- Volume 84, Issue 8, 2003
Volume 84, Issue 8, 2003
- Reviews
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bZIP proteins of human gammaherpesviruses
More LessThe human gammaherpesviruses Epstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) both infect lymphoid and epithelial cells and both are implicated in the development of cancer. The two viruses establish latency in B-lymphoid cells that, once disrupted, leads to a burst of virus replication during the lytic cycle. A basic leucine zipper (bZIP) transcription factor encoded by EBV, Zta (also known as BZLF1 and ZEBRA), is key to the disruption of EBV latency. KSHV encodes a related protein, K-bZIP (also known as RAP and K8α). Recent developments in our understanding of the structures and functions of these two viral bZIP proteins have led to the conclusion that they are not homologues. Two important features of Zta are its ability to interact directly with DNA and to induce EBV replication whereas K-bZIP is not known to interact directly with DNA or to induce KSHV replication. Despite these differences, the ability to disrupt cell cycle control is conserved in both Zta and K-bZIP. The interactions of Zta and K-bZIP with cellular genes will be reviewed here.
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Molecular biology of umbraviruses: phantom warriors
More LessThe genomes of umbraviruses differ from those of most other viruses in that they do not encode a coat protein, and thus no virus particles are formed in infected plants. Protection of umbraviral RNA outside the host plant, during vector transmission, utilizes the coat protein of an assistor luteovirus, but this review focuses on the mechanisms that compensate for the lack of a coat protein in processes within the host plant. As well as an RNA-dependent RNA polymerase, umbravirus genomes encode two other proteins from almost completely overlapping open reading frames. One of these is a cell-to-cell movement protein that can mediate the transport of homologous and heterologous viral RNAs through plasmodesmata without the participation of a coat protein. The other, the ORF3 protein, binds to viral RNA to form filamentous ribonucleoprotein particles that have elements of helical structure. It serves to stabilize the RNA and facilitates its transport through the vascular system of the plant. It may also be involved in protection of the viral RNA from the plant's defensive RNA-silencing response, although it is not a suppressor of silencing. The ORF3 protein also enters the cell nucleus, specifically targeting the nucleolus. Although the function of this localization is unknown, the ORF3 protein may provide a valuable tool for investigating plant nucleolar function.
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- Animal
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- RNA viruses
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Receptor use by vesicular stomatitis virus pseudotypes with glycoproteins of defective variants of measles virus isolated from brains of patients with subacute sclerosing panencephalitis
The vaccine or Vero cell-adapted strains of measles virus (MV) have been reported to use CD46 as a cell entry receptor, while lymphotropic MVs preferentially use the signalling lymphocyte activation molecule (SLAM or CD150). In contrast to the virus obtained from patients with acute measles, little is known about the receptor that is used by defective variants of MV isolated from patients with subacute sclerosing panencephalitis (SSPE). The receptor-binding properties of SSPE strains of MV were analysed using vesicular stomatitis virus pseudotypes expressing the envelope glycoproteins of SSPE strains of MV. Such pseudotype viruses could use SLAM but not CD46 for entry. The pseudotype viruses with SSPE envelope glycoproteins could enter Vero cells, which do not express SLAM. In addition, their entry was not blocked by the monoclonal antibody to CD46, pointing to another entry receptor for SSPE strains on Vero cells. Furthermore, the unknown receptor(s), distinct from SLAM and CD46, may be present on cell lines derived from lymphoid and neural cells. Biochemical characterization of the receptor present on Vero cells and SK-N-SH neuroblastoma cells was consistent with a glycoprotein. Identification of additional entry receptors for MV will provide new insights into the mechanism of spread of MV in the central nervous system and possible reasons for differences between MVs isolated from patients with acute measles and SSPE.
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Successful mucosal immunization of cotton rats in the presence of measles virus-specific antibodies depends on degree of attenuation of vaccine vector and virus dose
After passive transfer of measles virus (MV)-specific antibodies, vaccine-induced seroconversion and subsequent protection is inhibited in cotton rats (Sigmodon hispidus). In this system, an attenuated, recombinant vesicular stomatitis virus expressing the MV haemagglutinin (VSV-H) was found previously to induce neutralizing antibodies and protection against MV challenge after intranasal (i.n.) immunization. Here it is demonstrated that, after i.n. immunization, VSV-H is found in both lung and brain tissue in the absence of clinical signs. Intratracheal inoculation, which does not lead to infection of the brain, proved that immunization via the lung mucosa is sufficient to protect. To reduce or eliminate infection of the brain after i.n. inoculation, stepwise-attenuated VSV-H mutants with truncated cytoplasmic tails of the G protein were tested in cotton rats. A mutant with 9 aa in the G cytoplasmic tail was found at much lower levels in the brain and was protective in the absence or presence of MV-specific antibodies. A more attenuated mutant containing only 1 aa in its tail was not found in brain tissue after inoculation, but it still induced protective antibody to measles in the absence of MV-specific antibody. However, its ability to induce MV-neutralizing antibodies in the presence of passively transferred MV-specific antibodies and its protective capacity was abolished unless higher-dose immunizations were used. This study demonstrates that a lower degree of attenuation is required to be able to immunize in the presence of MV-specific antibodies.
