruses, it seems likely that the Golgi apparatus, mitochondria and lipid droplets are also involved. As for hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMVs have been identified. These pores presumably allow the supply of metabolites essential for viral replication within the DMV, together with the export of the newly synthesized viral RNA to form the genome of future virions. It remains unknown whether, as for HCV, DMVs with open pores can coexist with the fully sealed DMVs required for the storage of large amounts of viral RNA. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis and morphology between these two phylogenetically distant viruses. An understanding of the mechanisms of DMV formation and their role in the infectious cycle of SARS-CoV-2 may be essential for the development of new antiviral approaches against this pathogen or other coronaviruses that may emerge in the future.

How Structural Features Define Biogenesis and Function of Human Telomerase RNA Primary Transcript.

Telomerase RNA has been uncovered as a component of the telomerase enzyme, which acts as a reverse transcriptase and maintains the length of telomeres in proliferated eukaryotic cells. Telomerase RNA is considered to have major functions as a template for telomeric repeat synthesis and as a structural scaffold for telomerase. However, investigations of its biogenesis and turnover, as well as structural data, have provided evidence of functions of telomerase RNA that are not associated with telomerase activity. The primary transcript produced from the human telomerase RNA gene encodes for the hTERP protein, which presents regulatory functions related to autophagy, cellular proliferation, and metabolism. This review focuses on the specific features relating to the biogenesis and structure of human telomerase RNA that support the existence of an isoform suitable for functioning as an mRNA. We believe that further investigation into human telomerase RNA biogenesis mechanisms will provide more levels for manipulating cellular homeostasis, survival, and transformation mechanisms, and may contribute to a deeper understanding of the mechanisms of aging.

Data driven and biophysical insights into the regulation of trafficking vesicles by extracellular matrix stiffness

SummaryBiomechanical signals from remodeled extracellular matrix (ECM) promote tumor progression. Here, we show that cell-matrix and cell-cell communication may be inherently linked and tuned through mechanisms of mechanosensitive biogenesis of trafficking vesicles. Pan-cancer analysis of cancer cells' mechanical properties (focusing primarily on cell stiffness) on substrates of varied stiffness and composition elucidated a heterogeneous cellular response to mechanical stimuli. Through machine learning, we identified a fingerprint of cytoskeleton-related proteins that accurately characterize cell stiffness in different ECM conditions. Expression of their respective genes correlates with patient prognosis across different tumor types. The levels of selected cytoskeleton proteins indicated that cortical tension mirrors the increase (or decrease) in cell stiffness with a change in ECM stiffness. A mechanistic biophysical model shows that the tendency for curvature generation by curvature-inducing proteins has an ultrasensitive dependence on cortical tension. This study thus highlights the effect of ECM stiffness, mediated by cortical tension, in modulating vesicle biogenesis.

The role of ubiquitin signaling pathway on liver regeneration in rats.

The ubiquitin signalling pathway is a large system associated with numerous intracellular mechanisms. However, its function in the liver regeneration process remains unknown. This particular study investigates the intracellular effect mechanisms of the ubiquitin signalling pathway. It also determines the differences in the expression of 88 genes belonging to the ubiquitin pathway using the RT-PCR array method. To conduct this research, three genes-that differed in the expression analysis were selected. Moreover, their proteins were analysed by western blot analysis while using Ki67 immunohistochemical analysis that determines proliferation rates by hour. It was determined that BRCA1 and BARD1, which are effective in DNA repair, play an active role at PH24. Similarly, Ube2t expression, which belongs to the Fanconi anaemia pathway associated with DNA repair, was also found to be high at PH12-72h. In addition, it was revealed that the expressions of Anapc2, Anapc11, Cdc20 belonging to the APC/CCdc20 complex, which participate in cell cycle regulation, differed at different hours after PH. Expression of Mul1, which is involved in mitochondrial biogenesis and mitophagy mechanisms, peaked at PH12 under the observation. Considering the Mul1 gene expression difference, MUL1-mediated mitophagy and mitochondrial fission mechanism may be associated with liver regeneration. It was also determined that PARKIN-mediated mitophagy mechanisms are not active in 0-72h of liver regeneration since PARKIN expression did not show a significant change in PH groups.

