体外共表达的猪繁殖与呼吸综合征病毒GP5和M蛋白可形成异源二聚体

为了探讨猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因编码的GP5蛋白和ORF6编码的M蛋白体外共表达特性,分别构建了PRRSV ORF5、ORF6单基因或双基因共表达的真核表达质粒pCI-ORF5、pCI-ORF6和pCI-ORF5/ORF6,转染BHK-21细胞,Western blot检测证实共表达的GP5和M蛋白能够形成异源二聚体。同时,以绿色荧光蛋白(EGFP)和红色荧光蛋白(RFP)为示踪,发现当ORF5-EGFP和ORF6-RFP共表达时,能促进GP5蛋白从内质网向高尔基体转运,提示GP5-M异源二聚体的形成可能与GP5蛋白的翻译后修饰、转运、定位有关。     

A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.     

Recombinant infectious bursal disease virus carrying hepatitis C virus epitopes

The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P(BC) and P(HI)) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector.     

Length of the acyl carbonyl bond in acyl-serine proteases correlates with reactivity

Resonance Raman (RR) spectroscopy has been used to obtain the vibrational spectrum of the acyl carbonyl group in a series of acylchymotrypsins and acylsubtilisins at the pH of optimum hydrolysis. The acyl-enzymes, which utilize arylacryloyl acyl groups, include three oxyanion hole mutants of subtilisin BPN', Asn155Leu, Asn155Gln, and Asn155Arg, and encompass a 500-fold range of deacylation rate constants. For each acyl-enzyme a RR carbonyl band has been identified which arises from a population of carbonyl groups undergoing nucleophilic attack in the active site. As the deacylation rate (k3) increases through the series of acyl-enzymes, the carbonyl stretching band (vC = O) is observed to shift to lower frequency, indicating an increase in single bond character of the reactive acyl carbonyl group. Experiments involving the oxyanion hole mutants of subtilisin BPN' indicate that a shift of vC = O to lower frequency results from stronger hydrogen bonding of the acyl carbonyl group in the oxyanion hole. A plot of log k3 against vC = O is linear over the range investigated, demonstrating that the changes in vC = O correlate with the free energy of activation for the deacylation reaction. By use of an empirical correlation between carbonyl frequency (vC = O) and carbonyl bond length (rC = O) it is estimated that rC = O increases by 0.015 A as the deacylation rate increases 500-fold through the series of acyl-enzymes. This change in rC = O is about 7% of that expected for going from a formal C = O double bond in the acyl-enzyme to a formal C-O single bond in the tetrahedral intermediate for deacylation. The data also allow us to estimate the energy needed to extend the acyl carbonyl group along its axis to be 950 kJ mol-1 A-1.     

Comparative study of variola virus and monkeypox virus interferon-gamma-binding

DNA fragments containing genes for coding IFN-gamma-binding proteins (IFNgammaBPs) of variola virus (VARV) and monkeypox virus (MPXV) were obtained from viral genomes using PCR. Isolated genes coding desired proteins were expressed in the insect Sf21 cells using baculovirus expression system. Secreted recombinant IFNgammaBPs were isolated from culture medium of infected Sf21 cells through affinity chromatography procedure. SDS-PAAG and Western blot analysis of culture medium of infected insect cells and preparations of purified recombinant IFNgammaBPs indicated that recombinant viral proteins were dimerized even in the absence of ligand (hIFNgamma) unlike their cell (eucaryotic) analogs. Biological activity of the recombinant IFNgammaBPs were studied in the test of protective effect inhibition of hIFNgamma on L68 cells infected with murine encephalomyocarditis virus. It was shown that recombinant IFNgammaBPs had dose-dependent IFNgamma-inhibiting activity. A possibility of the elaboration of new therapeutics for anti-hIFNgamma therapy on the base of IFNgammaBPs is discussed.     

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.     