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Bovine parainfluenza virus type 3 (PIV3) expressing the respiratory syncytial virus (RSV) attachment and fusion proteins protects hamsters from challenge with human PIV3 and RSV
More LessParainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV) are the main causes of ubiquitous acute respiratory diseases of infancy and early childhood, causing 20–25 % of pneumonia and 45–50 % of bronchiolitis in hospitalized children. The primary goal of this study was to create an effective and safe RSV vaccine based on utilizing attenuated bovine PIV3 (bPIV3) as a virus vector backbone. bPIV3 had been evaluated in human clinical trials and was shown to be attenuated and immunogenic in children as young as 2 months of age. The ability of bPIV3 to function as a virus vaccine vector was explored further by introducing the RSV attachment (G) and fusion (F) genes into the bPIV3 RNA genome. The resulting virus, bPIV3/RSV(I), contained an insert of 2900 nt, comprising two translationally competent transcription units. Despite this increase in genetic material, the virus replicated to high titres in Vero cells. This recombinant virus expressed the RSV G and F proteins sufficiently to evoke a protective immune response in hamsters upon challenge with RSV or human PIV3 and to elicit RSV neutralizing and PIV3 haemagglutinin inhibition serum antibodies. In effect, a bivalent vaccine was produced that could protect vaccinees from RSV as well as PIV3. Such a vaccine would vastly reduce the respiratory disease burden, the associated hospitalization costs and, most importantly, decrease morbidity and mortality of infants, immunocompromised individuals and the elderly.
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Newcastle disease virus nucleocapsid protein: self-assembly and length-determination domains
More LessThe nucleocapsid protein (NP) of Newcastle disease virus expressed in E. coli assembled as ring- and herringbone-like particles. In order to identify the contiguous NP sequence essential for assembly of these particles, 11 N- or C-terminally deleted NP mutants were constructed and their ability to self-assemble was tested. The results indicate that a large part of the NP N-terminal end, encompassing amino acids 1 to 375, is required for proper folding to form a herringbone-like structure. In contrast, the C-terminal end covering amino acids 376 to 489 was dispensable for the formation of herringbone-like particles. A region located between amino acids 375 to 439 may play a role in regulating the length of the herringbone-like particles. Mutants with amino acid deletions further from the C-terminal end (84, 98, 109 and 114 amino acids) tended to form longer particles compared to mutants with shorter deletions (25 and 49 amino acids).
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Subgroup C avian metapneumovirus (MPV) and the recently isolated human MPV exhibit a common organization but have extensive sequence divergence in their putative SH and G genes
More LessThe genes encoding the putative small hydrophobic (SH), attachment (G) and polymerase (L) proteins of the Colorado isolate of subgroup C avian pneumovirus (APV) were entirely or partially sequenced. They all included metapneumovirus (MPV)-like gene start and gene end sequences. The deduced Colorado SH protein shared 26·9 and 21·7 % aa identity with its counterpart in human MPV (hMPV) and APV subgroup A, respectively, but its only significant aa similarities were to hMPV. Conserved features included a common hydrophobicity profile with an unique transmembrane domain and the conservation of most extracellular cysteine residues. The Colorado putative G gene encoded several ORFs, the longer of which encoded a 252 aa long type II glycoprotein with aa similarities to hMPV G only (20·6 % overall aa identity with seven conserved N-terminal residues). The putative Colorado G protein shared, at best, 21·0 % aa identity with its counterparts in the other APV subgroups and did not contain the extracellular cysteine residues and short aa stretch highly conserved in other APVs. The N-terminal end of the Colorado L protein exhibited 73·6 and 54·9 % aa identity with hMPV and APV subgroup A, respectively, with four aa blocks highly conserved among Pneumovirinae. Phylogenetic analysis performed on the nt sequences confirmed that the L sequences from MPVs were genetically related, whereas analysis of the G sequences revealed that among MPVs, only APV subgroups A, B and D clustered together, independently of both the Colorado isolate and hMPV, which shared weak genetic relatedness at the G gene level.
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Isolation and partial characterization of a novel paramyxovirus from the gills of diseased seawater-reared Atlantic salmon (Salmo salar L.)
