Cloning and Characterization of UL34 Gene of Pseudorabies Virus HB Isolate

In this study, China isolate HB of pseudorabies virus(PRV) was confirmed and genotypically characterized by amplifying and sequencing of partial UL34, a conservative gene involved in the egress of nucleocapsids from the nucleus, for phylogenetic analysis. The open reading frame(orf) of UL34 of PRV HB isolate is composed of 786 nucleotides, which encoded 262 amino acids. In addition, a potential transmembrane domain(241-260 aa) and 11 potential phosphorylation sites were also found in the UL34 of PRV HB isolate. Multiple amino acids alignment indicated that UL34 proteins of PRV strains derived from different geographic origins were highly conservative, but some mutations were also found. Phylogenetic analysis based on UL34 protein indicated that PRV HB strain was evolutionarily distinct from other recent China strains sequenced so far, forming a single clade within the phylogeny. Moreover, PRV HB isolate had close evolutionary relationship with Bo HV-1 and Bo HV-5 within the Alphaherpesvirinae. Taken together, these results indicated that PRV strains were in the progress of evolution. This study has expanded the knowledge of genetic profiles of PRV strains.     

Cloning and characterization of <Emphasis Type="Italic">UL</Emphasis>34 gene of pseudorabies virus HB isolate

In this study, China isolate HB of pseudorabies virus (PRV) was confirmed and genotypically characterized by amplifying and sequencing of partial UL34, a conservative gene involved in the egress of nucleocapsids from the nucleus, for phylogenetic analysis. The open reading frame (orf) of UL34 of PRV HB isolate is composed of 786 nucleotides, which encoded 262 amino acids. In addition, a potential transmembrane domain (241-260 aa) and 11 potential phosphorylation sites were also found in the UL34 of PRV HB isolate. Multiple amino acids alignment indicated that UL34 proteins of PRV strains derived from different geographic origins were highly conservative, but some mutations were also found. Phylogenetic analysis based on UL34 protein indicated that PRV HB strain was evolutionarily distinct from other recent China strains sequenced so far, forming a single clade within the phylogeny. Moreover, PRV HB isolate had close evolutionary relationship with BoHV-1 and BoHV-5 within the Alphaherpesvirinae. Taken together, these results indicated that PRV strains were in the progress of evolution. This study has expanded the knowledge of genetic profiles of PRV strains.     

Polymorphism of human cytomegalovirus （HCMV） UL144 gene in low passage clinical isolates

To explore the impact of gene polymorphism of human cytomegalovirus （HCMV） on virus virulence, the full-length UL144 gene and partial sequence of glycoprotein B （UL55） gene were sequenced and analyzed for 23 clinical strains of HCMV isolated from urine samples of pediatric patients with congenital or postnatal HCMV infection. Among the 23 isolates, 13 （57 %） were UL144 genotype 1A, 3（13 %） were UL144 genotypes 2 and 7（30 %） were UL144 genotype 3; geographic differences in geno- type distribution were found for both UL144 and gB gene. No UL144 genotypes 1B and 1C were found in this study while these two genotypes were common in HCMV strains isolated in the US. Our results also demonstrated that for all clinical strains of gB genotypes I, III and UL144 genotypes 1A, 2, and 3 found in this study, mother-to-fetus vertical transmission was possible.     

Karyopherin-mediated import of integral inner nuclear membrane proteins

Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. Here we show that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-alpha and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-alpha, karyopherin-beta1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. We also provide evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.     

An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection

Herpes simplex virus 2 (HSV-2) infection causes significant morbidity and is an important cofactor for the transmission of HIV infection. A microbicide to prevent sexual transmission of HSV-2 would contribute substantially to controlling the spread of HIV and other infections. Because RNA interference (RNAi) provides effective antiviral defence in plants and other organisms, several studies have focused on harnessing RNAi to inhibit viral infection. Here we show that vaginal instillation of small interfering RNAs (siRNAs) targeting HSV-2 protects mice from lethal infection. siRNAs mixed with lipid are efficiently taken up by epithelial and lamina propria cells and silence gene expression in the mouse vagina and ectocervix for at least nine days. Intravaginal application of siRNAs targeting the HSV-2 UL27 and UL29 genes (which encode an envelope glycoprotein and a DNA binding protein, respectively) was well tolerated, did not induce interferon-responsive genes or cause inflammation, and protected mice when administered before and/or after lethal HSV-2 challenge. These results suggest that siRNAs are attractive candidates for the active component of a microbicide designed to prevent viral infection or transmission.     

