The Human Interferon-Induced MxA Protein Inhibits Early Stages of Influenza A Virus Infection by Retaining the Incoming Viral Genome in the Cytoplasm

The induction of an interferon-induced antiviral state is a powerful cellular response against viral infection that limits viral spread. Here, we show that a preexisting antiviral state inhibits the replication of influenza A viruses in human A549 cells by preventing transport of the viral genome to the nucleus and that the interferon-induced MxA protein is necessary but not sufficient for this process. This represents a previously unreported antiviral function of MxA against influenza A virus infection.     

基于Semliki森林病毒复制子的新型RNAi质粒载体的构建

目的:以semliki森林病毒复制子为基础,构建一类可迅速高效表达shRNA的新型RNAi载体。方法:以Semliki森林病毒衍生的复制子载体pSFV1为骨架,用CMV IE启动子替换SP6启动子并在3′-UTR下游插入SV40 polyA转录终止子,在原26S亚基因组启动子后插入带有相应改良多克隆位点的shRNA表达元件,同时加入抗新霉素选择复合体,并去掉3′-UTR的重复序列。所获载体用于沉默EGFP基因,通过体外细胞转染、病毒颗粒制备、荧光显微镜观察、RT-PCR分析等初步验证、评估其效果。结果:构建了基于Semliki森林病毒复制子的新型RNAi质粒载体pSFV-RNAi Ready。经体外实验初步证实,该载体直接转染细胞,或与辅助载体共转染,制备成具有感染能力的重组病毒颗粒后使用,均可高水平表达shRNA,沉默目的基因。其中使用病毒颗粒抑抑效率可高达90%以上。结论:该载体的成功构建,可望显著拓宽SFV载体的应用范围,丰富RNAi实施手段,并用于相关科学研究及基因药物技术开发。     

基于DNA和RNA的双功能Semliki森林病毒复制子载体的构建

以semliki森林病毒衍生的复制子载体pSFV1和辅助载体pSFV-helper2为骨架, 用CMV IE和T7启动子替换SP6启动子并在3′ UTR下游插入BGH转录终止子,构建了基于DNA和RNA的复制子表达载体pSMCTA和辅助载体pSHCTA。在DNA和RNA二种递送方式上证实该表达载体可高水平表达外源基因,与辅助载体共转染可制备具有感染能力并能表达外源基因的重组病毒颗粒。构建的基于DNA和RNA的双功能复制子载体显著地提高SFV载体应用范围,在体外可用于高水平表达外源基因及大规模制备重组病毒颗粒,在体内也可用于研制复制子疫苗和基因治疗载体。     

Structural Requirements for the Antiviral Activity of the Human MxA Protein against Thogoto and Influenza A Virus

The interferon-induced dynamin-like MxA protein has broad antiviral activity against many viruses, including orthomyxoviruses such as influenza A and Thogoto virus and bunyaviruses such as La Crosse virus. MxA consists of an N-terminal globular GTPase domain, a connecting bundle signaling element, and the C-terminal stalk that mediates oligomerization and antiviral specificity. We previously reported that the disordered loop L4 that protrudes from the compact stalk is a key determinant of antiviral specificity against influenza A and Thogoto virus. However, the role of individual amino acids for viral target recognition remained largely undefined. By mutational analyses, we identified two regions in the C-terminal part of L4 that contribute to an antiviral interface. Mutations in the proximal motif, at positions 561 and 562, abolished antiviral activity against orthomyxoviruses but not bunyaviruses. In contrast, mutations in the distal motif, around position 577, abolished antiviral activity against both viruses. These results indicate that at least two structural elements in L4 are responsible for antiviral activity and that the proximal motif determines specificity for orthomyxoviruses, whereas the distal sequence serves a conserved structural function.     

Orchid Fleck Virus Structural Proteins N and P Form Intranuclear Viroplasm-Like Structures in the Absence of Viral Infection

Orchid fleck virus (OFV) has a unique two-segmented negative-sense RNA genome that resembles that of plant nucleorhabdoviruses. In infected plant cells, OFV and nucleorhabdoviruses induce an intranuclear electron-lucent viroplasm that is believed to be the site for virus replication. In this study, we investigated the molecular mechanism by which OFV viroplasms are produced in vivo. Among OFV-encoded proteins, the nucleocapsid protein (N) and the putative phosphoprotein (P) were present in nuclear fractions of OFV-infected Nicotiana benthamiana plants. Transient coexpression of N and P, in the absence of virus infection, was shown to be sufficient for formation of an intranuclear viroplasm-like structure in plant cells. When expressed independently as a fluorescent protein fusion product in uninfected plant cells, N protein accumulated throughout the cell, while P protein accumulated in the nucleus. However, the N protein, when coexpressed with P, was recruited to a subnuclear region to induce a large viroplasm-like focus. Deletion and substitution mutagenesis demonstrated that the P protein contains a nuclear localization signal (NLS). Artificial nuclear targeting of the N-protein mutant was insufficient for formation of viroplasm-like structures in the absence of P. A bimolecular fluorescence complementation assay confirmed interactions between the N and P proteins within subnuclear viroplasm-like foci and interactions of two of the N. benthamiana importin-α homologues with the P protein but not with the N protein. Taken together, our results suggest that viroplasm formation by OFV requires nuclear accumulation of both the N and P proteins, which is mediated by P-NLS, unlike nucleorhabdovirus viroplasm utilizing the NLS on protein N.     

