Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

关于脑心肌炎病毒2A蛋白的研究进展

脑心肌炎病毒(Encephalomyocarditis virus,EMCV)是一种无囊膜的单股RNA病毒,属于小RNA病毒科,能够引起多种哺乳动物乃至人的感染。其非结构蛋白2A是重要的毒力因子,能够通过阻断翻译起始复合物的形成、结合翻译起始复合物因子及核糖体40s小亚基等方式竞争性地抑制宿主细胞蛋白的合成,还可通过抑制宿主细胞凋亡促进病毒扩散,并通过激活NF-κB引起宿主发生强烈的炎症反应[1]。此外,根据EMCV 2A蛋白的生物学特性,近年来,细胞生物学、病毒学领域均将其作为真核细胞与病毒互作的生物学工具展开了深入研究。     

Imaging of the Alphavirus Capsid Protein during Virus Replication

Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid (NC) core contains a capsid protein lattice enclosing the plus-sense RNA genome, and it is surrounded by a lipid bilayer containing a lattice of the E1 and E2 envelope glycoproteins. Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma membrane (PM), but the traffic and assembly of viral components and the exit of virions from host cells are not well understood. To visualize the dynamics of capsid protein during infection, we developed a Sindbis virus infectious clone tagged with a tetracysteine motif. Tagged capsid protein could be fluorescently labeled with biarsenical dyes in living cells without effects on virus growth, morphology, or protein distribution. Live cell imaging and colocalization experiments defined distinct groups of capsid foci in infected cells. We observed highly motile internal puncta that colocalized with E2 protein, which may represent the transport machinery that capsid protein uses to reach the PM. Capsid was also found in larger nonmotile internal structures that colocalized with cellular G3BP and viral nsP3. Thus, capsid may play an unforeseen role in these previously observed G3BP-positive foci, such as regulation of cellular stress granules. Capsid puncta were also observed at the PM. These puncta colocalized with E2 and recruited newly synthesized capsid protein; thus, they may be sites of virus assembly and egress. Together, our studies provide the first dynamic views of the alphavirus capsid protein in living cells and a system to define detailed mechanisms during alphavirus infection.     

Inactivation of Encephalomyocarditis Virus in Aerosols: Fate of Virus Protein and Ribonucleic Acid

After aerosolization at relative humidities of 50% or lower, encephalomyocarditis virus is rapidly inactivated. In this process the protein coat of the virion is damaged. This appears as a loss of hemagglutination activity and loss of affinity for hemagglutination inhibiting antibodies. The ribonucleic acid of the virus retains its infectivity but it becomes susceptible to ribonuclease. It sediments in sucrose gradients when centrifuged at high speed with the same velocity as free infectious ribonucleic acid extracted with phenol from intact encephalomyocarditis virus.     

Nuclear Transport of the Major Capsid Protein Is Essential for Adeno-Associated Virus Capsid Formation

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.     

Preparative labeling of proteins with [35S]methionine.

The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.     

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics.     

Metabolism of sodium oestrone [35S]sulphate in the rat

Intraperitoneal, intravenous or oral administration of sodium oestrone [(35)S]-sulphate to male and female Medical Research Council hooded rats is followed by the rapid excretion of the bulk of the radioactivity in urine in the form of inorganic [(35)S]sulphate. Pre-treatment of rats with an antibiotic regimen does not affect the results except in the case of oral administration, when relatively large amounts of the dose are recovered as ester [(35)S]sulphate in faeces. Intravenous administration of the labelled ester to male and female rats with cannulae in bile duct and ureter gave results similar to those obtained with free-range animals. Only small amounts of radioactivity appeared in bile and this was mainly in the form of ester sulphate, including both oestrone [(35)S]sulphate and oestradiol-17beta 3[(35)S]-sulphate. Whole-body radioautography pinpointed the liver as the probable site of the desulphation of the sulphate ester and this was confirmed by liver and kidney perfusion experiments and by studies with rats in which kidney function had been eliminated by ligation of the renal pedicles.     

Synthesis of Capsid and Noncapsid Viral Proteins in Response to Encephalomyocarditis Virus Ribonucleic Acid in Animal Cell-Free Systems

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of (35)S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.     

Studies on Thin-Layer Chromatography after Incubation with [35S]Methionine as an Aid to Identification of Mycobacteria

Thin-layer chromatography (TLC) of ethyl ether-ethanol extracts of mycobacteria obtained after incubation with [³⁵S] methionine is useful for differentiation among mycobacterial species, as the distribution of radioactive spots in TLC shows a characteristic pattern except for a few species, including M. intracellulare and M. gordonae. Some supplementary studies have been carried out in the present investigation and the following results have been obtained.

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The metabolism of l-serylglycine O[35S]-sulphate in the rat

1. The preparation of potassium l-serylglycine O-sulphate and the corresponding ³⁵S-labelled ester is described. 2. Intraperitoneal injection of potassium l-serylglycine O[³⁵S]-sulphate to rats results in about 75% of the radioactivity of the dose appearing in the urine within 48hr. Almost 72% of the radioactivity recovered in the urine was in the form of inorganic [³⁵S]sulphate. 3. Analysis of urines by paper chromatography showed the presence of unchanged l-serylglycine O[³⁵S]-sulphate and several other unidentified ³⁵S-labelled materials. 4. It has been established that micro-organisms of the gastrointestinal tract do not play any significant role in the production of inorganic [³⁵S]sulphate from the injected ester. 5. l-Serylglycine O-sulphate was hydrolysed by crude dipeptidase preparations from rat kidney and intestine to yield l-serine O-sulphate and glycine as the sole products.     