人巨细胞病毒临床低传代病毒株UL97 基因的克隆与分析

分离人巨细胞病毒（HCMV）临床低传代GZ02病毒株,并根据GenBank提供的实验室标准病毒株HCMV AD169磷酸转移酶基因UL97 DNA序列及有关文献设计引物,从HCMV GZ02病毒株基因组DNA中通过PCR扩增UL97基因,并克隆至pGEM3Z质粒载体．重组质粒经测序鉴定,发现HCM VGZ02病毒株UL97基因序列保守区域与HCMV AD169 UL97长度完全一致,但发生G205A、G761A、T823C、T1173C、T1282C、T1334C、T2106C等7个位点的碱基突变,涉及到编码氨基酸E69K,S275P,C428R和F445S位点发生改变。     

核酶M1GS-T6对HCMV UL97基因RNA片段体外切割作用

目的:研究M1GS核酶对HCMV UL97mRNA的体外切割作用.方法:针对HCMV UL97 mRNA T6位点设计与之互补的引导序列(Guide Sequence,GS),将其共价结合至大肠杆菌核酶P催化亚基(M1 RNA)的3'末端,构建M1GS-T6核酶,并用其对UL97基因亚克隆片段转录产物进行体外靶向切割实验.结果:核酶M1GS-T6具备特异性切割靶分子UL97 mRNA的能力.结论:核酶M1GS-T6具备特异性切割活性,为进一步研究HCMV病毒基因功能和治疗提供了新的途径.     

The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity

Primate lentiviruses such as human immunodeficiency type 1 (HIV-1) have the capacity to infect non-dividing cells such as tissue macrophages. In the process, viral complementary DNA traverses the nuclear envelope to integrate within chromatin. Given the intimate association between chromatin and the nuclear envelope, we examined whether HIV-1 appropriates nuclear envelope components during infection. Here we show that emerin, an integral inner-nuclear-envelope protein, is necessary for HIV-1 infection. Infection of primary macrophages lacking emerin was abortive in that viral cDNA localized to the nucleus but integration into chromatin was inefficient, and conversion of viral cDNA to non-functional episomal cDNA increased. HIV-1 cDNA associated with emerin in vivo, and the interaction of viral cDNA with chromatin was dependent on emerin. Barrier-to-autointegration factor (BAF), the LEM (LAP, emerin, MAN) binding partner of emerin, was required for the association of viral cDNA with emerin and for the ability of emerin to support virus infection. Therefore emerin, which bridges the interface between the inner nuclear envelope and chromatin, may be necessary for chromatin engagement by viral cDNA before integration.     

Relationship between LTP and the nuclear protein induced by calcineurin

Results demonstrate that calcineurin plays a role in long term potentiatibn (LTP) in the hippocampus. A 45 ku enzyme was administered, which exhibits Ca²⁺ /calmodulin independent activity to rats, and the protein components of nuclear extracts from the cell-free system of rat hippocampus were tested. It is found that the level of a component significantly increased in nuclear extracts following the activation of calcineurin. The concentration of the component in the nucleus is also markedly increased following hippocampal LTP elicited by ginsenosides. These results suggest that the activation of calcineurin induces a preexisting protein component to translocate to the nucleus in the hippocampus. The nuclear translocation of the component may be required for LTP in the hippocampus.     

A novel nuclear export activity in HIV-1 matrix protein required for viral replication

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.     

