Virus-like particles from the yeast Yarrowia lipolytica

Summary Four out of the 24 strains of the yeast Yarrowia lipolytica we have checked for the presence of virus-like particles (VLPs) proved to contain encapsidated double-stranded RNA (dsRNA) molecules, 4.9 kb long. A major VLP polypeptide of MW 80,000 was observed in all 4 cases, and a second one of MW 77,000 in three cases. dsRNA from the VLPs harboring only the larger polypeptide showed little homology with the 3 others. We have found no homology between VLP dsRNAs and host DNA or dsRNAs from Saccharomyces cerevisiae, and no relationship between the presence of VLPs and a possible killer phenomenon in Y. lipolytica.     

Production of the matrix protein of Nipah virus in Escherichia coli: Virus-like particles and possible application for diagnosis

The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (M_r) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 °C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.     

TY3 GAG3 protein forms ordered particles in Escherichia coli

The yeast retrovirus-like element Ty3 GAG3 gene encodes a Gag3 polyprotein analogous to retroviral Gag. Gag3 lacks matrix, but contains capsid, spacer, and nucleocapsid domains. Expression of a Ty3 Gag3 or capsid domain optimized for expression in Escherichia coli was sufficient for Ty3 particle assembly. Virus-like ordered particles assembled from Gag3 were similar in size to immature particles from yeast and contained nucleic acid. However, particles assembled from the CA domain were variable in size and displayed much less organization than native particles. These results indicate that assembly can be driven through interactions among capsid subunits in the particle, but that the nucleocapsid domain, likely in association with RNA, confers order upon this process.     

Virus-like particles derived from major capsid protein VP1 of different polyomaviruses differ in their ability to induce maturation in human dendritic cells

As polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic, we studied their interaction with human dendritic cells (hDCs). Exposure of hDCs to VLPs originating from murine (MPyV) or hamster polyomavirus (HaPyV) induced hDC maturation. In contrast, exposure of hDCs to VLPs derived from human polyomaviruses (BK and JC) and simian virus 40 (SV40) only marginally induced DC maturation. The hDCs stimulated by HaPyV- or MPyV-derived VLPs readily produced interleukin-12 and stimulated CD8-positive T-cell responses in vitro. The highest frequencies of activated T cells were again observed after pulsing with HaPyV- and MPyV-derived VLPs. Monocyte-derived hDCs both bound and internalized the various tested polyomavirus VP1-derived VLPs with different levels of efficiency, partially explaining their individual maturation potentials. In conclusion, our data suggest a high variability in uptake of polyomavirus-derived VLPs and potency to induce hDC maturation.     

Presence of double-stranded RNA and virus-like particles in Phaffia rhodozyma

Four double-stranded RNA (dsRNA) molecules were isolated from Phaffia rhodozyma UCD 67-385. Their molecular sizes were approximately 4.3, 3.1, 0.9 and 0.75 kilobase pairs (kbp) as determined by agarose-gel electrophoresis and they were designated as L, M, S₁ and S₂, respectively. By differential centrifugation in sucrose gradients, these dsRNAs copurified with isometric virus-like particles 36 nm in diameter. A cured strain, UV-S2, lacking the S₂-dsRNA was obtained from P. rhodozyma UCD 67-385 by ultraviolet (UV) light treatment. UV-S2 strain contains identical virus-like particles to those from the wild-type strain, as determined by electron microscopy, suggesting that the S₂-dsRNA was not essential for the expression of mycovirus structural polypeptides. On the other hand, both the UCD 67-385 and UV-S2 strains were able to kill P. rhodozyma UCD 67-383, a strain without dsRNAs. These results suggest that the dsRNA molecules also encode a killer system. Finally, the UV-S2 strain maintains killer ability, which suggests that S₂-dsRNA is not involved in the killer phenotype expression.     

α-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant–pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X. fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with α-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed “cyanogenesis”, which catalyzes the dissociation of α-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X. fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X. fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs.     

Characterization of recombinant malarial RecQ DNA helicase

RecQ DNA gene of multi-drug resistant Plasmodium falciparum K1 (PfRecQ1) was cloned, and the recombinant C-terminal-decahistidine-tagged PfRecQ1 was expressed in Escherichia coli. The purified enzyme could efficiently unwind partial duplex DNA substrate in a 3′ to 5′ direction. The malarial RecQ1 could not unwind substrates with both 5′ and 3′ overhangs, those with a 5′ overhang, or blunt-ended DNA duplexes. Unwinding of DNA helicase activity was driven by the hydrolysis of ATP. The drug inhibitory effects of six compounds indicated that only doxorubicin and daunorubicin could inhibit the unwinding activity.     