Abstract 1581: Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC)

Abstract Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, which could be attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for developing effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are small extracellular vesicles (sEVs) of approximately 100-150 nm in diameter, often referred as exosomes. Previous report revealed that increased levels of exosomes in NSCLC patients correlated with lower overall survival. Indeed, secretion of sEVs by cancer cells can induce malignant transformation of non-cancerous cells nearby or at distant locations through influencing pre-metastatic niche formation. Therefore, biogenesis and release of cancer derived sEVs are determinant steps for the development of tumor metastasis. However, the mechanisms through which sEVs are produced and secreted by cancer cells are yet to be determined. Here, we isolated and characterized sEVs from a panel of NSCLC cell lines (H358, Calu6, H460, SKMES-1) and non-tumorigenic cells (BEAS-2B and HBEC) to identify mechanisms involved in the elevated secretion of EVs by cancer cells. Contrary to what has been previously reported in literature, our data demonstrated that not all cancer cell lines release high levels of sEVs. Our data suggests that elevated secretion of sEVs could be driven by specific mutations and oncogenic signaling in cancer cells. Among all the cell lines tested, H358 cells which harbor the infamous G12C mutation in KRAS, had the highest sEV release. Therefore, we hypothesize that the KRASG12C can drive high sEV production and secretion in H358 cells. Our preliminary data suggests that siRNA mediated knockdown of oncogenic KRAS signaling in H358 cells significantly decreases secretion of sEVs. Ongoing studies aim to pinpoint the detailed mechanisms by which cancer cells utilize KRAS oncogenic signaling to promote EV biogenesis pathways and tumorigenesis. The current study will provide the foundation to developed new therapeutic strategies against tumor sEVs-driven metastasis. Citation Format: Zulaida M. Soto Vargas, Ikjot Singh Sohal, Humna Hasan, David Arteaga, Andrea L. Kasinski. Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1581.

Human umbilical cord-derived mesenchymal stromal cells ameliorate aging-associated skeletal muscle atrophy and dysfunction by modulating apoptosis and mitochondrial damage in SAMP10 mice

BackgroundSkeletal muscle mass and function losses in aging individuals are associated with quality of life deterioration and disability. Mesenchymal stromal cells exert immunomodulatory and anti-inflammatory effects and could yield beneficial effects in aging-related degenerative disease.Methods and resultsWe investigated the efficacy of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) on sarcopenia-related skeletal muscle atrophy and dysfunction in senescence-accelerated mouse prone 10 (SAMP10) mice. We randomly assigned 24-week-old male SAMP10 mice to a UC-MSC treatment group and control group. At 12 weeks post-injection, the UC-MSC treatment had ameliorated sarcopenia-related muscle changes in performance, morphological structures, and mitochondria biogenesis, and it enhanced the amounts of proteins or mRNAs for myosin heavy chain, phospho-AMP-activated protein kinase, phospho-mammalian target of rapamycin, phospho-extracellular signal-regulated kinase1/2, peroxisome proliferator-activated receptor-γ coactivator, GLUT-4, COX-IV, and hepatocyte growth factor in both gastrocnemius and soleus muscles, and it reduced the levels of proteins or mRNAs for cathepsin K, cleaved caspase-3/-8, tumor necrosis factor-α, monocyte chemoattractant protein-1, and gp91phox mRNAs. The UC-MSC treatment retarded mitochondria damage, cell apoptosis, and macrophage infiltrations, and it enhanced desmin/laminin expression and proliferating and CD34+/Integrin α7+ cells in both types of skeletal muscle of the SAMP10 mice. In vitro, we observed increased levels of HGF, PAX-7, and MoyD mRNAs at the 4th passage of UC-MSCs.ConclusionsOur results suggest that UC-MSCs can improve sarcopenia-related skeletal muscle atrophy and dysfunction via anti-apoptosis, anti-inflammatory, and mitochondrial biogenesis mechanisms that might be mediated by an AMPK-PGC1-α axis, indicating that UC-MSCs may provide a promising treatment for sarcopenia/muscle diseases.

