Relative Neurotropism of a Recombinant Rhabdovirus Expressing a Green Fluorescent Envelope Glycoprotein

A new recombinant vesicular stomatitis virus (rVSV) that expresses green fluorescent protein (GFP) on the cytoplasmic domain of the VSV glycoprotein (G protein) was used in the mouse as a model for studying brain infections by a member of the Mononegavirales order that can cause permanent changes in behavior. After nasal administration, virus moved down the olfactory nerve, first to periglomerular cells, then past the mitral cell layer to granule cells, and finally to the subventricular zone. Eight days postinoculation, rVSV was eliminated from the olfactory bulb. Little sign of infection could be found outside the olfactory system, suggesting that anterograde or retrograde axonal transport of rVSV was an unlikely mechanism for movement of rVSV out of the bulb. When administered intracerebrally by microinjection, rVSV spread rapidly within the brain, with strong infection at the site of injection and at some specific periventricular regions of the brain, including the dorsal raphe, locus coeruleus, and midline thalamus; the ventricular system may play a key role in rapid rVSV dispersion within the brain. Thus, the lack of VSV movement out of the olfactory system was not due to the absence of potential for infections in other brain regions. In cultures of both mouse and human central nervous system (CNS) cells, rVSV inoculations resulted in productive infection, expression of the G-GFP fusion protein in the dendritic and somatic plasma membrane, and death of all neurons and glia, as detected by ethidium homodimer nuclear staining. Although considered a neurotropic virus, rVSV also infected heart, skin, and kidney cells in dispersed cultures. rVSV showed a preference for immature neurons in vitro, as shown by enhanced viral infection in developing hippocampal cultures and in the outer granule cell layer in slices of developing cerebellum. Together, these data suggest a relative affinity of rVSV for some neuronal types in the CNS, adding to our understanding of the long-lasting changes in rodent behavior found after transient VSV infection.     

Visualizing Bdellovibrio bacteriovorus by Using the tdTomato Fluorescent Protein

Bdellovibrio bacteriovorus is a Gram-negative bacterium that belongs to the delta subgroup of proteobacteria and is characterized by a predatory life cycle. In recent years, work has highlighted the potential use of this predator to control bacteria and biofilms. Traditionally, the reduction in prey cells was used to monitor predation dynamics. In this study, we introduced pMQ414, a plasmid that expresses the tdTomato fluorescent reporter protein, into a host-independent strain and a host-dependent strain of B. bacteriovorus 109J. The new construct was used to conveniently monitor predator proliferation in real time, in different growth conditions, in the presence of lytic enzymes, and on several prey bacteria, replicating previous studies that used plaque analysis to quantify B. bacteriovorus. The new fluorescent plasmid also enabled us to visualize the predator in liquid cultures, in the context of a biofilm, and in association with human epithelial cells.     

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties. This new preparation also allowed for access of perfusates to the synapse. We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity. This everted the larval body wall, like turning a sock inside-out. We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore. To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.     

Role of the cytoplasmic tail domains of Bunyamwera orthobunyavirus glycoproteins Gn and Gc in virus assembly and morphogenesis

The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.     

Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 10⁴ transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP⁺ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1.     

A Super-Ecliptic,pHluorin-mKate2, Tandem Fluorescent Protein-Tagged Human LC3 for the Monitoring of Mammalian Autophagy

Tandem fluorescent protein-tagged LC3s that were comprised of a protein tag that emits green fluorescence (e.g., EGFP or mWasabi) fused with another tag that emits red fluorescence (e.g. mCherry or TagRFP) were used for monitoring the maturation step of mammalian autophagosomes. A critical point for this tandem fluorescent-tagged LC3 was the sensitivity of green fluorescence at an acidic pH. EGFP and mWasabi continue to emit a weak, but significant, fluorescence at a pH of approximately 6. To overcome this issue, we focused on super-ecliptic pHluorin, which is a more pH-sensitive GFP variation. The green fluorescence of EGFP and mWasabi in the cells was still observed at weakly acidic levels (pH 6.0–6.5). In contrast, the fluorescence of pHluorin was more significantly quenched at pH 6.5, and was almost completely abolished at pH 5.5–6.0, indicating that pHluorin is more suitable for use in a tandem fluorescent protein-tag for monitoring autophagy. A pHluorin-mKate2 tandem fluorescence protein showed pH-sensitive green fluorescence and pH-resistant far-red fluorescence. We therefore generated expression plasmids for pHluorin-mKate2-tagged human LC3 (PK-hLC3), which could be used as a modifier for LC3-lipidation. The green and far-red fluorescent puncta of PK-hLC3 were increased under starvation conditions. Puncta that were green-negative, but far-red positive, were increased when autolysosomes accumulated, but few puncta of the mutant PK-hLC3ΔG that lacked the carboxyl terminal Gly essential for autophagy were observed in the cells under the same conditions. These results indicated that the PK-hLC3 were more appropriate for the pH-sensitive monitoring of the maturation step of autophagosomes.     

