RESUMEN
The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.
Asunto(s)
Cisteína Endopeptidasas , Virus de la Influenza A , Proteínas no Estructurales Virales , Replicación Viral , Animales , Perros , Humanos , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Enzimas Desubicuitinizantes/metabolismo , Células HEK293 , Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Gripe Humana/virología , ARN Viral/metabolismo , ARN Viral/genética , Ubiquitinación , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Replicación Viral/fisiología , Línea Celular , Células Vero , Chlorocebus aethiopsRESUMEN
BACKGROUND: Amoxicillin resistance in Helicobacter pylori is mainly associated with mutations in penicillin-binding protein-1A (PBP-1A). However, the specific amino acid substitutions in PBP-1A that confer amoxicillin resistance in H. pylori remain to be investigated. OBJECTIVE: This study aimed to investigate the molecular mechanism underlying amoxicillin resistance in patients with refractory H. pylori infection. METHODS: Esophagogastroduodenoscopy (EGD) was performed in patients with persistent H. pylori infection after at least two courses of H. pylori eradication therapy between January-2018 to March-2021. Refractory H. pylori was cultured from the gastric biopsy specimens. Antibiotic susceptibility testing was conducted to determine the minimum inhibitory concentrations (MICs). Sequence analysis of pbp-1A was performed for amoxicillin-resistant strains. RESULTS: Thirty-nine successfully cultured isolates were classified as refractory H. pylori isolates, and seventeen isolates were resistant to amoxicillin (MIC > 0.125 mg/L). Sequence analysis of resistant strains showed multiple mutations in the C-terminal region of PBP-1A that conferred amoxicillin resistance in H. pylori. However, the number of PBP-1A mutations did not correlate with the high MICs of amoxicillin-resistant isolates. Notably, some amino acid substitutions were identified in all Taiwanese isolates with history of eradication failure but not in published amoxicillin-susceptible strains, suggesting that the mutations may play a role in conferring antibiotic resistance to these strains. CONCLUSIONS: Our results show that amoxicillin resistance in refractory H. pylori is highly correlated with numerous PBP-1A mutations that are strain specific. Continuous improvements in diagnostic tools, particularly molecular analysis approaches, can help to optimize current antimicrobial regimens.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Humanos , Amoxicilina/farmacología , Amoxicilina/uso terapéutico , Proteínas de Unión a las Penicilinas/genética , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/genética , Sustitución de Aminoácidos , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana/genéticaRESUMEN
Influenza A virus (IAV) is widely disseminated across different species and can cause recurrent epidemics and severe pandemics in humans. During infection, IAV attaches to receptors that are predominantly located in cell membrane regions known as lipid rafts, which are highly enriched in cholesterol and sphingolipids. Following IAV entry into the host cell, uncoating, transcription, and replication of the viral genome occur, after which newly synthesized viral proteins and genomes are delivered to lipid rafts for assembly prior to viral budding from the cell. Moreover, during budding, IAV acquires an envelope with embedded cholesterol from the host cell membrane, and it is known that decreased cholesterol levels on IAV virions reduce infectivity. Statins are commonly used to inhibit cholesterol synthesis for preventing cardiovascular diseases, and several studies have investigated whether such inhibition can block IAV infection and propagation, as well as modulate the host immune response to IAV. Taken together, current research suggests that there may be a role for statins in countering IAV infections and modulating the host immune response to prevent or mitigate cytokine storms, and further investigation into this is warranted.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas , Virus de la Influenza A , Gripe Humana , Colesterol/metabolismo , Humanos , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Microdominios de Membrana/metabolismo , Esfingolípidos/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Helicobacter pylori infection is closely associated with various gastrointestinal diseases and poses a serious threat to human health owing to its increasing antimicrobial resistance. H. pylori possesses two major virulence factors, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), which are involved in its pathogenesis. Probiotics have recently been used to eradicate H. pylori infection and reduce the adverse effects of antibiotic-based therapies. Parabacteroides goldsteinii MTS01 is a novel next-generation probiotic (NGP) with activities that can alleviate specific diseases by altering the gut microbiota. However, the mechanism by which P. goldsteinii MTS01 exerts its probiotic effects against H. pylori infection remains unclear. Our results showed that administration of P. goldsteinii MTS01 to H. pylori-infected model mice altered the composition of the gut microbiota and significantly reduced serum cholesterol levels, which mitigated H. pylori-induced gastric inflammation. In addition, the pathogenic effects of H. pylori VacA and CagA on gastric epithelial cells were markedly abrogated by treatment with P. goldsteinii MTS01. These results indicate that P. goldsteinii MTS01 can modulate gut microbiota composition and has anti-virulence factor functions, and thus could be developed as a novel functional probiotic for reducing H. pylori-induced pathogenesis.