Shutoff on Neuroblastoma Cell Protein Synthesis by Semliki Forest Virus: Loss of Ability of Crude Initiation Factors to Recognize Early Semliki Forest Virus and Host mRNA's

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing “pH5” system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (±80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5′ terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M_r, 80,000) and cap-binding protein (M_r, 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.     

Human MxA Protein Protects Mice Lacking a Functional Alpha/Beta Interferon System against La Crosse Virus and Other Lethal Viral Infections

The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-alpha/beta). MxA inhibits the multiplication of several RNA viruses in cell culture. However, its antiviral potential in vivo has not yet been fully explored. We have generated MxA-transgenic mice that lack a functional IFN system by crossing MxA-transgenic mice constitutively expressing MxA with genetically targeted (knockout) mice lacking the beta subunit of the IFN-alpha/beta receptor (IFNAR-1(-/-) mice). These mice are an ideal animal model to investigate the unique antiviral activity of human MxA in vivo, because they are unable to express other IFN-induced proteins. Here, we show that MxA confers resistance to Thogoto virus, La Crosse virus, and Semliki Forest virus. No Thogoto virus progeny was detectable in MxA-transgenic mice, indicating an efficient block of virus replication at the primary site of infection. In the case of La Crosse virus, MxA restricted invasion of the central nervous system. In contrast, Semliki Forest virus multiplication in the brain was detectable in both MxA-expressing and nonexpressing IFNAR-1(-/-) mice. However, viral titers were clearly reduced in MxA-transgenic mice. Our results demonstrate that MxA does not need the help of other IFN-induced proteins for activity but is a powerful antiviral agent on its own. Moreover, the results suggest that MxA may protect humans from potential fatal infections by La Crosse virus and other viral pathogens.     

人乳铁蛋白基因在烟草中的表达及抗细菌与病毒的能力

从人血液中性白细胞中分离细胞总ＲＮＡ,用ｏｌｉｇｏ（ｄＴ） 为引物进行ｃＤＮＡ 第１ 条链合成,再用乳铁蛋白ｃＤＮＡ 的特异性引物进行聚合酶链反应（ＰＣＲ） ,分两段合成并拼成完整的乳铁蛋白ｃＤＮＡ。核苷酸序列测定证明,该ｃＤＮＡ 所编码的氨基酸与前人论证的人乳铁蛋白的氨基酸序列完全一致。将该ｃＤＮＡ 构建成植物表达载体ｐ３５ＳｈＬＦｃ,经根癌农杆菌ＬＢＡ４４０４ 感染烟草叶盘,获得具有卡那霉素抗性的烟草植株,ＰＣＲ检测和Ｓｏｕｔｈｅｒｎ杂交表明,人乳铁蛋白基因已整合到烟草基因组中。Ｎｏｒｔｈｅｒｎ 杂交与Ｗｅｓｔｅｒｎ 免疫印迹分析,检测到人乳铁蛋白基因在转化烟草中表达。体外实验证明,人乳铁蛋白基因在烟草中的表达产物对植物致病菌烟草火叶病菌、水稻白叶枯病菌有一定抑制作用     

