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Proceedings of the National Academy of... Mar 2024A number of endogenous genes in the human genome encode retroviral -like proteins, which were domesticated from ancient retroelements. The paraneoplastic Ma antigen...
A number of endogenous genes in the human genome encode retroviral -like proteins, which were domesticated from ancient retroelements. The paraneoplastic Ma antigen (PNMA) family members encode a -like capsid domain, but their ability to assemble as capsids and traffic between cells remains mostly uncharacterized. Here, we systematically investigate human PNMA proteins and find that a number of PNMAs are secreted by human cells. We determine that PNMA2 forms icosahedral capsids efficiently but does not naturally encapsidate nucleic acids. We resolve the cryoelectron microscopy (cryo-EM) structure of PNMA2 and leverage the structure to design engineered PNMA2 (ePNMA2) particles with RNA packaging abilities. Recombinantly purified ePNMA2 proteins package mRNA molecules into icosahedral capsids and can function as delivery vehicles in mammalian cell lines, demonstrating the potential for engineered endogenous capsids as a nucleic acid therapy delivery modality.
Topics: Animals; Humans; RNA, Messenger; Capsid; Cryoelectron Microscopy; Nerve Tissue Proteins; Mammals; Antigens, Neoplasm
PubMed: 38437549
DOI: 10.1073/pnas.2307812120 -
AIDS Research and Therapy Sep 2023Human immunodeficiency virus type 1 (HIV-1) is the primary epidemic strain in China. Its genome contains two regulatory genes (tat and rev), three structural genes (gag,... (Review)
Review
Human immunodeficiency virus type 1 (HIV-1) is the primary epidemic strain in China. Its genome contains two regulatory genes (tat and rev), three structural genes (gag, pol, and env), and four accessory genes (nef, vpr, vpu, and vif). Long terminal repeats (LTRs) in thegenome regulate integration, duplication, and expression of viral gene. The permissibility of HIV-1 infection hinges on the host cell cycle status. HIV-1 replicates by exploiting various cellular processes via upregulation or downregulation of specific cellular proteins that also control viral pathogenesis. For example, HIV-1 regulates the life cycle of p53, which in turn contributes significantly to HIV-1 pathogenesis. In this article, we review the interaction between HIV-1-associated factors and p53, providing information on their regulatory and molecular mechanisms, hinting possible directions for further research.
Topics: Humans; HIV-1; Tumor Suppressor Protein p53; HIV Infections; China; Genes, Viral
PubMed: 37691100
DOI: 10.1186/s12981-023-00563-7 -
Nature Communications Nov 2023Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of...
Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.
Topics: Humans; HIV-1; Sphingomyelins; Cell Membrane; Gene Products, gag; Cholesterol; Membrane Microdomains
PubMed: 37990014
DOI: 10.1038/s41467-023-42994-w -
Proceedings of the National Academy of... Jul 2023Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. and its close relatives harbor several families of...
Retrotransposons and retroviruses shape genome evolution and can negatively impact genome function. and its close relatives harbor several families of LTR-retrotransposons, the most abundant being Ty1 in several laboratory strains. The cytosolic foci that nucleate Ty1 virus-like particle (VLP) assembly are not well understood. These foci, termed retrosomes or T-bodies, contain Ty1 Gag and likely Gag-Pol and the Ty1 mRNA destined for reverse transcription. Here, we report an intrinsically disordered N-terminal ion-ike omain (PrLD) within Gag that is required for transposition. This domain contains amino acid composition similar to known yeast prions and is sufficient to nucleate prionogenesis in an established cell-based prion reporter system. Deleting the Ty1 PrLD results in dramatic VLP assembly and retrotransposition defects but does not affect Gag protein level. Ty1 Gag chimeras in which the PrLD is replaced with other sequences, including yeast and mammalian prionogenic domains, display a range of retrotransposition phenotypes from wild type to null. We examine these chimeras throughout the Ty1 replication cycle and find that some support retrosome formation, VLP assembly, and retrotransposition, including the yeast Sup35 prion and the mouse PrP prion. Our interchangeable Ty1 system provides a useful, genetically tractable in vivo platform for studying PrLDs, complete with a suite of robust and sensitive assays. Our work also invites study into the prevalence of PrLDs in additional mobile elements.
Topics: Animals; Mice; Saccharomyces cerevisiae; Retroelements; RNA, Messenger; Gene Products, gag; Virus Assembly; Mammals
PubMed: 37459521
DOI: 10.1073/pnas.2303358120 -
Frontiers in Immunology 2023Osteoarthritis (OA) affects a large percentage of the population worldwide. Current surgical and nonsurgical concepts for treating OA only result in symptom-modifying...