表达绿色荧光蛋白嵌合狂犬病病毒HEP-GFP株的拯救

狂犬病毒(Rabies Virus,RV)是人和犬、猫等多种动物狂犬病的病原,其基因组是单股负链RNA,基因组结构为3'-核蛋白(N)基因-磷蛋白(P)基因-基质蛋白(M)基因-糖蛋白(G)基因-大蛋白(L)基因-5'.     

表达狂犬病病毒糖蛋白的重组犬2型腺病毒的构建

本试验以犬2型腺病毒全基因组重组质粒pPolyⅡCAV 2及其E3 区重组质粒pVAX E3 为基础,通过DraⅢ和SspⅠ双酶切,缺失第25097bp 26141bp共1044bp的E3区片段,按与编码链相同转录方向插入由CMV启动子、狂犬病病毒SRV9 株糖蛋白基因、SV40 polyA基因构成的总长2424bp的表达盒,获得重组基因组质粒pPolyⅡCAV 2 CGS(34.7kb)。以AscⅠ和ClaⅠ双酶切,游离重组基因组(32.7kb),在脂质体LipofectamineTM 2000 介导下,转染MDCK细胞系,获得了E3 缺失区携带狂犬病病毒糖蛋白表达盒的重组犬2 型腺病毒CAV 2 CGS。Western印迹试验表明,CAV 2 CGS表达了狂犬病病毒糖蛋白。初步接种试验显示,重组病毒可以诱导犬产生狂犬病病毒特异性抗体。     

表达绿色荧光蛋白的重组CVI988病毒的构建及特性

提取马立克氏病毒Ⅰ型疫苗毒株CVI988的总DNA为模板,利用PCR技术扩增出病毒生长非必需的US2基因并克隆入T—easy载体。将CMV启动子和增强子控制的含GFP基因表达盒克隆入US2基因中,成功构建了含GFP基因的转移质粒载体pGUS2GFP。用脂质体将其与CVI988株共转染CEF细胞,用96孔板稀释法得到纯化的表达绿色荧光蛋白的重组CVI988病毒株rCVIGFP,并分别测定其在体内和体外的生长情况。表达EGFP基因的重组病毒在细胞上生长曲线与亲本毒CVI988类似,体外实验表明,1日龄腹腔接种该重组毒后,可以从鸡体内分离到表达绿色荧光的病毒。     

伪狂犬病病毒糖蛋白G基因的结构分析及其原核表达

利用PCR技术扩增了伪狂犬病毒湖北株(PRV HB)糖蛋白G(gG)基因,进行了序列测定和分析.结果显示扩增和测序片段长1804bp,G+C含量68.78%.gG基因ORF长1500bp,编码500个氨基酸组成的多肽.与PRV Rice 株gG基因比较,两者核苷酸及推导的氨基酸序列同源性分别为98%、84.1%.320～380位之间的氨基酸序列存在较大差异.根据序列分析结果,选取gG基因长短不同的两个片段分别克隆到原核表达载体pET28a(+)进行表达.经SDS-PAGE和Dot-ELISA分析证实,表达出分子量大小分别约为55kD和63kD的特异性gG多肽,这为深入阐明PRV gG基因结构与功能及研制gG-ELISA诊断试剂盒奠定了基础.     

伪狂犬病病毒糖蛋白G基因的结构分析及其原核表达

利用PCR技术扩增了伪狂犬病毒湖北株 (PRVHB)糖蛋白G(gG)基因 ,进行了序列测定和分析。结果显示扩增和测序片段长 180 4bp ,G C含量 6 8.78%。gG基因ORF长 15 0 0bp ,编码 5 0 0个氨基酸组成的多肽。与PRVRice株 gG基因比较 ,两者核苷酸及推导的氨基酸序列同源性分别为 98%、84.1%。 32 0～ 380位之间的氨基酸序列存在较大差异。根据序列分析结果 ,选取 gG基因长短不同的两个片段分别克隆到原核表达载体 pET2 8a( )进行表达。经SDS PAGE和Dot ELISA分析证实 ,表达出分子量大小分别约为 5 5kD和 6 3kD的特异性gG多肽 ,这为深入阐明PRV gG基因结构与功能及研制 gG ELISA诊断试剂盒奠定了基础     

Replication of Deoxyribonucleic Acid During the Division Cycle of Salmonella typhimurium

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.     

Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in ΔdatA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.Initiation of DNA replication is highly regulated to coordinate with cell proliferation. It begins with a series of events in which the replication machinery is assembled at the replication origin of the chromosomal DNA (15, 26, 28, 38). Central to this process are the initiator proteins that bind to the origin of replication and eventually lead to the unwinding of the origin and to helicase loading on the unwound region. Previous biochemical studies and recent structural studies of the bacterial initiator protein DnaA have proposed the molecular mechanism of the action of ATP-DnaA in forming a large oligomeric complex to remodel the unique origin, oriC, and trigger duplex melting (12, 26). However, it is still not clear how the timing of initiation is controlled so that it takes place at a fixed time in the cell cycle. It has been reported that a basal level of DnaA molecules is bound by high-affinity DnaA binding sites (DnaA boxes R1, R2, and R4) at oriC throughout the cell cycle (9, 37). It is also suggested that noncanonical ATP-DnaA binding sites within oriC are occupied at elevated levels of the initiator molecules prior to the initiation event (18, 25). Thus, regulation of the activity and availability of DnaA is an important factor for the initiation control.At least three schemes are known to prevent untimely initiations in Escherichia coli. First, oriC is subject to sequestration, a process that prevents reinitiation, possibly by blocking ATP-DnaA from binding to newly replicated oriC (8, 24). E. coli oriC contains 11 GATC sites that are normally methylated on both strands by Dam methyltransferase. Immediately after passage of the replication fork, GATC sites are in a hemimethylated state, with the newly synthesized strands remaining unmethylated. SeqA binds specifically to such sites and, at oriC, protects these regions from reinitiation for about one-third of the cell cycle (6, 39). Second, in a process termed regulatory inactivation of DNA (RIDA), ATP-DnaA molecules are converted to an inactive ADP-bound form after initiation by the combined action of a β subunit of DNA polymerase III holoenzyme and Hda (16, 17). Newly synthesized DnaA molecules are able to bind ATP for the next initiation event, since its cellular concentration is much higher than that of ADP. ATP-DnaA is also regenerated from the inactive ADP-DnaA later in the cell cycle (21). Finally, the chromosomal segment datA serves to reduce the level of free DnaA protein by titrating a large number of DnaA molecules after replication of the site close to oriC (20).Cytological studies would be very useful for developing our understanding of the regulation mechanisms associated with the initiation step. In the present study, we tagged E. coli DnaA with a fluorescent protein in order to monitor its behavior in live cells. Microscopic observation revealed that DnaA is distributed all over the nucleoid. Remarkably, the majority of cells bore distinct foci that emitted brighter fluorescence against a weak fluorescent background on the nucleoid. We analyzed the behavior of these foci during the cell cycle with respect to oriC and datA.     

Gene Therapy of Lung Adenocarcinoma using Herpes Virus Expressing a Fusogenic Membrane Glycoprotein

The aim of this study is to observe the in vitro-targeted destruction of lung adenocarcinoma using recombinant Type I herpes simplex virus (HSV-I)-mediated gibbon ape leukemia virus envelope glycoprotein (GALV.fus), controlled by UL38 promoter and cytomegalovirus promoter (CMVP). A recombinant HSV-I plasmid encoding the GALV.fus was transfected into green monkey kidney cells, the lung adenocarcinoma line A549, and the human fetal fibroblast cell line HFL-I GNHu5 in various doses. The effects and expression of in vitro GALV.fus were observed using an inverted microscope. Enhanced green fluorescence protein expression served as the contro1 for GALV.fus. Recombinant HSV-I virus was produced. Fusogenic recombinant virus infection led to cell fusions in A549 in a dose-dependent manner. Nonfusogenic viruses only produced conventional cytotoxic effects. Recombinant HSV-I with the CMVP initiated cell fusions in HFL-1 GNHu5 cells with arrested cell cycles or as quiescence. HSV-I regulated by UL38p caused cell fusion only in growing cells. Protein expression of GALV.fus was confirmed by Western Blot in infected A549 and HFL-1 GNHu5. Delivery and tumor-specific expression of GALV.fus gene can selectively and safely target lung cancer in vitro, and may prove to be a novel gene therapy for lung cancer.