More LessA formerly undescribed virus has been isolated from the gills of farmed Atlantic salmon post-smolts in Norway suffering from gill disease. Cytopathic effects appeared in RTgill-W1 cells 9 weeks post-inoculation with gill tissue material. Virus production continued for an extended period thereafter. Light and electron microscopic examination revealed inclusions and replication in the cytoplasm. The viral nucleocapsid consisted of approximately 17 nm thick filaments in a herringbone pattern. Certain areas of the plasma membrane were thickened by the alignment of nucleocapsids on the internal surface and projections of 10 nm long viral glycoprotein spikes on the external surface. Virus assembly and release was achieved by budding through the modified plasma membrane. Negatively stained virions were spherical and partly pleomorphic with a diameter of 150–300 nm as seen by electron microscopy. The virus was sensitive to chloroform, heat and low and high pH, and replication was not inhibited by Br-dU or IdU indicating an RNA genome. Both haemagglutination and receptor-destroying enzyme activity were associated with the virions and the formation of syncytia in infected cultures indicated fusion activity. The receptor-destroying enzyme was identified as neuraminidase. The virus contained five major structural polypeptides with estimated molecular masses of 70, 62, 60, 48 and 37 kDa. Its buoyant density was 1·18–1·19 g ml−1 in CsCl gradients. From the observed properties we conclude that this new virus belongs to the Paramyxoviridae and suggest the name Atlantic salmon paramyxovirus (ASPV). Furthermore, replication occurred at 6–21 °C, suggesting a host range confined to cold-blooded animals.
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Dengue virus type 2 NS3 protease and NS2B-NS3 protease precursor induce apoptosis
More LessApoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17·5 and 51·5 % of cells expressing NS3 serine protease and NS2B-NS3185 serine protease precursor protein [NS2B-NS3185(pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3185(pro). NS2B-NS3185(pro) was detected as NS2B-NS3185(pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His51 with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3185(pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3185(pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.
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Hantaviruses induce cytopathic effects and apoptosis in continuous human embryonic kidney cells
More LessHantaviruses are maintained in nature in persistently infected rodents and can also persistently infect cultured mammalian cells, causing little or no cytopathology. An unexpected outcome of this study was the observation of cytopathic effects (CPE) in the hantavirus-infected human embryonic kidney cell line HEK293. It was confirmed that hantaviruses induce apoptosis in HEK293 cells, although apoptosis appeared mostly in uninfected, bystander cells and rarely in infected HEK293 cells. Although studies by others suggest that the nucleocapsid protein of Puumala virus interacts with the Fas-mediated apoptosis enhancer Daxx at the gene expression level, it was determined that members of the TNF receptor superfamily did not contribute to the apoptosis observed in infected HEK293 cells. The observation of CPE in HEK293 cells might lead to a better understanding of the mechanisms of persistence and pathogenesis in hantavirus infections.
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Analysis of the cloverleaf element in a human rhinovirus type 14/poliovirus chimera: correlation of subdomain D structure, ternary protein complex formation and virus replication
More LessRNA genomes of enteroviruses and rhinoviruses contain a 5′-terminal structure, the cloverleaf (CL), which serves as signal in RNA synthesis. Substitution of the poliovirus [PV1(M)] CL with that of human rhinovirus type 2 (HRV2) was shown previously to produce a viable chimeric PV, whereas substitution with the HRV14 CL produced a null phenotype. Fittingly, the HRV14 CL failed to form a complex with PV-specific proteins 3CDpro–3AB or 3CDpro–PCBP2, considered essential for RNA synthesis. It was reported previously ( Rohll et al., J Virol 68, 4384–4391, 1994 ) that the major determinant for the null phenotype of a PV/HRV14 chimera resides in subdomain Id of the HRV14 CL. Using a chimeric PV/HRV14 CL in the context of the PV genome, stem–loop Id of HRV14 CL was genetically dissected. It contains the sequence C57 UAU 60-G, the underlined nucleotides forming the loop that is shorter by 1 nt when compared to the corresponding PV structure (UUGC 60 GG). Insertion of a G nucleotide to form a tetra loop (C57 UAU 60 GG61) did not rescue replication of the chimera. However, an additional mutation at position 60 (C57 UAC 60 GG61) yielded a replicating genome. Only the mutant PV/HRV14 CL with the UAC 60 G tetra loop formed ternary complexes efficiently with either PV proteins 3CDpro–3AB or 3CDpro–PCBP2. Thus, in the context of PV RNA synthesis, the presence of a tetra loop in subdomain D of the CL per se is not sufficient for function. The sequence and, consequently, the structure of the tetra loop plays an essential role. Biochemical assays demonstrated that the function of the CL element and the function of the cis-acting replication element in the 3Dpol–3CDpro-dependent uridylylation of VPg are not linked.