Progress in circular RNAs of plants

With the development of high-throughput sequencing technology, circular RNAs (circRNAs) have gradually become a hotspot in the research on non-coding RNA. CircRNAs are produced by the covalent circularization of a downstream 3' splice donor and an upstream 5' splice acceptor through backsplicing, and they are pervasive in eukaryotic cells. CircRNAs used to be considered byproducts of false splicing, whereas an explosion of related studies in recent years has disproved this misconception. Compared with the rich studies of circRNAs in animals, the study of circRNAs in plants is still in its infancy. In this review, we introduced the discovery of plant circRNAs, the discovery of plant circRNAs, the circularization feature, expression specificity, conservation, and stability of plant circRNAs and expounded the identification tools, main types, and biogenesis mechanisms of circRNAs. Furthermore, we summarized the potential roles of plant circRNAs as microRNA (miRNA) sponges and translation templates and in response to biotic/abiotic stress, and briefed the degradation and localization of plant circRNAs. Finally, we discussed the challenges and proposed the future directions in the research on plant circRNAs.

Candida albicans Hyphal Extracellular Vesicles Are Different from Yeast Ones, Carrying an Active Proteasome Complex and Showing a Different Role in Host Immune Response.

ABSTRACTCandida albicans is the principal causative agent of lethal fungal infections, predominantly in immunocompromised hosts. Extracellular vesicles (EVs) have been described as crucial in the interaction of microorganisms with their host. Since the yeast-to-hypha transition is an important virulence trait with great impact in invasive candidiasis (IC), we have addressed the characterization of EVs secreted by hyphal cells (HEVs) from C. albicans, comparing them to yeast EVs (YEVs). YEVs comprised a larger population of bigger EVs with mainly cell wall proteins, while HEVs were smaller, in general, and had a much higher protein diversity. YEVs were able to rescue the sensitivity of a cell wall mutant against calcofluor white, presumably due to the larger amount of cell wall proteins they contained. On the other hand, HEVs also contained many cytoplasmic proteins related to protein metabolism and intracellular protein transport and the endosomal sorting complexes required for transport (ESCRT) pathway related to exosome biogenesis, pointing to an intracellular origin of HEVs. Interestingly, an active 20S proteasome complex was secreted exclusively in HEVs. Moreover, HEVs contained a greater number of virulence-related proteins. As for their immunogenic role, both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis; however, under our conditions, only HEVs showed a cytotoxic effect on human macrophages and could elicit the release of tumor necrosis factor alpha (TNF-α) by these macrophages.IMPORTANCE This first analysis of HEVs of C. albicans has shown clear differences between them and the YEVs of C. albicans, showing their relevance and possible use in the discovery of new diagnostic markers and treatment targets against C. albicans infections. The data obtained point to different mechanisms of biogenesis of YEVs and HEVs, as well as different involvements in cell biology and host interaction. YEVs played a more relevant role in cell wall maintenance, while HEVs were more closely related to virulence, as they had greater effects on human immune cells. Importantly, an active 20S proteosome complex was described as a fungal-EV cargo. A deeper study of its role and those of many other proteins exclusively detected in HEVs and involved in different relevant biological processes of this fungus could open up interesting new areas of research in the battle against C. albicans.

Tunneling Nanotube-Mediated Communication: A Mechanism of Intercellular Nucleic Acid Transfer.

