RESUMO
An amphiphilic segmented polyurethane (F-PU-S), with pendant sulfate groups and a flexible hydrocarbon backbone, exhibits intrachain H-bonding-reinforced folding and hierarchical assembly, producing an anionic polymersome with efficient display of sulfate groups at the surface. It shows an excellent antiviral activity against Sendai virus (SV) by inhibiting its entry to the cells. Mechanistic investigation suggests fusion of the SV and the polymersome to produce larger particles in which neither the folded structure of the polymer nor the fusogenic property of the SV exists anymore. In sharp contrast, a structurally similar polymer R-PU-S, in which the chain folding pathway is blocked by replacing the flexible C6 chain with a rigid cyclohexane chain in the backbone, cannot form a similar polymersome structure and hence does not exhibit any antiviral activity. On the other hand, the third polymer (F-PU-C), which is similar to F-PU-S except for the pendant anionic groups (carboxylate instead of sulfate), also fails to exhibit any antiviral activity against SV, confirming the essential role of the chain folding as well as the pendant sulfate groups for the fusion-induced antiviral activity of F-PU-S, which provides an important structural guideline for developing new antiviral polymers.
Assuntos
Polímeros , Polímeros/farmacologia , Estrutura Secundária de ProteínaRESUMO
CUL4A is an ubiquitin ligase deregulated in numerous pathologies including cancer and even hijacked by viruses for facilitating their survival and propagation. However, its role in Human papilloma virus (HPV)-mediated cervical carcinogenesis remains elusive. The UALCAN and GEPIA datasets were analyzed to ascertain the transcript levels of CUL4A in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) patients. Subsequently, various biochemical assays were employed to explore the functional contribution of CUL4A in cervical carcinogenesis and to shed some light on its involvement in Cisplatin resistance in cervical cancer. Our UALCAN and GEPIA datasets analyses reveal elevated CUL4A transcript levels in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) patients that correlate with adverse clinicopathological parameters such as tumor stage and lymph node metastasis. Kaplan-Meier plot and GEPIA assessment depict poor prognosis of CESC patients having high CUL4A expression. Varied biochemical assays illustrate that CUL4A inhibition severely curtails hallmark malignant properties such as cellular proliferation, migration, and invasion of cervical cancer cells. We also show that CUL4A knockdown in HeLa cells causes increased susceptibility and better apoptotic induction toward Cisplatin, a mainstay drug used in cervical cancer treatment. More interestingly, we find reversion of Cisplatin-resistant phenotype of HeLa cells and an augmented cytotoxicity towards the platinum compound upon CUL4A downregulation. Taken together, our study underscores CUL4A as a cervical cancer oncogene and illustrates its potential as a prognosis indicator. Our investigation provides a novel avenue in improving current anti-cervical cancer therapy and overcoming the bottle-neck of Cisplatin resistance.
RESUMO
Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo ß-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo ß-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo ß-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.
Assuntos
Terapia Biológica/métodos , Búfalos/genética , Expressão Gênica/genética , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Leite/química , Transgenes/genética , Animais , Caseínas/genética , Feminino , Humanos , Regiões Promotoras Genéticas/genética , Vírus Sendai/genéticaRESUMO
BACKGROUND: Placental like alkaline phosphatase (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues [corrected]. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting. METHODS: PLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties. RESULTS: Reduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region. CONCLUSION: A combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.
Assuntos
Fosfatase Alcalina/genética , Inativação Gênica , Técnicas de Transferência de Genes , Isoenzimas/genética , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Anticorpos de Cadeia Única/metabolismo , Virossomos/metabolismo , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA/genética , Fator de Transcrição E2F1/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 16/metabolismo , Humanos , Cinética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: A specific targeting modality for hepatocellular carcinoma (HCC) could ideally encompass a liver cell specific delivery system of a transcriptional unit that is active only in neoplastic cells. Sendai virosomes, derived from Sendai viral envelopes, home to hepatocytes based on the liver specific expression of asialoglycoprotein receptors (ASGPRs) which are recognized by the Sendai virosomal fusion (F) proteins. As reported earlier by us and other groups, transcriptional gene silencing (TGS) does not require continuous presence of the effector siRNA/shRNA molecule and is heritable, involving epigenetic modifications, leading to long term transcriptional repression. This could be advantageous over conventional gene therapy approaches, since continuous c-Myc inactivation is required to suppress hepatocarcinoma cells. METHODS: Exploiting such virosomal delivery, the alpha-fetoprotein (AFP) promoter, in combination with various tumour specific enhancers, was used to drive the expression of shRNA directed against ME1a1 binding site of the proto-oncogene c-Myc P2 promoter, in order to induce TGS in neoplastic liver cells. RESULTS: The dual specificity achieved by the Sendai virosomal delivery system and the promoter/enhancer guided expression ensured that the shRNA inducing TGS was active only in liver cells that had undergone malignant transformation. Our results indicate that such a bimodal therapeutic system induced specific activation of apoptosis in hepatocarcinoma cells due to heterochromatization and increased DNA methylation of the CpG islands around the target loci. CONCLUSIONS: The Sendai virosomal delivery system, combined with AFP promoter/enhancer expression machinery, could serve as a generalized mechanism for the expression of genes deleterious to transformed hepatocarcinoma cells. In this system, the epigenetic suppression of c-Myc could have an added advantage for inducing cell death in the targeted cells.