基于DNA的Semliki森林病毒复制子载体体内外高水平表达外源基因

基于DNA的复制子载体显著地提高了复制子载体的应用范围,在体外可用于高水平表达外源基因,大规模制备重组病毒颗粒,在体内可用于复制子疫苗和基因治疗载体研究.将复制子RNA的cDNA置于RNA聚合酶Ⅱ启动子和转录终止子控制下时,基于RNA的复制子载体可转变为基于DNA的复制子载体.当DNA载体转染细胞后,第一阶段,RNA聚合酶Ⅱ启动子在核内起始合成RNA,经过加工和修饰后运输到细胞质中,相当于复制子RNA,第二阶段,该RNA自我复制及表达外源基因,相当于病毒RNA复制循环.在成功地构建了基于DNA和RNA的双功能复制子表达载体pSCTA和辅助载体pSHCTA的基础上,为了获得高效的基于DNA的复制子载体,对其进行改进而构建了共4种不同的基于DNA的semliki森林病毒复制子,通过对表达载体和相应的辅助载体表达报告基因及对制备重组病毒颗粒的能力进行比较,获得了复制能力提高的复制子载体pSCAR和pSHCAR.该表达载体pSCAR可高水平表达外源基因,与辅助载体pSHCAR共转染可制备高滴度的重组病毒颗粒,并且也能表达抗原基因.另外,报告基因在DNA复制子载体注射的小鼠体内得到了高水平表达,并且DNA免疫小鼠后也诱导产生高滴度特异性抗体以及细胞免疫反应.结果表明,经过改造SFV复制子载体,获得了高效的基于DNA的SFV复制子载体.该复制子载体增强了原复制子载体应用能力,并有潜力作为复制子疫苗和基因治疗载体.     

Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.     

猪2型圆环病毒ORF2基因在塞姆利基森林病毒RNA复制子衍生的新型真核表达载体中的表达

猪 2型圆环病毒 (porcinecircovirus 2 ,PCV2 )是断乳仔猪多系统衰竭综合征 (postweaningmultisystemicwastingsyndrome,PMWS)的原发性病原。PCV2的ORF2编码病毒唯一的结构蛋白Cap。根据GenBank中公布的PCV2JXL株的序列设计一对引物 ,应用PCR方法从该毒株感染的PK 15细胞中扩增出完整的ORF2基因 ,将此基因克隆于本实验室此前构建的塞姆利基森林病毒 (SemlikiForestvirus,SFV)RNA复制子衍生的新型真核表达载体Psfv1cs中的BamHⅠ位点 ,获得重组质粒pSFV1CS Cap。用pSFV1CS-Cap分别转染BHK-21细胞和293T细胞 ,经间接免疫荧光试验检测表明 ,PCV2 ORF2基因在转染细胞中得到表达。小鼠接种试验表明 ,该重组质粒能诱导小鼠产生特异性抗体.     

Expression of Amino-Terminal Portions or Full-Length Viral Replicase Genes in Transgenic Plants Confers Resistance to Potato Virus X Infection

The first open reading frame (ORF 1) of potato virus X (PVX) encodes a putative replicase gene. Transgenic tobacco lines expressing ORF 1 are resistant to PVX infection when inoculated with either PVX or PVX RNA. Analyses of lines containing various portions of the ORF 1 gene demonstrated that resistance is conferred to plants by expressing approximately the first half of the ORF 1 gene. One line expressing the untranslated leader and first 674 codons of ORF 1 is highly resistant to PVX infection. Conversely, lines expressing either approximately the third or fourth quarter of the ORF 1 gene, which contain the conserved nucleotide triphosphate (NTP) binding motif and Gly-Asp-Asp (GDD) motif, respectively, are not protected from PVX infection. In the resistant full-length and amino-terminal lines, lower numbers of local lesions were observed, and the virus accumulation in the inoculated and upper leaves was reduced when compared with the nontransformed control. When the performance of the most resistant ORF 1 line was compared with the most resistant coat protein (CP) line in a resistance test, the best ORF 1 line was more resistant to PVX infection than the best transgenic line expressing the PVX CP gene. These findings define a promising new approach for controlling plant viral infection.     

Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5′- and 3′-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5′-NCR of the positive-sense genomic RNA - 5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA - 3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.     

HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

Nef is an important player for viral infectivity and AIDS progression, but the mechanisms involved are not completely understood. It was previously demonstrated that Nef interacts with GagPol through p6*-Protease region. Because p6* and Protease are involved in processing, we explored the effect of Nef on viral Protease activity and virion assembly. Using in vitro assays, we observed that Nef is highly capable of inhibiting Protease activity. The IC50 for nef-deficient viruses in drug susceptibility assays were 1.7- to 3.5-fold higher than the wild-type counterpart varying with the type of the Protease inhibitor used. Indicating that, in the absence of Nef, Protease is less sensitive to Protease inhibitors. We compared the protein content between wild-type and nef-deficient mature viral particles by gradient sedimentation and observed up to 2.7-fold reduction in the Integrase levels in nef-deficient mature particles. This difference in levels of Integrase correlated with the difference in infectivity levels of wild type and nef-deficient viral progeny. In addition, an overall decrease in the production of mature particles was detected in nef-deficient viruses. Collectively, our data support the hypothesis that the decreased infectivity typical of nef-deficient viruses is due to an abnormal function of the viral Protease, which is in turn associated with less mature particles being produced and the loss of Integrase content in these particles, and these results may characterize Nef as a regulator of viral Protease activity.