Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template

Full-length poliovirus complementary DNA (cDNA) was synthesized by assembling oligonucleotides of plus and minus strand polarity. The synthetic poliovirus cDNA was transcribed by RNA polymerase into viral RNA, which translated and replicated in a cell-free extract, resulting in the de novo synthesis of infectious poliovirus. Experiments in tissue culture using neutralizing antibodies and CD155 receptor-specific antibodies and neurovirulence tests in CD155 transgenic mice confirmed that the synthetic virus had biochemical and pathogenic characteristics of poliovirus. Our results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence.     

Phospholipase-induced crystallization of channels in mitochondrial outer membranes

When outer membranes from Neurospora crassa mitochondria are treated with low levels of phospholipase A2 under continuous dialysis, two-dimensional crystalline arrays of the pore protein component of these membranes are formed.     

Membranous structures associated with translation and transcription of poliovirus RNA

Poliovirus RNA and proteins are synthesized in association with distinct membranous structures that were separated by means of Isopycnic centrifugation of cytoplasmic extracts in discontinuous sucrose-density gradients. Viral RNA is replicated in a structure that contains rapidly labeled replicative intermediate RNA and viral RNA polymerase associated with the smooth membrane fraction. In sucrose gradients this viral RNA replication complex is distributed at densities in the range of 1.12 to 1.18 grams per cubic centimeter. Viral proteins are synthesized on polyribosomes bound to membranes and sediment with polyribosomes at densities of less than 1.25 grams per cubic centimeter.     

Detergent-solubilized RNA polymerase from cells infected with foot-and-mouth disease virus

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.     

Cloned poliovirus complementary DNA is infectious in mammalian cells

A complete, cloned complementary DNA copy of the RNA genome of poliovirus was constructed in the Pst I site of the bacterial plasmid pBR322. Cultured mammalian cells transfected with this hybrid plasmid produced infectious poliovirus. Cells transfected with a plasmid which lacked the first 115 bases of the poliovirus genome did not produce virus.     

Specific inhibition of viral ribonucleic acid replication by Gliotoxin

Gliotoxin inhibits intracellular replication of poliovirus in HeLa cells at a stage subsequent to adsorption and penetration of virus. The sensitive step is synthesis of viral RNA: synthesis of viral protein is unaffected except as a consequence of blockade of RNA synthesis. Concentrations of gliotoxin sufficient to block viral RNA synthesis completely do not affect cellular RNA synthesis.     

Translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region

The central portion (region P) of the 742-nucleotide noncoding 5' end of poliovirus allows the RNA to initiate protein synthesis in the absence of the usual 5' 7-methylguanosine capping group. Poliovirus 5' noncoding region was fused to a reporter gene and transfected into cells. There was extensive augmentation of the expression of this gene by poliovirus-mediated inhibition of cap-dependent protein synthesis. That the construct initiated in a cap-independent manner was verified through in vitro experiments. Small lesions throughout region P blocked its initiation function, implying that a coherent functional unit, hundreds of nucleotides long, is responsible for cap-independent initiation by poliovirus RNA.     

膜片钳技术及其应用

膜片钳技术是在电压钳基础上发展的，将尖端为1μm的玻璃微电极吸附到细胞表面，使微电极与细胞膜形成高阻封接，从而可记录到膜上pA级的离子通道电流，目前已发展了多种记录模式，对细胞进行电压和电流钳制，以观察各种离子通道及其调控。此外膜片钳技术与胞内纤维荧光测钙技术、膜电容监测技术、碳纤维电极局部电化学微量检测技术、逆转录多聚酶链式反应技术（PCR）结合，在药理学、病理学、神经科学、脑科学、细胞生物学和分子生物学等生物科学方面，得到越来越广泛的应用。     

Intestinal microbiota promote enteric virus replication and systemic pathogenesis

Intestinal bacteria aid host health and limit bacterial pathogen colonization. However, the influence of bacteria on enteric viruses is largely unknown. We depleted the intestinal microbiota of mice with antibiotics before inoculation with poliovirus, an enteric virus. Antibiotic-treated mice were less susceptible to poliovirus disease and supported minimal viral replication in the intestine. Exposure to bacteria or their N-acetylglucosamine-containing surface polysaccharides, including lipopolysaccharide and peptidoglycan, enhanced poliovirus infectivity. We found that poliovirus binds lipopolysaccharide, and exposure of poliovirus to bacteria enhanced host cell association and infection. The pathogenesis of reovirus, an unrelated enteric virus, also was more severe in the presence of intestinal microbes. These results suggest that antibiotic-mediated microbiota depletion diminishes enteric virus infection and that enteric viruses exploit intestinal microbes for replication and transmission.     