Tunneling nanotubes (TNTs) are thin, F-actin-based membranous protrusions that connect distant cells and can provide e a novel mechanism for intercellular communication. By establishing cytoplasmic continuity between interconnected cells, TNTs enable the bidirectional transfer of nuclear and cytoplasmic cargo, including organelles, nucleic acids, drugs, and pathogenic molecules. TNT-mediated nucleic acid transfer provides a unique opportunity for donor cells to directly alter the genome, transcriptome, and metabolome of recipient cells. TNTs have been reported to transport DNA, mitochondrial DNA, mRNA, viral RNA, and non-coding RNAs, such as miRNA and siRNA. This mechanism of transfer is observed in physiological as well as pathological conditions, and has been implicated in the progression of disease. Herein, we provide a concise overview of TNTs’ structure, mechanisms of biogenesis, and the functional effects of TNT-mediated intercellular transfer of nucleic acid cargo. Furthermore, we highlight the potential translational applications of TNT-mediated nucleic acid transfer in cancer, immunity, and neurological diseases.

Current Knowledge on Exosome Biogenesis, Cargo-Sorting Mechanism and Therapeutic Implications.

Extracellular vesicles (EVs) are nanoscale membrane vesicles released by donor cells that can be taken up by recipient cells. The study of EVs has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and disease. Exosomes, with an average diameter of ≈100 nanometers, are a subset of EVs. Different molecular families have been shown to be involved in the formation of exosomes and subsequent secretion of exosomes, which largely leads to the complexity of the form, structure and function of exosomes. In addition, because of their low immunogenicity and ability to transfer a variety of bioactive components to recipient cells, exosomes are regarded as effective drug delivery systems. This review summarizes the known mechanisms of exosomes biogenesis, cargo loading, exosomes release and bioengineering, which is of great importance for further exploration into the clinical applications of EVs.

The polyene antifungal candicidin is selectively packaged into membrane vesicles in Streptomyces S4

In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of environments and niches. While the precise mechanism of biogenesis is unknown, it is focused on the modification of the Gram-negative cell wall to facilitate blebbing at sites of weakness in and around the characteristically thin peptidoglycan layer within the periplasm. Here, we investigate the biogenesis of membrane vesicles (MVs) in the Gram-positive organism Streptomyces albus S4 (Seipke et al. J Bacteriol 193:4270–4271, 2011 and Fazal et al. Antonie Van Leeuwenhoek 113:511–520, 2020). The S. albus S4 strain is an antifungal (candicidin and antimycin) producing organism that was isolated from attine ants (Barke et al. BMC Biol 8:109, 2010). The biogenesis and characterisation of S. albus S4 MVs is demonstrated using the wild-type (WT) and mutant strains ΔantC (no antimycin production) ΔfscC (no candicidin production) and ΔantC ΔfscC (produces neither antimycin nor candicidin). Here, we have shown that the S. albus S4 strain produces MVs and that these are comprised of both specific protein profiles and secondary metabolites, with a clear demonstration of the ability to selectively package one antifungal (candicidin) but not the other (antimycin).

Cotranslational Mechanisms of Protein Biogenesis and Complex Assembly in Eukaryotes.

The formation of protein complexes is crucial to most biological functions. The cellular mechanisms governing protein complex biogenesis are not yet well understood, but some principles of cotranslational and posttranslational assembly are beginning to emerge. In bacteria, this process is favored by operons encoding subunits of protein complexes. Eukaryotic cells do not have polycistronic mRNAs, raising the question of how they orchestrate the encounter of unassembled subunits. Here we review the constraints and mechanisms governing eukaryotic co- and posttranslational protein folding and assembly, including the influence of elongation rate on nascent chain targeting, folding, and chaperone interactions. Recent evidence shows that mRNAs encoding subunits of oligomeric assemblies can undergo localized translation and form cytoplasmic condensates that might facilitate the assembly of protein complexes. Understanding the interplay between localized mRNA translation and cotranslational proteostasis will be critical to defining protein complex assembly in vivo.

Microbial Metagenomics of the Extubated Lacrimal Stents Following Dacryocystorhinostomy: The Lacriome Paper 4.