肠道病毒71型病毒样颗粒在汉逊酵母中的表达

目的应用汉逊酵母表达系统进行肠道病毒71型（EV71）病毒样颗粒（VLP）的表达。方法将经过汉逊酵母密码子优化的人EV71的 P1和3CD基因片段克隆到汉逊酵母表达载体PMV上,获得重组表达质粒PMV-P1-3CD,转化汉逊酵母宿主菌AU0501,PCR方法及稳定传代培养筛选整合P1和3 CD基因的重组菌株。重组菌种接种在含有1％甲醇的培养基中进行诱导表达,对表达产物进行SDS-PAGE、Western blot检测。挑选优胜表达菌株进行30 L发酵罐发酵培养,发酵产物经过粗略纯化后进行电镜分析。结果筛选获得EV71重组表达菌株;SDS-PAGE检测结果显示在相对分子质量（Mr）为26×103、33×103、35×103处有明显的VP3、VP1、VP0蛋白条带,Mr 大小与预期的目的蛋白大小一致;Western blot检测结果显示表达产物与EV71-VP1单克隆抗体在M r 为33×103处有较为明显的VP1反应条带,表达产物具有良好的免疫反应性;表达菌株发酵表达量可达200 mg/L,电镜分析可见24～30 nm的VLP,且颗粒结构完好。结论应用汉逊酵母表达系统成功表达了EV71 VLP,为今后研制EV71 VLP疫苗奠定基础。     

Isolation and characterization of subgenomic DNAs encapsidated in "single" T = 1 isometric particles of Maize streak virus

"Single" T = 1 isometric particles of Maize streak virus (MSV) have been isolated from infected maize leaves. Biochemical and genetic characterizations show that these particles contain subgenomic (sg) MSV DNA encapsidated by the MSV coat protein. The largest sg DNA is 1.56 kb, slightly larger than half genome size, although sg DNAs as small as 0.2 kb were also cloned. The sg DNAs are not infectious, and they do not appear to play a role in the pathogenicity of MSV. This is the first report of sg DNAs for MSV and, to our knowledge, the first time that encapsidated sg DNAs have been characterized at the sequence level for any geminivirus. These data will assist in our investigations into the role of genomic DNA in the formation of the unique geminate capsid architecture of the Geminiviridae.     

Virus-like vesicles and extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales

Cultures of hyperthermophilic archaea (order Thermococcales) have been analyzed by electron microscopy and epifluorescence staining for the presence of virus-like particles. We found that most strains of Thermococcus and Pyrococcus produce various types of spherical membrane vesicles and unusual filamentous structures. Cellular DNA can be strongly associated with vesicles and appears as fluorescent dots by epifluorescence microscopy, suggesting that some particles assumed to be viruses in ecological studies might instead be vesicles associated with extracellular DNA. DNA in vesicle preparations is remarkably resistant to DNase treatment and thermodenaturation, indicating that association with vesicles could be an important factor determining DNA stability in natural environments.     

Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120

In order to improve the immunogenicity of HIV-1 envelope glycoproteins, we have fused gp120 to a carrier protein, hepatitis B surface antigen (HBsAg), which is capable of spontaneous assembly into virus-like particles. The HBsAg-gp120 hybrid proteins assembled efficiently into 20-30 nm particles. The particles resemble native HBsAg particles in size and density, consistent with a lipid composition of about 25% and a gp120 content of about 100 per particle. Particulate gp120 folds in its native conformation and is biologically active, as shown by high affinity binding of CD4. The particles express conformational determinants targeted by a panel of broadly cross-reactive neutralizing antibodies, and they show tight packing of gp120. Because the particles are lipoprotein micelles, an array of gp120 on their surface closely mimics gp120 on the surface of HIV-1 virions. These gp120-rich particles can enhance the quality, as well as quantity, of antibodies elicited by a gp120 vaccine.     

Induction of insert-specific immune response in mice by hamster polyomavirus VP1 derived virus-like particles carrying LCMV GP33 CTL epitope

Hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) carrying model GP33 CTL epitope derived from Lymphocytic choriomeningitis virus (LCMV) were generated in yeast and examined for their capability to induce CTL response in mice. Chimeric VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo and induced antigen-specific CD8+ T cell proliferation. Mice immunized only once with VP1-GP33 VLPs without adjuvant developed an effective GP33-specific memory T cell response: 70% were fully and 30% partially protected from LCMV infection. Moreover, aggressive growth of tumors expressing GP33 was significantly delayed in these mice in vivo. Therefore, HaPyV VP1-derived VLP harboring CTL epitopes are attractive vaccine candidates for the induction of insert-specific CTL immune response.     

Expression and characterization of human group C rotavirus virus-like particles in insect cells

Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpC RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.     