A kinetic framework for a mammalian RNA polymerase in vivo

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.     

Poliovirus mutants resistant to neutralization with soluble cell receptors

Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.     

Multienzyme systems of DNA replication

Replication is accomplished by multienzyme systems whose operations are usefully considered in respect to three stages of the process: initiation, elongation, anid termination. 1) Initiation entails synthesis of a short RNA fragment that serves as primer for the elongation step of DNA synthesis. This stage, probed by SS phage DNA templates, reveals three distinctive and highly specific systems in E. coli. The Ml3 DNA utilizes RNA polymerase in a manner that may reflect how plasmid elements are replicated in the cell. The ?X174 DNA does not rely on RNA-polymerase, but requires instead five distinctive proteins which may belong to an apparatus for initiating a host chromosome replication cycle at the origin. The G4 DNA, also independent of RNA polymerase, needs simply the dnaG protein for its distinctive initiation and may thus resemble the system that initiates the replication fragments at the nascent growing fork. In each case it is essential that in vitro the DNA-unwinding protein coat the viral DNA and influence its structure. 2) Elongation is achieved in every case by the multisubunit, holoenzyme form of DNA polymerase III. Copolymerase III, which is an enzyme subunit, and adenosine triphosphate are required to form a proper complex with the primer template but appear dispensable for the ensuing chain growth by DNA polymerase (33). 3) Termination requires excision of the RNA priming fragment, filling of gaps and sealing of interruptions to produce a covalently intact phosphodiester backbone. DNA polymerase I has the capacity for excision and gapfilling and DNA ligase is required for sealing. What once appeared to be a simple DNA polymerase-mediated conversion of a single-strand to a duplex circle (34) is now seen as a complex series of events in which diverse multienzyme systems function. Annoyance with the difficulties in resolving and reconstituting these systems is tempered by the conviction that these are the very systems used ,by the cell in replicating its chromosome and extrachromosomal elements. Thus, understanding of the regulation of replication events in the cell, their localization at membrane surfaces and integration with cell division, and their coordination with phage DNA maturation and particle assembly will all be advanced by knowledge of the components of the replicative machinery.     

Computer simulations of self-assembled membranes

Molecular dynamics simulations in three dimensions of particles that self-assemble to form two-dimensional, membrane-like objects are presented. Anisotropic, multibody forces, chosen so as to mimic real interactions between amphiphilic molecules, generate a finite rigidity and compressibility of the assembled membranes, as well as a finite line tension at their free edges. This model and its generalizations can be used to study a large class of phenomena taking place in fluctuating membranes. For instance, both fluid and solid-like phases, separated by a phase transition, are obtained and some of the large-scale properties of these membranes studied. In particular, thermal undulations of quasi-spherical fluid vesicles are analyzed, in a manner similar to recent experiments in lipid systems.     

Crystal structure of the rabies virus nucleoprotein-RNA complex

Negative-strand RNA viruses condense their genome into a helical nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA polymerase complex. The crystal structure of a recombinant rabies virus nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by nucleoproteins is likely a common mechanism used by negative-strand RNA viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.     

植物表观遗传中的RNA介导的DNA甲基化

植物基因组对基因的表达调控不仅表现在转录水平上,也表现在染色质结构的变化上。在植物中存在着特有的RNA 聚合酶Ⅳ (RNA Pol Ⅳ) 和 RNA聚合酶Ⅴ (RNA Pol Ⅴ),它们与RNA 聚合酶Ⅱ(RNA Pol Ⅱ)相似。RNA Pol Ⅳ和RNA Pol Ⅴ的转录产物包括参与表观遗传调控的长链非编码RNA (lncRNAs)和干扰小RNA (siRNAs)。这类非编码RNA广泛地参与了基因组上发生的胞嘧啶甲基化,去甲基化以及甲基化扩散。近年来,研究已经发现RNA介导的染色质水平的基因沉默涉及植物的生长发育、胁迫应答、表观遗传多态性,并对植物的表型多样化、生理适应性以及植物进化具有显著影响。