Vaccinia virus interleukin-18-binding protein promotes virulence by reducing gamma interferon production and natural killer and T-cell activity

Interleukin-18 (IL-18) is a proinflammatory cytokine that promotes natural killer (NK) and T-cell activation. Several poxviruses, including vaccinia virus (VV), encode a soluble IL-18-binding protein (IL-18bp). The role of the VV IL-18bp (gene C12L) in vivo was studied with wild-type (vC12L), deletion mutant (vDeltaC12L), and revertant (vC12L-rev) viruses in a murine intranasal model of infection. The data show that vDeltaC12L was markedly attenuated, characterized by a mild weight loss and reduced virus titers in lungs, brain, and spleen. Three days after infection, NK cytotoxic activity was augmented in the lung, spleen, and mediastinal lymph nodes (MLNs) of vDeltaC12L-infected mice compared to controls. Seven days after infection, vDeltaC12L-infected mice displayed heightened VV-specific cytotoxic T-lymphocyte (CTL) responses in the lungs, spleen, and MLNs. Gamma interferon (IFN-gamma) levels were also dramatically elevated in lavage fluids and cells from lungs of mice infected with vDeltaC12L. Finally, we demonstrate that IL-18 is produced in vitro and in vivo after VV infection. Taken together, these data demonstrate a role for the vIL-18bp in counteracting IL-18 in both the innate and the specific immune response to VV infection and indicate that the ability of IL-18 to promote vigorous T-cell responses (cytotoxic activity and IFN-gamma production) is a critical factor in the accelerated clearance of the vDeltaC12L mutant.     

Human T lymphotrophic virus type I may not be associated with multiple sclerosis in Japan.

To study the possible involvement of human T lymphotrophic virus type I (HTLV-I) or a related retrovirus in Japanese cases of multiple sclerosis (MS), we first performed a Western blot analysis with purified Ag of HTLV-I. Ten out of 31 MS patients (32.2%), 19 of 66 patients (28.8%) with other neurologic diseases, and 2 of 64 healthy blood donors (3.1%) had antibodies reactive with Ag corresponding to the group-specific Ag (gag) proteins (p15, p19, p24) on their sera. There were no significant differences between MS and other neurologic diseases concerning the patterns and the frequency. Second, we tried to establish T cell lines from PBMC of 22 MS patients with crude IL-2 without accessory cells, because HTLV-I-infected T cells can be immortalized in a high ratio under those conditions. Only one T cell line (MS-14C), however, could be maintained in long term culture. MS-14C and cultured T cells for 3 to 5 wk derived from MS patients were examined by Southern blot analysis under both stringent and low stringent conditions with HTLV-I as a probe. No HTLV-I related bands could be detected. By polymerase chain reaction examination, we also could not detect HTLV-I provirus genome in the fresh PBMC from 20 MS patients, although some of them had gag-reactive antibodies. Our data do not favor the hypothesis of HTLV-I or an HTLV-I-related human retrovirus in the etiology of MS.     

RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA.

The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3' end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3' noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3' NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3' NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3' NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis.     

Murine leukemia virus R Peptide inhibits influenza virus hemagglutinin-induced membrane fusion

The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.     

Genetic diversity of Usutu virus

Usutu virus is a mosquito-borne virus first isolated from Culex naevei in South Africa in 1959. The first emergence of Usutu virus outside Africa was recorded in Austria. Here, a phylogenetic analysis targeting the E5 and NS5 genes was carried out on the viral strains circulating in Europe. The NS5 gene tree showed two main clades, one of which included the Italian sequences. In the E gene tree all sequences grouped into the same main clade, with sequences from Austria divided into two separate clusters. Only sites under negative selective pressure were found in E and NS5 proteins. The results suggest that Usutu virus circulating in Europe has a degree of genetic diversity higher than expected and that infection may arise from different sources.     

Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs.

We have characterized the proteins and mRNAs of bovine respiratory syncytial (BRS) virus strain 391-2 and constructed cDNA clones corresponding to 9 of the 10 BRS virus mRNAs. The proteins of BRS virus-infected cells were compared with the proteins from human respiratory syncytial (HRS) virus-infected cells. Nine proteins specific to BRS virus-infected cells, corresponding to nine HRS virus proteins, were identified. Only a BRS virus polymerase protein remains to be identified. The BRS virus G glycoprotein showed major antigenic differences from the HRS virus G glycoprotein by immunoprecipitation and Western (immuno-) blot analysis, whereas the BRS virus F, N, M, and P proteins showed antigenic cross-reactivity with their HRS virus counterparts. Analysis of RNAs from BRS virus-infected cells showed virus-specific RNAs which had electrophoretic mobilities similar to those of mRNAs of HRS virus but which hybridized poorly or not at all with HRS virus-specific probes in Northern (RNA) blot analysis. To analyze the BRS virus RNAs further, cDNA clones to the BRS virus mRNAs were generated. Nine separate groups of clones were identified and shown to correspond to nine BRS virus mRNAs by Northern blot analysis. A 10th BRS virus large mRNA was identified by analogy with the HRS virus polymerase mRNA. These data show that like HRS virus, BRS virus has 10 genes coding for 10 mRNAs.     