INTRODUCTION
Osteoarthritis (OA) affects a large percentage of the population worldwide. Current surgical and nonsurgical concepts for treating OA only result in symptom-modifying effects. However, there is no disease-modifying therapy available. Extracellular vesicles released by mesenchymal stem/stromal cells (MSC-EV) are promising agents to positively influence joint homeostasis in the osteoarthritic surroundings. This pilot study aimed to investigate the effect of characterized MSC-EVs on chondrogenesis in a 3D chondrocyte inflammation model with the pro-inflammatory cytokine TNFα.
METHODS
Bovine articular chondrocytes were expanded and transferred into pellet culture at passage 3. TNFα, human MSC-EV preparations (MSC-EV batches 41.5-EV and 84-EV), EVs from human platelet lysate (hPL-EV), or the combination of TNFα and EVs were supplemented. To assess the effect of MSC-EVs in the chondrocyte inflammation model after 14 days, DNA, glycosaminoglycan (GAG), total collagen, IL-6, and NO release were quantified, and gene expression of anabolic (COL-II, aggrecan, COMP, and PRG-4), catabolic (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), dedifferentiation (COL-I), hypertrophy (COL-X, VEGF), and inflammatory (IL-8) markers were analyzed; histological evaluation was performed using safranin O/Fast Green staining and immunohistochemistry of COL I and II. For statistical evaluation, nonparametric tests were chosen with a significance level of p < 0.05.
RESULTS
TNFα supplementation resulted in catabolic stimulation with increased levels of NO and IL-6, upregulation of catabolic gene expression, and downregulation of anabolic markers. These findings were supported by a decrease in matrix differentiation (COL-II). Supplementation of EVs resulted in an upregulation of the chondrogenic marker PRG-4. All MSC-EV preparations significantly increased GAG retention per pellet. In contrast, catabolic markers and IL-8 expression were upregulated by 41.5-EV. Regarding protein levels, IL-6 and NO release were increased by 41.5-EV. Histologic and immunohistochemical evaluations indicated a higher differentiation potential of chondrocytes treated with 84-EV.
DISCUSSION
MSC-EVs can positively influence chondrocyte matrix production in pro-inflammatory surroundings, but can also stimulate inflammation. In this study MSC-EV 41.5-EV supplementation increased chondrocyte inflammation, whereas MSC-84-EV supplementation resulted a higher chondrogenic potential of chondrocytes in 3D pellet culture. In summary, the selected MSC-EVs exhibited promising chondrogenic effects indicating their significant potential for the treatment of OA; however, the functional heterogeneity in MSC-EV preparations has to be solved.
Topics: Animals; Cattle; Humans; Chondrocytes; Tumor Necrosis Factor-alpha; Interleukin-6; Interleukin-8; Pilot Projects; Cells, Cultured; Inflammation; Osteoarthritis; Glycosaminoglycans; Mesenchymal Stem Cells; Extracellular Vesicles
PubMed: 37564645
DOI: 10.3389/fimmu.2023.1198198 -
International Journal of Molecular... Jun 2023Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) enzyme, leading to the...
Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) enzyme, leading to the accumulation of glycosaminoglycans (GAG), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), mainly in cartilage and bone. This lysosomal storage disorder (LSD) is characterized by severe systemic skeletal dysplasia. To this date, none of the treatment options for the MPS IVA patients correct bone pathology. Enzyme replacement therapy with elosulfase alpha provides a limited impact on bone growth and skeletal lesions in MPS IVA patients. To improve bone pathology, we propose a novel gene therapy with a small peptide as a growth-promoting agent for MPS IVA. A small molecule in this peptide family has been found to exert biological actions over the cardiovascular system. This work shows that an AAV vector expressing a C-type natriuretic (CNP) peptide induces bone growth in the MPS IVA mouse model. Histopathological analysis showed the induction of chondrocyte proliferation. CNP peptide also changed the pattern of GAG levels in bone and liver. These results suggest the potential for CNP peptide to be used as a treatment in MPS IVA patients.
Topics: Animals; Mice; Mucopolysaccharidosis IV; Keratan Sulfate; Glycosaminoglycans; Cartilage; Bone Development
PubMed: 37373036
DOI: 10.3390/ijms24129890 -
Bone Oct 2023Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by pathogenic variants in the SLC26A2 gene encoding for a cell membrane sulfate/chloride antiporter...
Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by pathogenic variants in the SLC26A2 gene encoding for a cell membrane sulfate/chloride antiporter crucial for sulfate uptake and glycosaminoglycan (GAG) sulfation. Research on a DTD animal model has suggested possible pharmacological treatment approaches. In view of future clinical trials, the identification of non-invasive biomarkers is crucial to assess the efficacy of treatments. Urinary GAG composition has been analyzed in several metabolic disorders including mucopolysaccharidoses. Moreover, the N-terminal fragment of collagen X, known as collagen X marker (CXM), is considered a real-time marker of endochondral ossification and growth velocity and was studied in individuals with achondroplasia and osteogenesis imperfecta. In this work, urinary GAG sulfation and blood CXM levels were investigated as potential biomarkers for individuals affected by DTD. Chondroitin sulfate disaccharide analysis was performed on GAGs isolated from urine by HPLC after GAG digestion with chondroitinase ABC and ACII, while CXM was assessed in dried blood spots. Results from DTD patients were compared with an age-matched control population. Undersulfation of urinary GAGs was observed in DTD patients with some relationship to the clinical severity and underlying SLC26A2 variants. Lower than normal CXM levels were observed in most patients, even if the marker did not show a clear pattern in our small patient cohort because CXM values are highly dependent on age, gender and growth velocity. In summary, both non-invasive biomarkers are promising assays targeting various aspects of the disorder including overall metabolism of sulfated GAGs and endochondral ossification.
Topics: Animals; Anion Transport Proteins; Sulfate Transporters; Achondroplasia; Glycosaminoglycans; Biomarkers; Collagen; Sulfates
PubMed: 37454964
DOI: 10.1016/j.bone.2023.116838 -
Frontiers in Neuroscience 2023DYT- dystonia is a neurological disorder characterized by involuntary muscle contractions and abnormal movements. It is a severe genetic form of dystonia caused by... (Review)
Review
DYT- dystonia is a neurological disorder characterized by involuntary muscle contractions and abnormal movements. It is a severe genetic form of dystonia caused by mutations in the gene. TorsinA is a member of the AAA + family of adenosine triphosphatases (ATPases) involved in a variety of cellular functions, including protein folding, lipid metabolism, cytoskeletal organization, and nucleocytoskeletal coupling. Almost all patients with -related dystonia harbor the same mutation, an in-frame GAG deletion (ΔGAG) in the last of its 5 exons. This recurrent variant results in the deletion of one of two tandem glutamic acid residues (i.e., E302/303) in a protein named torsinA [torsinA(△E)]. Although the mutation is hereditary, not all carriers will develop DYT- dystonia, indicating the involvement of other factors in the disease process. The current understanding of the pathophysiology of DYT- dystonia involves multiple factors, including abnormal protein folding, signaling between neurons and glial cells, and dysfunction of the protein quality control system. As there are currently no curative treatments for DYT- dystonia, progress in research provides insight into its pathogenesis, leading to potential therapeutic and preventative strategies. This review summarizes the latest research advances in the pathogenesis, diagnosis, and treatment of DYT- dystonia.
PubMed: 37638318
DOI: 10.3389/fnins.2023.1216929 -
Journal of Virology Jun 2023HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other...
HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV-) sequences from a subset of 14 participants. We detected HIV DNA () in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain ( < 0.01). Levels of HIV between BG and FC were similar ( > 0.2), while OCC had the lowest levels ( = 0.01). Females had higher HIV DNA levels in tissues than males (, = 0.03; 2-LTR, = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV- sequences ( = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies.
Topics: Female; Humans; Male; Brain; DNA, Viral; HIV Infections; Proviruses; Spleen; Middle Aged; Lymphoid Tissue; env Gene Products, Human Immunodeficiency Virus; HIV-1
PubMed: 37184401
DOI: 10.1128/jvi.00543-23 -
Retrovirology Jan 2024The p6 domain of the Gag precursors (Gag p6) in human immunodeficiency virus type 1 (HIV-1) plays multifunctional roles in the viral life cycle. It utilizes the... (Review)
Review
The p6 domain of the Gag precursors (Gag p6) in human immunodeficiency virus type 1 (HIV-1) plays multifunctional roles in the viral life cycle. It utilizes the endosomal sorting complex required for transport (ESCRT) system to facilitate viral budding and release from the plasma membrane through the interactions with the ESCRT-I component tumor susceptibility gene 101 (TSG101) and with the ALG-2 interacting protein X (ALIX). Moreover, Gag p6 contributes to viral replication by a range of posttranslational modifications such as SUMOylation, ubiquitination and phosphorylation. Additionally, Gag p6 also mediates the incorporation of the accessory protein Vpr into virions, thereby promoting Vpr-induced viral replication. However, less attention is focused on Gag p6 as therapeutic intervention. This review focuses on the structures and diverse functions of Gag p6 in viral replication, host cells, and pathogenesis. Additionally, several challenges were also discussed in studying the structure of Gag p6 and its interactions with partners. Consequently, it concludes that the Gag p6 represents an attractive target for the development of antiretroviral drugs, and efforts to develop p6-targeted antiretrovirals are expected to undergo significant growth in the forthcoming years.
Topics: Humans; HIV-1; Anti-Retroviral Agents; Biological Transport; Cell Membrane; Endosomal Sorting Complexes Required for Transport
PubMed: 38263239
DOI: 10.1186/s12977-024-00633-2