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A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy
Despite numerous studies on human immunodeficiency virus type 1 (HIV-1) fitness, many key conceptual and technical questions are still unsolved. For example, the proper system to determine virus fitness of HIV-1 is still unknown. In this study, an assay was developed to estimate HIV-1 fitness based on growth competition experiments and TaqMan real-time PCR. This novel technique was compared with several methods (i.e. virus growth kinetics, growth competition/heteroduplex-tracking analysis and single-cycle replication capacity assay) in order to analyse the impact of various genomic regions and overall genetic background on virus fitness. HIV-1 primary isolates and three different sets of recombinant viruses [i.e. recombinant clones carrying protease (PR), reverse transcriptase (RT) or the 3′ end of Gag, PR and RT (3′Gag/PR/RT), sequences amplified by PCR from the same primary isolates)] were evaluated. Here, it is demonstrated that, in spite of intrinsic differences, both growth competition/TaqMan and single-cycle replication assays detected a significant reduction in HIV-1 fitness as a consequence of drug-resistant mutations in pol. However, this new assay, based on HIV-1 isolates, may be useful to quantify replicative fitness in viruses from patients treated simultaneously with antiretroviral drugs targeting different genomic regions of HIV-1 (e.g. pol and env).
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Analysis of transcriptional regulatory sequences in the human endogenous retrovirus W long terminal repeat
More LessThe U3 region of the human endogenous retrovirus W long terminal repeat (HERV-W LTR) contains several putative regulatory sequences that might not only regulate transcription of viral genes but also influence the expression of neighbouring cellular genes. In this study, we analysed the U3 region in detail in order to understand the transcriptional regulatory mechanism of HERV-W. Two transcription factor (TF) binding sites for Oct-1 and C/EBP were important as a silencer and an enhancer, respectively, for transcriptional regulation. Furthermore, it was possible to divide the HERV-W LTR isolates into two groups depending on their promoter strength, which might be determined by the integrity of the two TF binding sites. However, neither the Oct-1 binding site nor the CAAT-box was required for the cell type-specific activity of the HERV-W LTR. Instead, the 3′ terminus of U3 from 191 to 260, which includes a TATA box, was sufficient for specificity, suggesting that the efficiency of assembly of basic transcription machinery at the TATA box of HERV-W LTR might determine the cell type specificity.
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DNA vaccination of macaques by a full-genome simian/human immunodeficiency virus type 1 plasmid chimera that produces non-infectious virus particles
A DNA vaccination regime was investigated previously in rhesus macaques using a full-genome human immunodeficiency virus type 1 (HIV-1) plasmid, which, due to mutations in the nucleocapsid (NC) proteins, produced only non-infectious HIV-1 particles ( Akahata et al., Virology 275, 116–124, 2000 ). In that study, four monkeys were injected intramuscularly 14 times with the plasmid. All of them showed immunological responses against HIV-1 and partial protection from challenge with a simian immunodeficiency virus/HIV (SHIV) chimeric virus. To improve this DNA vaccination regime, the plasmid used for vaccination was changed. In the present study, four macaques were injected intramuscularly eight times with a full-genome SHIV plasmid that produces non-infectious SHIV particles. CTL activities were higher than those observed in monkeys vaccinated previously with the HIV-1 plasmid. In all macaques vaccinated, peak plasma virus loads after homologous challenge with SHIV were two to three orders of magnitude lower than those of the naive controls, and virus loads fell below the level of detection at 6 weeks post-challenge. This suggested that the vaccination regime in this study was partially effective and better than the previous regime.
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Characterization of enzootic nasal tumour virus of goats: complete sequence and tissue distribution
The complete genome sequence of a new isolate of enzootic nasal tumour virus (ENTV-2), associated with enzootic nasal adenocarcinoma (ENA) of goats, was determined. The genome exhibits a genetic organization characteristic of β-retroviruses. ENTV-2 is closely related to the retrovirus (ENTV-1) associated with enzootic adenocarcinoma of sheep, and to jaagsiekte retrovirus. The main sequence differences between these viruses reside in orfX, the U3 LTR, two small regions in gag and the transmembrane (TM) region of env. Sequence analysis of the TM region of env from several sheep and goats naturally affected by ENA suggested that ENTV-1 and ENTV-2 are distinct viruses rather than geographical variants. Although both viruses transform secretory epithelial cells of the ethmoid turbinate, the study of their tissue distribution using specific PCRs showed that ENTV-2 establishes a disseminated lymphoid infection whereas ENTV-1 is mainly confined to the tumour.