To study the metagenome of the microbes present on the extubated lacrimal stents following a dacryocystorhinostomy. A prospective study was performed on 10 consecutive extubated lacrimal stents obtained for the metagenomic analysis from the patients following an endoscopic dacryocystorhinostomy. The stents were extubated at 4 weeks postoperatively under endoscopic guidance and immediately transported on ice to the laboratory. Following DNA extraction and library preparation, a whole shotgun metagenome sequencing was performed on the Illumina platform. The downstream processing and bioinformatics of the samples were performed using multiple software packaged in SqueezeMeta pipeline or MG-RAST pipeline. The taxonomic hit distribution across the stent samples showed that bacteria were the most common isolates (mean, 69.70%), followed by viruses (mean, 0.02%) and archaea (0.003%). The 3 major phyla identified were Firmicutes, Actinobacteria, and Proteobacteria. The prevalent organisms include Pseudomonas aeruginosa, Staphylococcus aureus, Corynebacterium accolens, Dolosigranulum pigrum, Citrobacter koserii, Staphylococcus epidermidis, E. coli, and Hemophilus influenza . The functional subsystem profiling demonstrated microbial genes associated with metabolism, cellular, and information processing. The functional subsystem categories were metabolism involving carbohydrates, amino acids, DNA and RNA, cell wall or cell capsule biogenesis, membrane transport, virulence, and defense mechanisms. The present study is the first whole metagenome sequencing of the microbes isolated from the extubated lacrimal stents. The stents harbor diverse microbial communities with distinct ecosystem dynamics. Further studies on microbes-host interactions in the early postoperative period would provide valuable insights.

Long Non-Coding RNAs in Pancreatic Cancer: Biologic Functions, Mechanisms, and Clinical Significance.

Simple SummaryPancreatic cancer (PC) is a highly aggressive malignant tumor with a high mortality rate. Growing evidence shows that long non-coding RNAs (lncRNAs) might participate in the pathogenesis of PC. This review presents the biogenesis mechanism, classifications, and modes of action of lncRNAs, especially the functions and mechanisms of lncRNAs in PC. It also discusses the clinical significance of lncRNAs in PC.Despite tremendous efforts devoted to research in pancreatic cancer (PC), the mechanism underlying the tumorigenesis and progression of PC is still not completely clear. Additionally, ideal biomarkers and satisfactory therapeutic strategies for clinical application in PC are still lacking. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) might participate in the pathogenesis of diverse cancers, including PC. The abnormal expression of lncRNAs in PC is considered a vital factor during tumorigenesis that affects tumor cell proliferation, migration, invasion, apoptosis, angiogenesis, and drug resistance. With this review of relevant articles published in recent years, we aimed to summarize the biogenesis mechanism, classifications, and modes of action of lncRNAs and to review the functions and mechanisms of lncRNAs in PC. Additionally, the clinical significance of lncRNAs in PC was discussed. Finally, we pointed out the questions remaining from recent studies and anticipated that further investigations would address these gaps in knowledge in this field.

Research progress on the molecular regulatory mechanism of exosome biogenesis

Exosomes are small vesicles with a diameter of about 40-160 nm actively secreted by cells. They participate in a variety of pathophysiological processes and are closely related to the occurrence and development of diseases. As a newly carrier of intercellular communication and circulating biomarkers of disease diagnosis and prognosis, exosomes have attracted great attention for their potential clinical applications. However, many aspects such as exosome biogenesis, targeted transport, and mechanism of action are still unclear. This paper focuses on the exosome biogenesis, summarizes the exosome biogenesis pathways, and describes relevant molecular modulation mechanisms of importance. And this review provides a theoretical basis for disease treatment based on regulating exosome production.

Fission Yeast Autophagy Machinery.

Autophagy is a conserved process that delivers cytoplasmic components to the vacuole/lysosome. It plays important roles in maintaining cellular homeostasis and conferring stress resistance. In the fission yeast Schizosaccharomyces pombe, autophagy is important for cell survival under nutrient depletion and ER stress conditions. Experimental analyses of fission yeast autophagy machinery in the last 10 years have unveiled both similarities and differences in autophagosome biogenesis mechanisms between fission yeast and other model eukaryotes for autophagy research, in particular, the budding yeast Saccharomyces cerevisiae. More recently, selective autophagy pathways that deliver hydrolytic enzymes, the ER, and mitochondria to the vacuole have been discovered in fission yeast, yielding novel insights into how cargo selectivity can be achieved in autophagy. Here, we review the progress made in understanding the autophagy machinery in fission yeast.