Asunto(s)
Microbioma Gastrointestinal , Infecciones por Helicobacter , Helicobacter pylori , Animales , Antibacterianos/farmacología , Antígenos Bacterianos/genética , Bacteroidetes , Colesterol , Citotoxinas , Infecciones por Helicobacter/complicaciones , Humanos , Ratones , Factores de Virulencia/genéticaRESUMEN
Influenza A virus (IAV) has caused recurrent epidemics and severe pandemics. In this study, we adapted an MS2-MCP live-cell imaging system to visualize IAV replication. A reporter plasmid, pHH-PB2-vMSL, was constructed by replacing a part of the PB2-coding sequence in pHH-PB2 with a sequence encoding 24 copies of a stem-loop structure from bacteriophage MS2 (MSL). Binding of MS2 coat protein (MCP) fused to green fluorescent protein (GFP) to MSL enabled the detection of vRNA as fluorescent punctate signals in live-cell imaging. The introduction of pHH-PB2-vMSL into A549 cells transduced to express an MCP-GFP fusion protein lacking the nuclear localization signal (MCP-GFPdN), subsequently allowed tracking of the distribution and replication of PB2-vMSL vRNA after IAV PR8 infection. Spatial and temporal measurements revealed exponential increases in vRNA punctate signal intensity, which was only observed after membrane blebbing in apoptotic cells. Similar signal intensity increases in apoptotic cells were also observed after MDCK cells, transduced to express MCP-GFPdN, were infected with IAV carrying PB2-vMSL vRNA. Notably, PB2-vMSL vRNA replication was observed to occur only in apoptotic cells, at a consistent time after apoptosis initiation. There was a lack of observable PB2-vMSL vRNA replication in non-apoptotic cells, and vRNA replication was suppressed in the presence of apoptosis inhibitors. These findings point to an important role for apoptosis in IAV vRNA replication. The utility of the MS2-imaging system for visualizing time-sensitive processes such as viral replication in live host cells is also demonstrated in this study.
RESUMEN
Conventionally, statins are used to treat high cholesterol levels. They exhibit pleiotropic effects, such as the prevention of cardiovascular disease and decreased cancer mortality. Gastric cancer (GC) is one of the most common cancers, ranking as the third leading global cause of cancer-related deaths, and is mainly attributed to chronic Helicobacter pylori infection. During their co-evolution with hosts, H. pylori has developed the ability to use the cellular components of the host to evade the immune system and multiply in intracellular niches. Certain H. pylori virulence factors, including cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA), and cholesterol-α-glucosyltransferase (CGT), have been shown to exploit host cholesterol during pathogenesis. Therefore, using statins to antagonize cholesterol synthesis might prove to be an ideal strategy for reducing the occurrence of H. pylori-related GC. This review discusses the current understanding of the interplay of H. pylori virulence factors with cholesterol and reactive oxygen species (ROS) production, which may prove to be novel therapeutic targets for the development of effective treatment strategies against H. pylori-associated GC. We also summarize the findings of several clinical studies on the association between statin therapy and the development of GC, especially in terms of cancer risk and mortality.
RESUMEN
BGLF2 is a tegument protein of the Epstein-Barr virus (EBV). This study finds that BGLF2 is expressed in the late stage of the EBV lytic cycle. Microscopic investigations reveal that BGLF2 is present in both the nucleus and the cytoplasm and colocalized with BBLF1 and gp350 at juxtanuclear regions in the cytoplasm. This study also finds that the basic KKK69 motif of BGLF2 and acidic DYEE31 motif of BBLF1 are crucial for the interaction between BGLF2 and BBLF1, which is required for the recruitment of BGLF2 to the BBLF1 that is anchored on the trans-Golgi-network (TGN). In addition, BGLF2 in a density gradient is co-sedimented with un-enveloped capsids, revealing that BGLF2 associates with the EBV capsid before the final envelopment. The knockout of BGLF2 expression is demonstrated to reduce the numbers of infectious virions that are released into the culture medium, but they do not affect the expression of lytic proteins and viral DNA replication. The production of infectious viral particles by a BGLF2-knockout mutant can be rescued by exogenously expressed BGLF2 but only partially rescued by BGLF2-3KA, which is a mutant with reduced ability to interact with BBLF1 but does not affect its ability to activate the MAPK pathway and the expression of the EBV lytic proteins, suggesting that the interaction of BGLF2 with BBLF1 is important to the efficient production of infectious viral particles during the maturation. The results of this study improve our understanding of how BGLF2 promotes EBV viral production.