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.     

Metabolism of sodium oestrone [35S]sulphate in the guinea pig

Intraperitoneal administration of sodium oestrone [(35)S]sulphate to male and female free-ranging guinea pigs is followed by excretion of most of the radioactivity mainly as inorganic [(35)S]sulphate in the urine within 72h. The remainder of the radioactivity in the urine was found in oestrone [(35)S]sulphate, two unidentified metabolites (A and B) and traces of oestradiol-17beta 3-[(35)S]sulphate. When injected intraperitoneally into animals with bile-duct and bladder cannulae, most of the dose was excreted in the bile. Unchanged oestrone [(35)S]sulphate was the main biliary component excreted in males and females, but the latter also excreted appreciable amounts of oestradiol-17beta 3-[(35)S]sulphate and metabolites A and B. The urine from these animals also contained these metabolites, inorganic [(35)S]sulphate and also oestrone [(35)S]sulphate, but in small amounts. Metabolite A was present only in samples from males. Whole body radioautography pinpointed the liver and kidney as the possible sites of metabolism of the ester. The ester underwent little desulphation in the isolated perfused female guinea-pig liver and in animals in which kidney function had been eliminated, and was excreted unchanged in the bile. These results and the observed low oestrogen sulphatase and arylsulphatase C activities found in guinea-pig liver and kidney support the view that the two enzymes are identical.     

Studies on the Topography of Reovirus and Bluetongue Virus Capsid Proteins

Protein labeling experiments confirm the surface location of proteins 2 and 5 in bluetongue virus, and proteins sigma3 and mu2 in reovirus. Lambda 2 is the major surface component of the reovirus core, and proteins 1, 3, and 4 appear to be the outer components of the bluetongue virus subviral particle.     

Labelling of proteins with [35S]methionine in monolayer cultures of rat hepatocytes

Optimal conditions for the labelling of proteins with [35S]methionine in monolayers of rat hepatocytes have been established. The ability to incorporate the radioactive amino acid was constant for at least 26 h and independent of whether the medium was buffered with CO2/HCO3 or with 4-(2-hydroxyethyl)-1-piper-azineethanesulphonic acid (Hepes). Preincubation in methionine-free medium for up to 30 min yielded increasing, and from 60 to 180 min decreasing, rates of incorporation. An apparent Km value of 0.06 mM was obtained for the incorporation reaction in cells preincubated for 40 min.     

Identification of a Varicella-Zoster Virus Replication Inhibitor That Blocks Capsid Assembly by Interacting with the Floor Domain of the Major Capsid Protein

A novel anti-varicella-zoster virus compound, a derivative of pyrazolo[1,5-c]1,3,5-triazin-4-one (coded as 35B2), was identified from a library of 9,600 random compounds. This compound inhibited both acyclovir (ACV)-resistant and -sensitive strains. In a plaque reduction assay under conditions in which the 50% effective concentration of ACV against the vaccine Oka strain (V-Oka) in human fibroblasts was 4.25 μM, the 50% effective concentration of 35B2 was 0.75 μM. The selective index of the compound was more than 200. Treatment with 35B2 inhibited neither immediate-early gene expression nor viral DNA synthesis. Twenty-four virus clones resistant to 35B2 were isolated, all of which had a mutation(s) in the amino acid sequence of open reading frame 40 (ORF40), which encodes the major capsid protein (MCP). Most of the mutations were located in the regions corresponding to the “floor” domain of the MCP of herpes simplex virus 1. Treatment with 35B2 changed the localization of MCP in the fibroblasts infected with V-Oka but not in the fibroblasts infected with the resistant clones, although it did not affect steady-state levels of MCP. Overexpression of the scaffold proteins restored the normal MCP localization in the 35B2-treated infected cells. The compound did not inhibit the scaffold protein-mediated translocation of MCP from the cytoplasm to the nucleus. Electron microscopic analysis demonstrated the lack of capsid formation in the 35B2-treated infected cells. These data indicate the feasibility of developing a new class of antivirals that target the herpesvirus MCPs and inhibit normal capsid formation by a mechanism that differs from those of the known protease and encapsidation inhibitors. Further biochemical studies are required to clarify the precise antiviral mechanism.     

风疹病毒衣壳蛋白与宿主细胞相互作用蛋白的初步筛选及分析

风疹病毒(Rubella virus,RV)的衣壳蛋白(Capsid protein,CP)不仅是病毒颗粒的重要组成部分,而且还可以通过与宿主蛋白之间的相互作用来调控病毒的转录和复制.为了系统研究衣壳蛋白与宿主蛋白之间的相互作用关系,我们从RV基因组中克隆获得衣壳蛋白基因序列,将该序列导入含有eXact-6His串联亲和纯化标签的慢病毒表达载体中,并构建了稳定表达eXact-6His-Capsid融合蛋白的293T细胞系.通过eXact和6His标签的两次亲和纯化获得衣壳蛋白相互作用蛋白复合物,质谱检测并筛选后发现22个可能与衣壳蛋白相互作用的宿主蛋白.随后构建衣壳蛋白的相互作用网络并进行功能学分析,发现其相互作用蛋白主要参与病毒感染、RNA剪切、细胞凋亡及酶相关通路等过程.