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Mutational analysis of the R peptide cleavage site of Moloney murine leukaemia virus envelope protein
More LessMoloney murine leukaemia virus (MoMLV) enters host cells by membrane fusion between the viral envelope and the host cell membrane. The cytoplasmic tail (R peptide) of the MoMLV envelope protein (Env) is cleaved by the viral protease during virion maturation. R peptide-truncated Env induces syncytia in susceptible cells but R peptide-containing Env does not, indicating that the R peptide inhibits membrane fusion. To examine the function of amino acid residues at the R peptide cleavage site in virus entry, mutant Env expression plasmids containing amino acid substitutions at these cleavage site residues were constructed. Some of these mutants induced syncytia in NIH 3T3 cells, even though they expressed the R peptide, indicating the importance of these residues for membrane fusion inhibition by the R peptide. Some mutants in which R peptide cleavage was detected had comparable transduction efficiency to wild-type Env, but mutants in which R peptide cleavage was not detected had lower transduction efficiency than wild-type Env. This result strongly supports that R peptide cleavage is required for virus entry.
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- DNA viruses
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IL-12 and IL-18 act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system
More LessDevelopment of a protective host response against intracellular pathogens requires innate and cell-mediated immune responses, with cytokines playing an important role in host defences. Different studies in mice have shown that IL-12 can promote protective immunity to a variety of viruses but, during virus infection, little is known about the in vivo function of IL-18 alone or in combination with IL-12. Using recombinant vaccinia viruses (rVVs) expressing IL-12 and IL-18, the antiviral role of both cytokines in mice has been analysed. The specific anti-VV immune response elicited and the persistence of the virus in target tissues were compared in BALB/c mice inoculated with rVVs expressing IL-12 and IL-18 either singly or in combination. Delivery of IL-12 and IL-18 by rVVs in mice induced a significant enhancement in virus clearance from ovaries and spleen, greater than that expected from the sum of action of both cytokines. Virus clearance involved NK and T cells, as demonstrated in mice depleted of NK cells and in immunodeficient SCID animals. Th1 parameters (CD8+ T cell response and IgG2a : IgG1 ratios) were increased in mice inoculated with rVVs expressing both IL-12 and IL-18 as compared to those animals receiving a single cytokine. These findings indicate that when IL-12 and IL-18 are delivered by rVVs, different mechanisms involving both the innate and specific arms of the immune system act as mediators in the synergistic action of IL-12 and IL-18, leading to VV clearance. These results are of interest for the design of prophylactic as well as therapeutic VV-based strategies.
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A kinetic analysis of immune mediators in the lungs of mice infected with vaccinia virus and comparison with intradermal infection
More LessThe early inflammatory response to a virus may be critical in restricting infection and in shaping the subsequent adaptive immune response. In this study we have examined the early inflammatory response of mice following infection with vaccinia virus (VV) strain Western Reserve (WR). Respiratory challenge of BALB/c mice with VV led to early virus replication in the lung and upper respiratory tract followed by dissemination of virus to other visceral organs and to the brain. The number of inflammatory cells, largely macrophages and T lymphocytes, recovered from bronchoalveolar lavage (BAL) fluid increased markedly during infection and coincided with the expression of CC chemokine ligands (CCL) 3, 2 and 11 and CXC chemokine ligands (CXCL) 1 and 2/3 in BAL. The peak of the inflammatory response occurred around day 10 and declined thereafter. The antiviral cytokines IFN-γ and TNF-α, and the reactive nitrogen intermediate nitric oxide (NO), were also detected in BAL from VV-infected mice. A markedly different inflammatory response was observed after intradermal inoculation of WR into the ear pinnae of mice. Intradermal challenge was followed by highly localized virus replication and by a cellular influx, consisting largely of neutrophils and T lymphocytes, into the dermal compartment of the infected ear. Together these findings highlight differences in the pathogenesis and in the cellular inflammatory response to WR following intranasal and intradermal inoculation of mice.
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Evaluation of lumpy skin disease virus, a capripoxvirus, as a replication-deficient vaccine vector
More LessLumpy skin disease virus (LSDV), a capripoxvirus with a host range limited to ruminants, was evaluated as a replication-deficient vaccine vector for use in non-ruminant hosts. By using the rabies virus glycoprotein (RG) as a model antigen, it was demonstrated that recombinant LSDV encoding the rabies glycoprotein (rLSDV-RG) was able to express RG in both permissive (ruminant) and non-permissive (non-ruminant) cells. The recombinant LSDV, however, replicated to maturity only in permissive but not in non-permissive cells. Recombinant LSDV-RG was assessed for its ability to generate immunity against RG in non-ruminant hosts (rabbits and mice). Rabbits inoculated with rLSDV-RG produced rabies virus (RV) neutralizing antibodies at levels twofold higher than those reported by the WHO to be protective. BALB/c mice immunized with rLSDV-RG elicited levels of RV-specific cellular immunity (T-cell proliferation) comparable with those of mice immunized with a commercial inactivated rabies vaccine (Verorab; Pasteur Merieux). Most importantly, mice immunized with rLSDV-RG were protected from an aggressive intracranial rabies virus challenge.