RESUMEN
Cholesterol-α-glucosyltransferase (CGT) encoded by the type 1 capsular polysaccharide biosynthesis protein J (capJ) gene of Helicobacter pylori converts cellular cholesterol into cholesteryl glucosides. H. pylori infection induces autophagy that may increase bacterial survival in epithelial cells. However, the role of H. pylori CGT that exploits lipid rafts in interfering with autophagy for bacterial survival in macrophages has not been investigated. Here, we show that wild-type H. pylori carrying CGT modulates cholesterol to trigger autophagy and restrain autophagosome fusion with lysosomes, permitting a significantly higher bacterial burden in macrophages than that in a capJ-knockout (∆CapJ) mutant. Knockdown of autophagy-related protein 12 impairs autophagosome maturation and decreases the survival of internalised H. pylori in macrophages. These results demonstrate that CGT plays a crucial role in the manipulation of the autophagy process to impair macrophage clearance of H. pylori.
Asunto(s)
Autofagia/fisiología , Colesterol/metabolismo , Glucosiltransferasas/metabolismo , Helicobacter pylori/metabolismo , Macrófagos/microbiología , Animales , Autofagosomas/metabolismo , Proteína 12 Relacionada con la Autofagia/genética , Proteína 12 Relacionada con la Autofagia/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Inactivación de Genes , Glucosiltransferasas/genética , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Lisosomas/metabolismo , Lisosomas/microbiología , Microdominios de Membrana/metabolismo , RatonesRESUMEN
The human tumor viruses that replicate as plasmids (we use the term plasmid to avoid any confusion in the term episome, which was coined to mean DNA elements that occur both extrachromosomally and as integrated forms during their life cycles, as does phage lambda) share many features in their DNA synthesis. We know less about their mechanisms of maintenance in proliferating cells, but these mechanisms must underlie their partitioning to daughter cells. One amazing implication of how these viruses are thought to maintain themselves is that while host chromosomes commit themselves to partitioning in mitosis, these tumor viruses would commit themselves to partitioning before mitosis and probably in S phase shortly after their synthesis.
Asunto(s)
Replicación del ADN/genética , ADN Viral/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Papillomaviridae/genética , Plásmidos/genética , ADN Viral/biosíntesis , Humanos , Mitosis/genética , Replicón/genéticaRESUMEN
Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi's sarcoma-associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning and viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.
Asunto(s)
Cromosomas , Replicación del ADN , ADN Viral/genética , Genoma Viral , Inestabilidad Genómica , Herpesvirus Humano 8/genética , Replicación Viral , Antígenos Virales/genética , Antígenos Virales/metabolismo , Simulación por Computador , ADN Viral/biosíntesis , Evolución Molecular , Regulación Viral de la Expresión Génica , Células HEK293 , Células HeLa , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/crecimiento & desarrollo , Herpesvirus Humano 8/metabolismo , Interacciones Huésped-Patógeno , Humanos , Hibridación Fluorescente in Situ , Microscopía Confocal , Microscopía por Video , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Factores de Tiempo , Imagen de Lapso de Tiempo , TransfecciónRESUMEN
The intrinsic properties of different viruses have driven their study. For example, the capacity for efficient productive infection of cultured cells by herpes simplex virus 1 has made it a paradigm for this mode of infection for herpesviruses in general. Epstein-Barr virus, another herpesvirus, has two properties that have driven its study: It causes human cancers, and it exhibits a tractable transition from its latent to its productive cycle in cell culture. Here, we review our understanding of the path Epstein-Barr virus follows to move from a latent infection to and through its productive cycle. We use information from human infections to provide a framework for describing studies in cell culture and, where possible, the molecular resolutions from these studies. We also pose questions whose answers we think are pivotal to understanding this path, and we provide answers where we can.
