HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4(+) T cells and either uninfected human CD4(+) T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread.
The root system is essential for the stable growth of plants. Roots help anchor plants in the soil and play a crucial role in water uptake, mineral nutrient absorption and endogenous phytohormone formation. Root-restriction (RR) cultivation, a powerful technique, confines plant roots to a specific soil space. In the present study, roots of one-year-old "Muscat Hamburg" grapevine under RR and control (nR) treatments harvested at 70 and 125 days after planting were used for transcriptome sequencing, and in total, 2031 (nR7 vs. nR12), 1445 (RR7 vs. RR12), 1532 (nR7 vs. RR7), and 2799 (nR12 vs. RR12) differentially expressed genes (DEGs) were identified. Gene Ontology (GO) enrichment analysis demonstrated that there were several genes involved in the response to different phytohormones, including abscisic acid (ABA), auxin (IAA), ethylene (ETH), gibberellins (GAs), and cytokinins (CTKs). Among them, multiple genes, such as PIN2 and ERF113, are involved in regulating vital plant movements by various phytohormone pathways. Moreover, following RR cultivation, DEGs were enriched in the biological processes of plant-type secondary cell wall biosynthesis, the defense response, programmed cell death involved in cell development, and the oxalate metabolic process. Furthermore, through a combined analysis of the transcriptome and previously published microRNA (miRNA) sequencing results, we found that multiple differentially expressed miRNAs (DEMs) and DEG combinations in different comparison groups exhibited opposite trends, indicating that the expression levels of miRNAs and their target genes were negatively correlated. Furthermore, RR treatment indeed significantly increased the ABA content at 125 days after planting and significantly decreased the IAA content at 70 days after planting. Under RR cultivation, most ABA biosynthesis-related genes were upregulated, while most IAA biosynthesis-related genes were downregulated. These findings lay a solid foundation for further establishing the network through which miRNAs regulate grapevine root development through target genes and for further exploring the molecular mechanism through which endogenous ABA and IAA regulate root architecture development in grapevine.
Monoterpenes are a class of volatile organic compounds that play crucial roles in imparting floral and fruity aromas to Muscat-type grapes. However, our understanding of the regulatory mechanisms underpinning monoterpene biosynthesis in grapes, particularly following abscisic acid (ABA) treatment, remains elusive. This study aimed to explore the impact of exogenous ABA on monoterpene biosynthesis in Ruiduhongyu grape berries by employing Headspace Solid-Phase Micro-Extraction Gas Chromatography–Mass Spectrometry (HS-SPME/GC–MS) analysis and transcriptome sequencing. The results suggested significant differences in total soluble solids (TSS), pH, and total acid content. ABA treatment resulted in a remarkable increase in endogenous ABA levels, with concentrations declining from veraison to ripening stages. ABA treatment notably enhanced monoterpene concentrations, particularly at the E_L37 and E_L38 stages, elevating the overall floral aroma of grape berries. According to the variable gene expression patterns across four developmental stages in response to ABA treatment, the E_L37 stage had the largest number of differential expressed genes (DEGs), which was correlated with a considerable change in free monoterpenes. Furthermore, functional annotation indicated that the DEGs were significantly enriched in primary and secondary metabolic pathways, underlining the relationship between ABA, sugar accumulation, and monoterpene biosynthesis. ABA treatment upregulated key genes involved in the methylerythritol phosphate (MEP) pathway, enhancing carbon allocation and subsequently impacting terpene synthesis. This study also identified transcription factors, including MYB and AP2/ERF families, potentially modulating monoterpene and aroma-related genes. Weighted gene co-expression network analysis (WGCNA) linked ABA-induced gene expression to monoterpene accumulation, highlighting specific modules enriched with genes associated with monoterpene biosynthesis; one of these modules (darkgreen) contained genes highly correlated with most monoterpenes, emphasizing the role of ABA in enhancing grape quality during berry maturation. Together, these findings provide valuable insights into the multifaceted effects of exogenous ABA on monoterpene compounds and grape berry flavor development, offering potential applications in viticulture and enology.
An HIV-1 strain, SH01, isolated by coculture of peripheral blood mononuclear cells (PBMC) was easily passaged in MT4 cell line and multinuclear giant syncytia could be induced in MT2 cells. It was a syncytium-inducing (SI) T-cell-tropic isolate. Electron microscopy showed this isolate shared the typical features of HIV-1 in morphology, such as conical core, variation in size and budding release from cell membrane. A distinct fact was that HIV particles could be found in cytoplasmic vacuoles in MT4 cells infected at 7th day or trasformed persistently with HIV-1 SH01, but cell debris or lysosomes were seen at the same time. So these vacuoles were phagosomes or endocytic vacuoles rather than dilated vesicles of Golgi apparatus or endoplasmic reticulum. Furthermore, a large number of HIV particles were also found by chance in phagolysosomes in HIV-1 SH01-infected MT4 cells at 7th day, but it was quite common in HIV-1 SH01-transformed MT4 cells and the remnants of HIV particles were located in some phagolysosomes, yet the dissolution of phagolysosome membrane could be demonstrated simultaneously. Conclusively, it is evident that HIV particles were taken in by MT4 cells through endocytosis and digested by lysosomes, so the role of lysosomes should be paid attention to more extensively in the clearance of HIV in human body. In addition, a kind of virus-like particles was located in cytoplasmic matrix rather than in vacuoles or vesicles, which were more diversified in size with cores bigger, circular or oval and possibly produced from Golgi apparatus, but their characteristics needs to be further determined.