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Epstein–Barr virus latent membrane protein-1 (LMP-1) and lytic LMP-1 localization in plasma membrane-derived extracellular vesicles and intracellular virions
More LessEpstein–Barr virus (EBV) is a human herpesvirus associated with a number of malignancies. EBV establishes a latent infection in human B cells in vitro, and infected lymphoblastoid cells proliferate indefinitely as a result of virus activation of cellular signalling pathways. Latently infected cells express a viral oncoprotein called the latent membrane protein-1 (LMP-1). LMP-1 signals both proliferative and survival signals to the infected B cell. The switch from latency to lytic replication is associated with upregulation of an N-terminally truncated LMP-1, called lytic LMP-1 (lyLMP-1). To understand better the relationship between LMP-1 protein function and the virus life cycle, LMP-1 and lyLMP-1 were precisely localized in infected B cells. Immunoelectron microscopy of latently infected cells revealed LMP-1 localized in discrete patches in the plasma membrane. Unexpectedly, immunogold-labelled LMP-1 was found in vesicles budding from the plasma membrane into the extracellular space and in small membrane vesicles accumulating in conditioned medium from infected cells. LyLMP-1 immunolabelling was observed only in B95-8 cells harbouring detectable intracellular virus particles and was abundant in the nuclear membrane early, and in the plasma membrane late, following lytic cycle induction. LyLMP-1 immunoreactivity was also observed at sites of virus budding and associated with intracellular virions, suggesting that lyLMP-1 might be incorporated into cytoplasmic virions when budding through the nuclear membrane.
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The vascular lesions of a cow and bison with sheep-associated malignant catarrhal fever contain ovine herpesvirus 2-infected CD8+ T lymphocytes
More LessMalignant catarrhal fever (MCF) is a herpesvirus disease syndrome of ruminants. The microscopic pathology of MCF is characterized by lymphoid proliferation and infiltration, necrotizing vasculitis and epithelial necrosis. Because previous attempts to detect viral antigen or nucleic acids in lesions have been unsuccessful, the pathogenesis of the lesions in acute MCF has been speculated to involve mechanisms of autoimmunity and lymphocyte dysregulation. In this study, the vascular lesions in the brains of a cow and a bison with acute MCF were evaluated by in situ PCR and immunohistochemistry. The results demonstrated that the predominant infiltrating cell type in these lesions was CD8+ T lymphocytes and that large numbers of these cells were infected with ovine herpesvirus 2. The lesions also contained macrophages, but no detectable CD4+ or B lymphocytes.
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The latency-associated transcript promoter of pseudorabies virus directs neuron-specific expression in trigeminal ganglia of transgenic mice
More LessThe latency-associated transcript (LAT) promoter of pseudorabies virus (PRV) is unique among viral promoters in that it remains active in trigeminal ganglia during the latent state. It is not known which the viral or host proteins regulate expression of the PRV LAT gene in latently infected neurons. To determine whether host transcriptional proteins in neurons can regulate the PRV LAT promoter in vivo, three transgenic mouse lines containing the PRV LAT promoter (LAP; LAP1 and LAP2) linked to the chloramphenicol acetyltransferase (CAT) gene were generated. All of the transgenic mouse lines, in the absence of the viral proteins, displayed strong expression of the transgene in trigeminal ganglia in addition to other neuronal tissues such as cerebral cortex, cerebellum, hippocampus and olfactory bulb. Expression of the transgene in neurons of trigeminal ganglia was demonstrated by in situ hybridization. These data provide direct evidence that neuronal transcription factors are sufficient to activate the PRV LAP in vivo and that the promoter is neuron-specific.
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Bombyx mori nucleopolyhedrovirus-based surface display system for recombinant proteins
More LessWe describe here the development of a ‘eukaryotic display system’ for heterologous proteins on the viral and host cell surfaces using Bombyx mori nucleopolyhedrovirus (BmNPV). The reporter gene gfp (green fluorescent protein) was fused to either the gp64 gene encoding the full-length BmNPV envelope protein GP64 or to its 5′ region encoding only the N-terminal domain harbouring the signal sequence, and recombinant viruses expressing the corresponding fusion proteins under the strong viral polyhedrin promoter were generated. On infection of the host insect B. mori or the host-derived BmN cells with the full-length GP64–GFP virus, abundant expression of the recombinant protein and its display on the cell surface were achieved. The fusion protein was also a component of the budded virions. Thus, the BmNPV-based display system provides an alternative to the previously established Autographa californica multinucleocapsid nucleopolyhedrovirus display system. The recombinant virus expressing GFP has also been used in preliminary pathological investigations on virus infection in B. mori and provides a simple method for screening for antiviral agents.