Asunto(s)
Herpesvirus Humano 4/crecimiento & desarrollo , Activación Viral/genética , Latencia del Virus/genética , Replicación Viral/genética , Células Cultivadas , ADN Viral/metabolismo , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Regulación Viral de la Expresión Génica/genética , Herpesvirus Humano 4/genética , Humanos , Ensamble de Virus/genéticaRESUMEN
Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit "true" late kinetics-their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other ß- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these "ßγ" genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt) was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Herpesvirus Humano 4/genética , Origen de Réplica/genética , Replicación Viral/genética , Replicación del ADN/genética , ADN Viral/genética , Genes Virales/genética , Células HEK293 , Humanos , Immunoblotting , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
Epstein-Barr virus and human papillomaviruses are human tumor viruses that infect and replicate in upper aerodigestive tract epithelia and cause head and neck cancers. The productive phases of both viruses are tied to stratified epithelia highlighting the possibility that these viruses may affect each other's life cycles. Our lab has established an in vitro model system to test the effects of EBV and HPV co-infection in stratified squamous oral epithelial cells. Our results indicate that HPV increases maintenance of the EBV genome in the co-infected cells and promotes lytic reactivation of EBV in upper layers of stratified epithelium. Expression of the HPV oncogenes E6 and E7 were found to be necessary and sufficient to account for HPV-mediated lytic reactivation of EBV. Our findings indicate that HPV increases the capacity of epithelial cells to support the EBV life cycle, which could in turn increase EBV-mediated pathogenesis in the oral cavity.
Asunto(s)
Herpesvirus Humano 4/fisiología , Queratinocitos/virología , Papillomaviridae/fisiología , Simbiosis , Línea Celular Transformada , Células Cultivadas , Humanos , Mucosa Bucal/virología , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/clasificación , Técnicas de Cultivo de Tejidos , Activación Viral , Latencia del Virus , Replicación ViralRESUMEN
Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three-dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Epitelio/virología , Papillomaviridae/crecimiento & desarrollo , Infecciones por Papillomavirus/virología , Cultivo de Virus/métodos , Humanos , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificaciónRESUMEN
Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.
Asunto(s)
Autofagia , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Herpesvirus Humano 4/inmunología , Proteínas Inmediatas-Precoces/fisiología , Transactivadores/fisiología , Secuencia de Bases , Cartilla de ADN , Células HEK293 , Humanos , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
The spontaneous transition of Epstein-Barr virus (EBV) from latency to productive infection is infrequent, making its analysis in the resulting mixed cell populations difficult. We engineered cells to support this transition efficiently and developed EBV DNA variants that could be visualized and measured as fluorescent signals over multiple cell cycles. This approach revealed that EBV's productive replication began synchronously for viral DNAs within a cell but asynchronously between cells. EBV DNA amplification was delayed until early S phase and occurred in factories characterized by the absence of cellular DNA and histones, by a sequential redistribution of PCNA, and by localization away from the nuclear periphery. The earliest amplified DNAs lacked histones accompanying a decline in four histone chaperones. Thus, EBV transits from being dependent on the cellular replication machinery during latency to commandeering both that machinery and nuclear structure for its own reproductive needs.
Asunto(s)
Replicación del ADN , ADN Viral/metabolismo , Herpesvirus Humano 4/fisiología , Histonas/metabolismo , Línea Celular , Herpesvirus Humano 4/genética , Humanos , Activación ViralRESUMEN
Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies (1-3). These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres (4-8). Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell. Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest (9). The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed. This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase(10). The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes(11). In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.
Asunto(s)
Replicación del ADN , Microscopía Fluorescente/métodos , Plásmidos/química , Células HeLa , Infecciones por Herpesviridae/patología , Herpesvirus Humano 8/genética , Humanos , Operón Lac , Plásmidos/genética , Plásmidos/metabolismo , Secuencias Repetidas en TándemRESUMEN
Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV's life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV's miRNAs.
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Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , MicroARNs/genética , Complejo Silenciador Inducido por ARN/metabolismo , Línea Celular , Biología Computacional , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , Inmunoprecipitación , MicroARNs/metabolismo , Unión Proteica , Proteómica , ARN Viral , Reproducibilidad de los ResultadosRESUMEN
Epstein-Barr virus (EBV) BBLF1 shares 13 to 15% amino acid sequence identities with the herpes simplex virus 1 UL11 and cytomegalovirus UL99 tegument proteins, which are involved in the final envelopment during viral maturation. This study demonstrates that BBLF1 is a myristoylated and palmitoylated protein, as are UL11 and UL99. Myristoylation of BBLF1 both facilitates its membrane anchoring and stabilizes it. BBLF1 is shown to localize to the trans-Golgi network (TGN) along with gp350/220, a site where final envelopment of EBV particles takes place. The localization of BBLF1 at the TGN requires myristoylation and two acidic clusters, which interact with PACS-1, a cytosolic protein, to mediate retrograde transport from the endosomes to the TGN. Knockdown of the expression of BBLF1 during EBV lytic replication reduces the production of virus particles, demonstrating the requirement of BBLF1 to achieve optimal production of virus particles. BBLF1 is hypothesized to facilitate the budding of tegumented capsid into glycoprotein-embedded membrane during viral maturation.