A physical link between the pseudorabies virus capsid and the nuclear egress complex

Following their assembly, herpesvirus capsids exit the nucleus by budding at the inner nuclear membrane. Two highly conserved viral proteins are required for this process, pUL31 and pUL34. In this report, we demonstrate that the pUL31 component of the pseudorabies virus nuclear egress complex is a conditional capsid-binding protein that is unmasked in the absence of pUL34. The interaction between pUL31 and capsids was confirmed through fluorescence microscopy and Western blot analysis of purified intranuclear capsids. Three viral proteins were tested for their abilities to mediate the pUL31-capsid interaction: the minor capsid protein pUL25, the portal protein pUL6, and the terminase subunit pUL33. Despite the requirement for each protein in nuclear egress, none of these viral proteins were required for the pUL31-capsid interaction. These findings provide the first formal evidence that a herpesvirus nuclear egress complex interacts with capsids and have implications for how DNA-containing capsids are selectively targeted for nuclear egress.     

Vertical slab gel electrophoresis on a large number of samples: an apparatus with the capacity for central cooling

Following horizontal electroelution, or blotting, of proteins from polyacrylamide gels to immobilizing matrices, such as nitrocellulose or Zeta-bind paper, the transferred proteins can be derivatized in situ with pyridoxal 5'-phosphate and sodium borohydride. After a quenching step to eliminate nonspecific binding of antibody to the protein-binding matrix, the blot is incubated with a solution containing a mouse monoclonal antibody specific for the 5'-phosphopyridoxyl group. The transferred proteins can then be located on the blot with second antibody staining procedures employing either a peroxidase-linked goat anti-mouse F(ab')2 antibody or a peroxidase-linked avidin/biotin system. The solid-phase enzyme-linked immunosorbent assay method described in this report is a mild, general, and sensitive immunochemical method for the detection of proteins on protein-binding matrices.     

Plasma proteomic analysis of patients infected with H1N1 influenza virus

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1‐infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano‐ultra performance LC‐MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1‐infected group as compared with the control group. Of 14 up‐ and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine‐rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.     

Evidence for mitochondrial localization of P5, a member of the protein disulphide isomerase family

This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.     

Expression of the nucleotide sequence for the M2e peptide of avian influenza virus in transgenic tobacco plants

The M2e peptide of the H5N1 A/Chicken/Kurgan/05/2005 avian influenza virus was successfully synthesized in transgenic tobacco plants. The amino-terminal segment of the M2 protein, comprised of 22, 30, or 43 amino acids, including the M2e peptide (M122, M130, and M143 variants, respectively), was translationally fused with the N-terminus of β-glucuronidase in the pBI121 plant expression vector. The nucleotide sequence of the target fragment was synthesized by ligation from synthetic oligonucleotides; its codon composition was adapted for expression in plants. Tobacco plants were successfully transformed with the obtained vectors (pBIM122, pBIM130, and pBIM143, respectively). In the plants transformed with pBIM143, the Ml43-β-glucuronidase fusion protein was not produced, probably due to the presence of the M2 protein transmembrane domain (25–43 aa of M2) in this construct. In the pBIM122- and pBIMl30-transformed plants, the target M2e peptide was expressed as a component of the Ml22-β-glucuronidase and M130-β-glucuronidase fusion proteins, respectively, as was detected by Western blot analysis. These proteins were detected as bands of the expected size without apparent degradation. As a result, the M2e peptide of the H5N1 avian influenza virus was successfully synthesized for the first time in nuclear-transformed transgenic plants. The results obtained in this study will be used for developing a transgenic plant-based edible antiinfluenza vaccine.