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Pathogenesis of Autographa californica M nucleopolyhedrovirus in fifth instar Spodoptera frugiperda
More LessWe have characterized infection and pathogenesis of an Autographa californica M nucleopolyhedrovirus recombinant, AcMNPV-hsp70/lacZ, carrying the lacZ reporter gene, in penultimate (fifth) instar Spodoptera frugiperda. Bioassays revealed that while <0·1 p.f.u. of budded virus was required to generate an LD50 by intrahaemocoelic injection, approximately 6000 occlusions were required orally to achieve the same mortality in newly moulted fifth instar (50) larvae. In pathogenesis experiments, 78 % of the 50 larvae inoculated orally with 6000 occlusions of AcMNPV-hsp70/lacZ were LacZ-positive at 8 h post-inoculation (p.i.) and 50 % had LacZ signals in tracheal cells indicating that in these larvae infection had been transmitted from the midgut to secondary target cells. At 24 h p.i., maximum numbers of midgut and midgut-associated tracheal foci were observed (mean of 35 foci per infected larva), and 88 % of the larvae were LacZ-positive. The extremely low foci-per-occlusion ratio (0·006) indicated that successful infection of midgut cells was the primary barrier to fatal infection. A second barrier involved the loss of infected tracheal cells associated with the midgut. At 24 h p.i., 88 % of the inoculated larvae had a systemic infection, but in bioassays only 51 % succumbed to polyhedrosis disease. The absence of melanized tracheal cells in the insects throughout the time-course suggested that the larvae that cleared their infections (38 %) did so by a mechanism other than a classical immune response.
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Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects
More LessInfection of cultured insect cells with Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) resulted in the generation of mutants with major genomic deletions. Some of the mutants lacked the ability to infect S. exigua larvae per os. The gene(s) responsible for this phenotype in SeMNPV was mapped within a contiguous sequence encoding ORFs 29–35. In this paper we have shown that SeMNPV ORFs 15–35 (including genes encoding cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP-1 and ARIF-1) are not essential for virus replication in cell culture or by in vivo intrahaemocoelic injection. By site-specific deletion mutagenesis of a full-length infectious clone of SeMNPV (bacmid) using ET recombination in E. coli, a series of SeMNPV bacmid mutants with increasing deletions in ORFs 15–35 was generated. Analyses of these mutants indicated that a deletion of SeMNPV ORF35 (Se35) resulted in loss of oral infectivity of polyhedral occlusion bodies. Reinsertion of ORF35 in SeMNPV bacmids lacking Se35 rescued oral infectivity. We propose the name pif-2 for Se35 and its baculovirus homologues (e.g. Autographa californica MNPV ORF22), by analogy to a different gene recently characterized in Spodoptera littoralis NPV, which was designated per os infectivity factor (pif). Similar to the p74 gene, which encodes an essential structural protein of the occlusion-derived virus envelope, pif and pif-2 belong to a group of 30 genes that are conserved among the Baculoviridae.
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Morphological and genomic characterization of the polydnavirus associated with the parasitoid wasp Glyptapanteles indiensis (Hymenoptera: Braconidae)
More LessGlyptapanteles indiensis polydnavirus (GiPDV) is essential for successful parasitization of the larval stage of the lepidopteran Lymantria dispar (gypsy moth) by the endoparasitic wasp Glyptapanteles indiensis. This virus has not been characterized previously. Ultrastructural studies of GiPDV showed that virions had a rod-like or rectangular form and each contained as many as ten nucleocapsids enclosed by a single unit membrane envelope. Field inversion gel electrophoresis (FIGE) analysis of the virus genomic DNA revealed that GiPDV had a segmented genome composed of 13 dsDNA segments, ranging in size from approximately 11 kb to more than 30 kb. Four genomic segments were present in higher molar concentration than the others. Further characterization of the GiPDV genome yielded several cDNA clones which derived from GiPDV-specific mRNAs, and Northern blot analysis confirmed expression of isolated cDNA clones in the parasitized host. Each was present on more than one GiPDV genomic DNA segment, suggesting the existence of related sequences among DNA segments. It has been proposed previously that in polydnavirus systems, genome segmentation, hypermolar ratio segments and segment nesting may function to increase the copy number of essential genes and to increase the levels of gene expression in the absence of virus replication. The present data support this notion and suggest that GiPDV morphology and genomic organization may be intrinsically linked to the function and evolutionary strategies of the virus.