Astrocytes are proposed to be a critical reservoir of HIV in the brain. However, HIV infection of astrocytes is inefficient in vitro except for cell-to-cell transmission from HIV-infected cells. Here, we explore mechanisms by which cell-free HIV bypasses entry and postentry barriers leading to a productive infection.HIV infection of astrocytes was investigated by a variety of techniques including transfection of CD4-expressing plasmid, treatment with lysosomotropic agents or using a transwell culture system loaded with HIV-infected lymphocytes. Infection was monitored by HIV-1 p24 in culture supernatants and integrated proviral DNA was quantified by Alu-PCR.Persistent HIV infection could be established in astrocytes by transfection of proviral DNA, transduction with VSV-G-pseudotyped viruses, transient expression of CD4 followed by HIV infection, or simultaneous treatment with lysosomotropic chloroquine or Tat-HA2 peptide with HIV infection. In absence of these treatments, HIV entered via endocytosis as seen by electronmicroscopy and underwent lysosomal degradation without proviral integration, indicating endocytosis is a dead end for HIV in astrocytes. Nevertheless, productive infection was observed when astrocytes were in close proximity but physically separated from HIV-infected lymphocytes in the transwell cultures. This occurred with X4 or dual tropic R5X4 viruses and was blocked by an antibody or antagonist to CXCR4.A CD4-independent, CXCR4-dependent mechanism of viral entry is proposed, by which immature HIV particles from infected lymphocytes might directly bind to CXCR4 on astrocytes and trigger virus--cell fusion during or after the process of viral maturation. This mechanism may contribute to the formation of brain HIV reservoirs.
Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein possesses a unique membrane-transduction property. Interestingly, Tat transduction could be dramatically increased 1000-fold based on LTR-transactivation assay when complexed with cationic liposomes (lipo-Tat), compared with Tat alone. Therefore, underlining mechanisms were explored further. Microscopy and flow cytometry showed that this effect was associated with enhanced membrane binding, large particle formation (1-2 μm) and increased intracellular uptake of Tat fluorescent proteins. Using pharmacological assays and immune colocalizations, it was found that lipid raft-dependent endocytosis and macropinocytosis were major pathways involved in lipo-Tat uptake, and actin-filaments played a major role in intracellular trafficking of lipo-Tat to the nucleus. Furthermore, we found that the Tat hydrophobic domain (aa 36–47) mediated formation of two positively charged molecules into lipo-Tat complexes via hydrophobic bonds, based on LTR-transactivation inhibition assay. Thus, the hydrophobic domain may play an important role in Tat protein uptake and be useful for intracellular delivery of biomacromolecules if coupled together with Tat basic peptide, a cell-penetrating peptide.—Li, G.-H., Li, W., Mumper, R. J., Nath, A. Molecular mechanisms in the dramatic enhancement of HIV-1 Tat transduction by cationic liposomes. FASEB J. 26, 2824–2834 (2012). www.fasebj.org
HIV-1 Tat protein can be secreted from infected cells and taken up by other target cells. Tat uptake and trafficking has been widely investigated. This property is also exploited for drug delivery. We initially confirmed that Tat mainly entered Hela cells by clathrin-dependent endocytosis and was degraded in lysosomes, whereas chloroquine could inhibit the degradation and enhanced its trafficking to the nucleus. Interestingly, a similar effect was noted in presence of chlorpromazine (CPZ). Most likely, the uptake of Tat was shifted to caveolar endocytosis after the clathrin pathway was blocked by CPZ. However, the LTR transactivation of Tat could be enhanced by at least 50 fold when a liposome reagent was introduced into the Tat delivery system. Further evidence showed that liposome reagents could switch the Tat uptake from clathrin-dependent endocytosis to lipid raft pathway and bypass the degradation in lysosomes. This was supported by both pharmacological assays using inhibitors of caveolae-mediated endocytosis (MβCD, etc.) or microtubules (cytochalasin D & lutrunculin A) and co-localization using Tat101venus and immunstaining with anti-clathrin1, anti-caveolin1, anti-EEA1, anti-Lamp1 or anti-Rab7. Subsequently, chemical molecules carrying “+” or “−” charges were tested for blocking the Tat uptake facilitated with the liposome reagent, including macromolecules (e.g., heparin, polybrene) and small molecules (e.g., spermine). Both “+” and “-” charged molecules could inhibit the uptake of Tat, but the “+” molecules actually attached to the membrane carrying “-” charges and the “-” molecules bound to the Tat-liposome complex with “+” charges. However, the Tat-liposome complex was probably formed by hydrophobic bonds linking liposome's lipid to Tat hydrophobic domain because both Tat and the liposome reagent carried “+”charges. A peptide containing partial hydrophobic domain of Tat (CFITKGLGISYGRKK) could competitively inhibit the uptake of Tat-liposome complex. In conclusion, the liposome reagent could facilitate Tat protein to traverse the membrane by changing endocytosis pathways and significantly enhance its delivery to the nucleus in a lysosome-independent manner. These observations have important implications for elucidating the mechanism of Tat uptake and trafficking and exploring drug delivery.
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