Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells

Background

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.

Methodology and Principal Findings

To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.

Conclusions/Significance

Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.     

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.     

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.     

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.     

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.     

狂犬病病毒糖蛋白哺乳动物细胞稳定表达细胞系建立

试验首先根据GenBank上所发表的狂犬病病毒CVS-24株糖蛋白基因序列,设计并合成一对特异性引物,通过反转录 聚合酶链式反应（RT-PCR）,获得糖蛋白全长cDNA,连接在pMD18-T载体上,测序证明克隆的正确性后,将其插入真核表达载体pIRES1neo,构建了糖蛋白单一表达载体pICG,表达质粒通过脂质体转染BHK-21细胞,在G418抗性压力下出现细胞克隆,通过PCR检测,确定启动子与糖蛋白基因在细胞基因组中共同整合。借助Western blot检测,证明所表达的糖蛋白与狂犬病抗血清有特异的反应性。采用间接ELISA法,筛选出3株高效表达糖蛋白的细胞株,分别命名为ICG1、ICG2、ICG3。     

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.     

Analysis of Receptor Usage by Ecotropic Murine Retroviruses, Using Green Fluorescent Protein-Tagged Cationic Amino Acid Transporters

Entry of ecotropic murine leukemia virus (MuLV) into host cells is initiated by interaction between the receptor-binding domain of the viral SU protein and the third extracellular domain (TED) of the receptor, cationic amino acid transporter 1 (CAT1). To study the molecular basis for the retrovirus-receptor interaction, mouse CAT1 (mCAT1) was expressed in human 293 cells as a fusion protein with jellyfish green fluorescent protein (GFP). Easily detected by fluorescence microscopy and immunoblot analysis with anti-GFP antibodies, the mCAT1-GFP fusion protein was expressed in an N-glycosylated form on the cell surface and in the Golgi apparatus, retaining the ecotropic receptor function. The system was applied to compare Friend MuLV (F-MuLV) and its neuropathogenic variant, PVC-211 MuLV, which exhibits a unique cellular tropism and host range, for the ability to use various CAT family members as a receptor. The results indicated that F-MuLV and PVC-211 MuLV could infect the cells expressing wild-type mCAT1 at comparable efficiencies and that rat CAT3, but not mCAT2, conferred a low but detectable level of susceptibility to F-MuLV and PVC-211 MuLV. The data also suggested that CAT proteins might be expressed in an oligomeric form. Further application of the system developed in this study may provide useful insights into the entry mechanism of ecotropic MuLV.     

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pPolyⅡ-CAV-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SRV\-9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pPolyⅡ-CAV-2-CGS(34.7kb).以AscⅠ和ClaⅠ双酶切,游离重组基因组(32.7kb),在脂质体Lipofectamine TM 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.     

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