Assuntos
Carcinoma Hepatocelular/genética , Hepatócitos/metabolismo , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , alfa-Fetoproteínas/genética , Animais , Células CHO , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Ilhas de CpG , Cricetulus , Metilação de DNA , Inativação Gênica , Terapia Genética , Células Hep G2 , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/patologia , Especificidade de Órgãos , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , Vírus Sendai/genética , VirossomosRESUMO
Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.
Assuntos
Peptídeos/farmacologia , Ribossomos/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ribossomos/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Negative-strand RNA viruses form cytoplasmic inclusion bodies (IBs) representing virus replication foci through phase separation or biomolecular condensation of viral and cellular proteins, as a hallmark of their infection. Alternatively, mammalian cells form stalled mRNA containing antiviral stress granules (SGs), as a consequence of phosphorylation of eukaryotic initiation factor 2α (eIF2α) through condensation of several RNA-binding proteins including TIA-1. Whether and how Chandipura virus (CHPV), an emerging human pathogen causing influenza-like illness, coma and death, forms IBs and evades antiviral SGs remain unknown. By confocal imaging on CHPV-infected Vero-E6 cells, we found that CHPV infection does not induce formation of distinct canonical SGs. Instead, CHPV proteins condense and co-localize together with SG proteins to form heterogeneous IBs, which ensued independent of the activation of eIF2α and eIF2α kinase, protein kinase R (PKR). Interestingly, siRNA-mediated depletion of PKR or TIA-1 significantly decreased viral transcription and virion production. Moreover, CHPV infection also caused condensation and recruitment of PKR to IBs. Compared to SGs, IBs exhibited significant rapidity in disassembly dynamics. Altogether, our study demonstrating that CHPV replication co-optimizes with SG proteins and revealing an unprecedented proviral role of TIA-1/PKR may have implications in understanding the mechanisms regulating CHPV-IB formation and designing antiviral therapeutics. Importance: CHPV is an emerging tropical pathogen reported to cause acute influenza-like illness and encephalitis in children with a very high mortality rate of ~70%. Lack of vaccines and an effective therapy against CHPV makes it a potent pathogen for causing an epidemic in tropical parts of globe. Given these forewarnings, it is of paramount importance that CHPV biology must be understood comprehensively. Targeting of host factors offers several advantages over targeting the viral components due to the generally higher mutation rate in the viral genome. In this study, we aimed at understanding the role of SGs forming cellular RNA-binding proteins in CHPV replication. Our study helps understand participation of cellular factors in CHPV replication and could help develop effective therapeutics against the virus.
Assuntos
Corpos de Inclusão Viral , Antígeno-1 Intracelular de Células T , Replicação Viral , eIF-2 Quinase , eIF-2 Quinase/metabolismo , eIF-2 Quinase/genética , Animais , Antígeno-1 Intracelular de Células T/metabolismo , Antígeno-1 Intracelular de Células T/genética , Chlorocebus aethiops , Células Vero , Corpos de Inclusão Viral/metabolismo , Humanos , Grânulos de Estresse/metabolismo , Corpos de Inclusão/metabolismo , Interações Hospedeiro-Patógeno , Grânulos Citoplasmáticos/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Separação de FasesRESUMO
Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.
Assuntos
Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Pequeno RNA não Traduzido/farmacologia , RNA Viral/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Animais , Sequência de Bases , Feminino , Hepacivirus/metabolismo , Fígado , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Pequeno RNA não Traduzido/química , Replicação Viral/efeitos dos fármacosRESUMO
Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy.