Carboxypeptidase A3 in the structure of the protease phenotype of mast cells: cytophysiological aspects

Carboxypeptidase A3 (CPA3) is a specific protease of mast cells (MC) with variable expression and appears to be one of the preformed components of the secretome. CPA3 is involved in regulation of the state of a specifi tissue microenvironment and components of the integrative-buffer metabolic environment in adaptive and pathological processes; it affects implementation of the innate immunity, mechanisms of angiogenesis, processes of the extracellular matrix remodeling, etc. CPA3 identification using protocols of multiplex immunohistochemistry allows specifying details of the organ-specific mast cell population features, including the protease phenotype, mechanisms of biogenesis with cytoand histotopographic criteria, and features of secretory pathways. Numerous biological effects of CPA3, including participation in the regulation of the pulmonary parenchyma and systemic blood flow, in biogenesis and remodeling of the fibrous component of the extracellular matrix, in epigenetic reprogramming, determine the importance of fundamental investigation of the physiological activity of protease and its involvement in the implementation of pathological processes. Further studies will contribute to the detection of the translational value of the mast cell CPA3 expression features as a prognostic factor and a promising molecular target for treatment of socially significant diseases.

Mechanisms of MicroRNA Biogenesis and Stability Control in Plants.

MicroRNAs (miRNAs), a class of endogenous, non-coding RNAs, which is 20–24 nucleotide long, regulate the expression of its target genes post-transcriptionally and play critical roles in plant normal growth, development, and biotic and abiotic stresses. In cells, miRNA biogenesis and stability control are important in regulating intracellular miRNA abundance. In addition, research on these two aspects has achieved fruitful results. In this review, we focus on the recent research progress in our understanding of miRNA biogenesis and their stability control in plants.

Carboxypeptidase A3 in the structure of the protease phenotype of mast cells: cytophysiological aspects

Carboxypeptidase A3 (CPA3) is a specific protease of mast cells (MC) with variable expression and appears to be one of the preformed components of the secretome. CPA3 is involved in regulation of the state of a specifi tissue microenvironment and components of the integrative-buffer metabolic environment in adaptive and pathological processes; it affects implementation of the innate immunity, mechanisms of angiogenesis, processes of the extracellular matrix remodeling, etc. CPA3 identification using protocols of multiplex immunohistochemistry allows specifying details of the organ-specific mast cell population features, including the protease phenotype, mechanisms of biogenesis with cyto- and histotopographic criteria, and features of secretory pathways. Numerous biological effects of CPA3, including participation in the regulation of the pulmonary parenchyma and systemic blood flow, in biogenesis and remodeling of the fibrous component of the extracellular matrix, in epigenetic reprogramming, determine the importance of fundamental investigation of the biological activity and regulation of pathological processes of CPA3. Further studies will contribute to the detection of the true value of the mast cell CPA3 expression features as a prognostic factor and a promising molecular target for treatment of socially significant diseases.

Human cytomegalovirus forms phase-separated compartments at viral genomes to facilitate viral replication.

SummaryHuman cytomegalovirus (HCMV) replicates its DNA genome in specialized replication compartments (RCs) in the host cell nucleus. These membrane-less organelles originate as spherical structures and grow in size over time. However, the mechanism of RC biogenesis has remained understudied. Using live-cell imaging and photo-oligomerization, we show that a central component of RCs, the UL112-113 proteins, undergo liquid-liquid phase separation (LLPS) to form RCs in the nucleus. We show that the self-interacting domain and large intrinsically disordered regions of UL112-113 are required for LLPS. Importantly, viral DNA induces local clustering of these proteins and lowers the threshold for phase separation. The formation of phase-separated compartments around viral genomes is necessary to recruit the viral DNA polymerase for viral genome replication. Thus, HCMV uses its UL112-113 proteins to generate RCs around viral genomes by LLPS to ensure the formation of a pro-replicative environment.