Activity of Novispirin G-10, a novel antimicrobial peptide against Chlamydia trachomatis and vaginosis-associated bacteria

We tested the activity of Novispirin G-10, a novel antimicrobial alpha-helical octadecapeptide structurally related to cathelicidins and other innate immunity peptides, against Chlamydia trachomatis serovars L2, D, and E and three organisms associated with bacterial vaginosis (BV). The peptide's activity against C. trachomatis was measured in 48-h shell vial assays with McCoy cell targets. Exposure to 100 micro g/ml of Novispirin G-10 reduced the infectivity of serovars D and E by 99.4-100% and serovar L2 by 91.7-99.1%. At the same concentration of 100 micro g/ml, Novispirin G-10 rapidly killed >99% of Mobiluncus curtisii, Gardnerella vaginalis, and Prevotella bivia, in standard colony-forming unit (CFU) assays. Given its simple structure and relative lack of cytotoxic and hemolytic activity, Novispirin G-10 may be a useful component of microbicide preparations designed to prevent chlamydial infection and/or remediate the abnormal vaginal flora associated with BV.     

Secretion of noninfectious dengue virus-like particles and identification of amino acids in the stem region involved in intracellular retention of envelope protein

DNA plasmids that express flavivirus premembrane/membrane (prM/M) and envelope (E) proteins in the form of virus-like particles (VLPs) have an excellent potential as DNA vaccine candidates against virus infection. The plasmid-expressed VLPs are also useful as safe, noninfectious antigens in serodiagnostic assays. We have constructed plasmids containing the prM/M and E gene regions for DENV-1, -3, and -4 that express and secrete VLPs when electroporated into Chinese hamster ovary cells. Constructs containing the full-length DENV-1 E protein gene did not secrete VLPs into tissue culture fluid effectively. However, a 16-fold increase in ELISA titers of DENV-1 VLPs was achieved after replacing the carboxy-terminal 20% region of DENV-1 E protein gene with the corresponding sequence of Japanese encephalitis virus (JEV). DENV-3 plasmids containing either the full-length DENV-3 E protein gene or the 20% JEV sequence replacement secreted VLPs to similarly high levels. Whereas DENV-4 VLPs were secreted to high levels by plasmids containing the full-length DENV-4 E protein gene but not by the chimeric plasmid containing 20% JEV E replacement. Domain substitutions by replacing prM/M protein stem-anchor region with the corresponding prM/M stem-anchor region of JEV or DENV-2 in the chimeric DENV-4 construct failed to promote the secretion of DENV-4 VLPs. Using the DENV-2 chimeric plasmid with carboxy-terminal 10% of JEV E gene, the sequence responsible for intracellular localization of E protein was mapped onto the E-H1 alpha-helix domain of DENV-2 E protein. Substitution of three amino acids from the DENV-2 sequence to the corresponding amino acids in the JEV sequence (I398L, M401A, and M412L) in the E-H1 was sufficient to promote extracellular secretion and resulted in detectable titers of DENV-2 VLP secretion.     

Purification of infectious myonecrosis virus (IMNV) in species of marine shrimp Litopenaeus vannamei in the State of Ceará

In Brazil, shrimp farming has been developed most intensely in the Northeast Region. Recently, however, exporters have become concerned over the appearance of Infectious Myonecrosis (IMN), the etiological agent of which is a virus called Infectious Myonecrosis Virus (IMNV). Although IMNV has been characterized extensively, purification methods are complicated to reproduce and very expensive. The objective of this study was to purify the IMNV virus using an easy reproductive method and to produce anti-IMNV antibodies to be used in diagnostic methods. Shrimp samples showing symptoms of IMN obtained from two aquaculture farms in Ceará were used for this purpose.IMNV-positive shrimps were macerated in phosphate buffer, pH 7.5, enriched with antioxidants, clarified with chloroform and the supernatant was submitted to differential centrifugation, precipitated using PEG and NaCl and finally loaded on a discontinuous gradient of sucrose. Purified IMNV was submitted to RT-PCR and electrophoresis either in agarose gel or SDS-PAGE, which revealed RNA and protein bands, characteristic of IMNV. IMNV induced humoral immune response in Swiss mice when administered subcutaneously. Anti-IMNV antibodies were identified by ELISA (enzyme-linked immunosorbent assay) and Western blotting methods and produced a response against purified IMNV and the crude extract obtained from the infected shrimp. However, antibodies specific to the crude extract obtained from uninfected shrimp were not detected. This is the first report of IMNV having been purified in Brazil and the first time that specific antibodies against IMNV proteins have been produced. These results suggest that easy methods can be developed to produce specific antiserum for viral diagnosis on a large scale.     

Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu²⁺, either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu²⁺ associated proenzymes. Cu²⁺ associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu²⁺ showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu²⁺ than rPPO2 did. Surprisingly, however, high concentration of Cu²⁺ (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2 < rPPO3 < rPPO1. If activated by ethanol, the Km and Vmax of each rPPO were lower than by 2-propanol. Due to the difficulty in obtaining functional PPOs via traditional purification methods, the method established in this study will be helpful to produce active insect recombinant PPOs for the study of PPO properties and functions in the future.