Assuntos
Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vírus Sendai/fisiologia , Transdução de Sinais , Internalização do Vírus , Linhagem Celular , Proteína HN/genética , Proteína HN/metabolismo , Hepatócitos/virologia , Humanos , Vírus Sendai/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
Chandipura virus (CHPV, a member of the Rhabdoviridae family) is an emerging pathogen that causes rapidly progressing influenza-like illness and acute encephalitis often leading to coma and death of the human host. Given several CHPV outbreaks in Indian sub-continent, recurring sporadic cases, neurological manifestation, and high mortality rate of this infection, CHPV is gaining global attention. The 'dark proteome' includes the whole proteome with special emphasis on intrinsically disordered proteins (IDP) and IDP regions (IDPR), which are proteins or protein regions that lack unique (or ordered) three-dimensional structures within the cellular milieu. These proteins/regions, however, play a number of vital roles in various biological processes, such as cell cycle regulation, control of signaling pathways, etc. and, therefore, are implicated in many human diseases. IDPs and IPPRs are also abundantly found in many viral proteins enabling their multifunctional roles in the viral life cycles and their capability to highjack various host systems. The unknown abundance of IDP and IDPR in CHPV, therefore, prompted us to analyze the dark proteome of this virus. Our analysis revealed a varying degree of disorder in all five CHPV proteins, with the maximum level of intrinsic disorder propensity being found in Phosphoprotein (P). We have also shown the flexibility of P protein using extensive molecular dynamics simulations up to 500 ns (ns). Furthermore, our analysis also showed the abundant presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs) in CHPV proteins. The identification of IDPs/IDPRs in CHPV proteins suggests that their disordered regions may function as potential interacting domains and may also serve as novel targets for disorder-based drug designs.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Infecções por Rhabdoviridae/metabolismo , Vesiculovirus/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral/genética , Humanos , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Infecções por Rhabdoviridae/virologia , Alinhamento de Sequência , Vesiculovirus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Understanding the structure-function of inclusion bodies (IBs) in the last two decades has led to the development of several mild solubilization buffers for the improved recovery of bioactive proteins. The recently developed freeze-thaw-based inclusion body protein solubilization method has received a great deal of attention due to its simplicity and cost-effectiveness. The present report investigates the reproducibility, efficiency, and plausible mechanism of the freeze-thaw-based IB solubilization. The percentage recovery of functionally active protein species of human growth hormone (hGH) and L-asparaginase from their IBs in Escherichia coli and the quality attributes associated with the freeze-thaw-based solubilization method were analyzed in detail. The overall yield of the purified hGH and L-asparaginase protein was found to be around 14 and 25%, respectively. Both purified proteins had functionally active species lower than that observed with commercial proteins. Biophysical and biochemical analyses revealed that the formation of soluble aggregates was a major limitation in the case of tough IB protein like hGH. On the other hand, the destabilization of soft IB protein like L-asparaginase led to the poor recovery of functionally active protein species. Our study provides insight into the advantages, disadvantages, and molecular-structural information associated with the freeze-thaw-based solubilization method.
RESUMO
Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine "switch" in HN that triggers fusion.
Assuntos
Proteína HN/fisiologia , Histidina , Vírus da Parainfluenza 3 Humana/fisiologia , Vírus Sendai/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Substituição de Aminoácidos/genética , Proteína HN/genética , Hepatócitos/virologia , Humanos , Mutagênese Sítio-Dirigida , Vírus da Parainfluenza 3 Humana/genética , Vírus Sendai/genéticaRESUMO
UNLABELLED: Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli beta-galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8 microg/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of Crigler-Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% +/- 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% +/- 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. CONCLUSION: FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.
Assuntos
Receptor de Asialoglicoproteína/administração & dosagem , Icterícia/terapia , Proteolipídeos/administração & dosagem , Transposases/administração & dosagem , Animais , Síndrome de Crigler-Najjar/terapia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética/métodos , Glucuronosiltransferase/administração & dosagem , Hepatócitos/efeitos dos fármacos , Humanos , Hiperbilirrubinemia/terapia , Ratos , Ratos Gunn , Proteínas Virais de Fusão/administração & dosagemRESUMO
COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.
Assuntos
Betacoronavirus/química , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Amino Açúcares/metabolismo , Betacoronavirus/fisiologia , Sítios de Ligação , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácido N-Acetilneuramínico/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores de Coronavírus , Receptores Virais/química , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , SARS-CoV-2 , Antígeno Sialil Lewis X/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Tropismo Viral , Internalização do VírusRESUMO
Ex vivo gene transfer into hepatocytes could serve several purposes in the context of gene therapy or cell transplantation: (1) isolated hepatocytes can be transduced in culture with therapeutic genes and then transplanted into the recipient; (2) marker genes can be introduced for subsequent identification of transplanted cells and their progeny; (3) gene transfer can be used for conditional immortalization of hepatocytes for expansion in culture; (4) immunomodulatory genes can be transferred into hepatocytes to prevent allograft rejection. Gene transfer into cultured hepatocytes can be achieved using DNA that is not incorporated into recombinant viruses. In such systems, transgene integration into the host cell genome can be enhanced using transposon systems, such as "sleeping beauty." In addition to using the conventional reagents, such as cationic liposomes, DNA transfer into hepatocytes can be achieved by Nucleofection or special hepatocyte-targeted carriers such as proteoliposomes containing galactose-terminated glycoproteins (e.g. the F protein of the Sendai virus). Alternatively, genes can be transferred using recombinant viruses, such as adenoviral vectors that are episomal or retroviral vectors (including lentiviruses) that permit integration of the transgene into the host genome. Gene transfer using lentiviral vectors has been achieved in both attached and suspended hepatocytes. Transduction efficiency of lentiviral vectors can be enhanced using magnetic nanoparticles (Magnetofection).