联合解耦上/下行链路级联和跨层双连接入技术的三层异构网设计和覆盖概率分析

研究了跨层双连接(dual-connectivity, DC)3层异构网络(heterogeneous network, HetNet)中上行链路(uplink, UL)和下行链路(downlink, DL)解耦级联(decoupled UL and DL association, DUDA)设计和网络覆盖概率问题。在该跨层DC DUDA模型中,一个用户的DL或UL同时与位于不同层的最佳和非最佳基站(base stations, BSs)级联,并且DL的接入基站可能与UL的不同。对此网络,获得了各种可能的跨层DC DUDA实现方案;利用实际的功率约束简化了每种案例的级联条件,获得了级联概率闭式解;利用随机几何方法获得了网络的覆盖概率。数值和仿真分析了系统参数对级联概率的影响,比较了耦合和解耦模型的级联概率和覆盖概率,发现提出的DUDA DC模型优于传统的耦合的UL/DL级联(coupled UL and DL association, CUDA)DC模型;给出了覆盖概率的数值分析和仿真比较,验证了文章所得结论。     

反复冻融次数和不同保存温度对伪狂犬病毒的神经环路标记效果的影响

伪狂犬病毒(pseudorabies virus, PRV)是常用于神经环路示踪研究的嗜神经病毒工具之一,然而目前缺乏 PRV的保存条件对其感染滴度以及在体神经环路示踪效果影响的研究.首先,将2 h内在－80℃及室温下同一批次的PRV分别进行1～4次冻融,利用噬斑法在BHK-21细胞上检测其滴度,研究结果显示1～3次冻融对PRV的滴度改变无显著影响,而第4次冻融使PRV病毒的滴度显著下降.其次,从－80℃冰箱中取出同一批次的两份PRV,将其冻融1次,分别在－80℃和－20℃保存两周后,利用噬斑法在BHK-21细胞上检测其滴度;同时,将两份PRV利用在体立体定位分别注射于小鼠齿状回(dentate gyrus, DG)区,取相同的感染时间点,对DG输入环路标记效果进行分析,研究得出不同温度下长期保存对PRV的滴度及标记效果有较大影响.该研究结果对于PRV病毒的保存及其在神经环路标记中的应用具有一定的参考意义.     

The nuclear lamina is a meshwork of intermediate-type filaments

The nuclear lamina, a protein meshwork lining the nucleoplasmic surface of the inner nuclear membrane, is thought to provide a framework for organizing nuclear envelope structure and an anchoring site at the nuclear periphery for interphase chromatin. In several higher eukaryotic cells, the lamina appears to be a polymer comprised mainly of one to three immunologically related polypeptides of relative molecular mass (Mr) 60,000-75,000 (60-70K) termed lamins. Three lamins (A, B, and C) are typically present in mammalian somatic cells. Previous studies on nuclear envelopes of rat liver and Xenopus oocytes suggested that the lamina has a fibrillar or filamentous substructure. Interestingly, protein sequences recently deduced for human lamins A and C from complementary DNA clones indicate that both of these polypeptides contain a region of approximately 350 amino acids very similar in sequence to the coiled-coil alpha-helical rod domain that characterizes all intermediate-type filament (IF) proteins. Here we analyse the supramolecular organization of the native nuclear lamina and the structure and assembly properties of purified lamins, and show that the lamins constitute a previously unrecognized class of IF polypeptides.     

Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2

Calcium is believed to control a variety of cellular processes, often with a high degree of spatial and temporal precision. For a cell to use Ca2+ in this manner, mechanisms must exist for controlling the ion in a localized fashion. We have now gained insight into such mechanisms from studies which measured Ca2+ in single living cells with high resolution using a digital imaging microscope and the highly fluorescent Ca2+-sensitive dye, Fura-2. Levels of Ca2+ in the cytoplasm, nucleus and sarcoplasmic reticulum (SR) are clearly different. Free [Ca2+] in the nucleus and SR was greater than in the cytoplasm and these gradients were abolished by Ca2+ ionophores. When external Ca2+ was raised above normal in the absence of ionophores, free cytoplasmic Ca2+ increased but nuclear Ca2+ did not. Thus, nuclear [Ca2+] appears to be regulated independently of cytoplasmic [Ca2+] by gating mechanisms in the nuclear envelope. The observed regulation of intranuclear Ca2+ in these contractile cells may thus be seen as a way to prevent fluctuation in Ca2+-linked nuclear processes during the rise in cytoplasmic [Ca2+] which triggers contraction. The approach described here offers the opportunity of following changes in Ca2+ in cellular compartments in response to a wide range of stimuli, allowing new insights into the role of local changes in Ca2+ in the regulation of cell function.