Assuntos
Técnicas de Transferência de Genes , Hepatócitos/metabolismo , Animais , Linhagem Celular Transformada/citologia , Terapia Genética/métodos , Hepatócitos/citologia , Hepatócitos/transplante , Humanos , Transplante de Fígado/métodos , Magnetismo/métodos , Modelos Biológicos , Nanopartículas/uso terapêutico , Coloração e Rotulagem/métodos , Condicionamento Pré-Transplante/métodosRESUMO
Connivance of cellular factors during virus-host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin-null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c-src. However, the villin RRI mutant, lacking actin-severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome-CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell-type dependent phosphorylation in regulating membrane fusion.
Assuntos
Membrana Celular/virologia , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Vírus Sendai/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Células CHO , Membrana Celular/fisiologia , Cricetulus , Interações Hospedeiro-Patógeno , Fusão de Membrana , Proteínas dos Microfilamentos/metabolismo , Mutação , Fosforilação , Tirosina/química , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Investigation of the potential association of single nucleotide polymorphisms (SNPs) at -308 G/A and -238 G/A of Tumor necrosis factor alpha (TNFalpha) with susceptibility to HPV-16 associated cervical cancer in Indian women. METHODS: The study included 165 histologically confirmed cases with 45 precancer and 120 cancer patients and an equal number (165) of healthy controls with normal cervical cytology. PCR-RFLP was employed to analyze TNFalpha promoter polymorphisms, which were confirmed by direct sequencing. Both patients and controls were screened for Human Papillomavirus (HPV) infection. RESULTS: The frequency of -308 A allele in TNFalpha was significantly higher in cases compared with control subjects (21% in cases vs. 9% in controls; p<0.01), with an odds ratio of 2.7 (95% CI = 1.41-5.15). Also, women carrying A allele for this locus presented 3 times increased susceptibility to HPV 16 infection as evident from carrier genotype distribution between HPV positive cases and control subjects (24% in HPV positive cases vs. 9% in controls; p<0.01; OR = 3.1; 95% CI = 1.60-6.03). No such association was found for TNFalpha-238 (G/A) polymorphism with the risk of development of cervical cancer. CONCLUSION: It suggests that SNP at -308 (G/A) of TNFalpha promoter may represent an increased risk for HPV infection and development of cervical cancer in Indian women.
Assuntos
Predisposição Genética para Doença/genética , Papillomavirus Humano 16/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Fator de Necrose Tumoral alfa/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Adenina , Sequência de Bases , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Genótipo , Guanina , Humanos , Índia/epidemiologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Lesões Pré-Cancerosas/genética , Prevalência , Regiões Promotoras Genéticas/genética , Neoplasias do Colo do Útero/epidemiologia , População Branca/genéticaRESUMO
Double-stranded RNA-mediated transcriptional gene silencing (TGS) has shown promising results over posttranscriptional gene silencing (PTGS) due to its long term and heritable nature. Various research groups have shed light on different mechanisms by which TGS operate. Some of these include histone modification, DNA methylation, or restriction of RNA polymerase binding onto the target gene's promoter. This serves as an added advantage since permanent c-Myc inactivation is critical for suppressing hepatocellular carcinoma (HCC). Inability to target cancer cells specifically, without affecting the normal cells, has been one of the biggest drawbacks of an effective cancer therapy. Therefore, we aimed to overcome this barrier by first generating tumor-specific transcriptional units expressing TGS inducing shRNAs against c-Myc's P2 promoter only in neoplastic liver cells. Secondly, we coupled this TGS inducing system with Sendai fusion virosomes for liver-specific delivery to minimize nonspecific side effects in vitro.
Assuntos
Carcinoma Hepatocelular/genética , Inativação Gênica , Técnicas de Transferência de Genes , Genes myc , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Células Cultivadas , Ilhas de CpG , Metilação de DNA , Humanos , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , VirossomosRESUMO
Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion-mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206-an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.