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Comparative analysis of the genome organization of human adenovirus 11, a member of the human adenovirus species B, and the commonly used human adenovirus 5 vector, a member of species C
More LessAdenovirus type 11 (Ad11), a member of the human adenovirus species B (HAdV-B), has a tropism for the urinary tract. The genome of Ad11 was found to comprise 34 794 bp and is 1141 bp shorter than the Ad5 genome of species HAdV-C. The G+C content of the Ad11 genome is 48·9 %, whereas that of Ad5 is 55·2 %. Ad11 and Ad5 share 57 % nucleotide identity and possess the same four early regions, but the E3 region of Ad11 could not be divided into E3A and E3B. The late genes of Ad11 and Ad5 are organized into six and five regions, respectively. Thirty-eight putative ORFs were identified in the Ad11 genome. The ORFs in the late regions, the E2B region and IVa2 show high amino acid identity between Ad11 and Ad5, whereas the ORFs in E1, E2A, E3 and E4, protein IX and the fibre protein show low amino acid identity. The highest and lowest identities were noted in the pre-terminal protein and fibre proteins: 85 % and 24·6 %, respectively. The E3 20·3K and 20·6K ORFs and the L6 agnoprotein were present in the Ad11 genome only, whereas the E3 11·6K cell death protein was identified only in Ad5. All ORFs but the E3 10·3K and L4 pVIII protein vary not only in composition but also in size. Ad11 may have a higher vector capacity than Ad5, since it has a shorter genome and a shorter fibre. Furthermore, in the E3 region, two additional ORFs can be deleted to give extra capacity for foreign DNA.
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Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins
More LessThe human MxA protein is an interferon (IFN)-inducible GTPase with proven antiviral activity against diverse viruses. IFN responsiveness is impaired in chronic hepatitis B virus (HBV) infection. Accordingly, initial experiments showed that, in contrast to parental HepG2 cells, when HepG2-derived 2.2.15 liver cells carrying the HBV genome were treated with IFN, they could not synthesize the MxA protein. Furthermore, MxA expression was reduced in HepG2 cells transiently transfected with the HBV genome. To assess whether HBV-encoded precore/core (preC/C) proteins interact with the IFN-signalling pathway, HepG2, Chang and HeLa cells were transfected with preC/C expression plasmids; the levels of signal transducers remained unaffected. Next, full-length and deletion mutants fused to the CAT reporter gene were tested to investigate whether MxA inhibition occurs at the promoter level. In co-transfection experiments, IFN-induced CAT activity was inhibited by preC/C expression in a dose-dependent manner. Analysis of deletion mutants showed that the region affected by the preC/C proteins comprises IFN-stimulated response elements 2 and 3, upstream of the putative start codon of the MxA promoter. In addition, HBV preC/C proteins interacted directly with the MxA promoter, as shown by electrophoretic mobility shift assays. These results demonstrate a mechanism that HBV probably uses to downregulate an element of the IFN-induced host antiviral responses, which accounts for the impairment observed in HBV-infected patients.
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The T1858 variant predisposing to the precore stop mutation correlates with one of two major genotype F hepatitis B virus clades
The precore mutation G1896→A occurs frequently in anti-HBe-positive carriers of HBsAg with T1858 in the stem of the encapsidation signal. Hepatitis B virus (HBV) genotype F, considered an Amerindian genotype, subdivides into two clades and the precore mutation occurs in Central American F strains. To investigate the relationship between substitutions at position 1858 and these clades, the precore and small S genes of 48 strains of HBV genotype F were subjected to phylogenetic analyses. Isolates of one clade, formed mainly of Central American strains, all had T1858 and Thr45 in the S gene, whereas in the other clade, formed mainly of South American strains and one strain from Polynesia, all had C1858 and Leu45. The latter strain was related to strains from Venezuela and Colombia, supporting an Amerindian contribution to the Polynesian population. The position of the Polynesian strain in the phylogenetic tree indicates that the two clades have resulted from an early split, showing a high degree of genetic stability of the stem of the HBsAg encapsidation signal.
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Human papillomavirus type 16 E2- and L1-specific serological and T-cell responses in women with vulval intraepithelial neoplasia
Human papillomavirus type 16 (HPV-16)-associated vulval intraepithelial neoplasia (VIN) is frequently a chronic, multifocal high-grade condition with an appreciable risk of progression to vulval cancer. The requirement to treat women with VIN has recently stimulated the use of immunotherapy with E6/E7 oncogene vaccines. Animal models have shown that E2 may also be a useful vaccine target for HPV-associated disease; however, little is known about E2 immunity in humans. This study investigated the prevalence of HPV-16 E2-specific serological and T-cell responses in 18 women with HPV-16-associated VIN and 17 healthy volunteers. E2 responses were determined by full-length E2–GST ELISA with ELISPOT and proliferation assays using E2 C-terminal protein. As positive controls, HPV-16 L1 responses were measured using virus-like particles (VLPs) and L1–GST ELISA with ELISPOT and proliferation using VLPs as antigen. The VIN patients all showed a strong serological response to L1 compared with the healthy volunteers by VLP (15/18 vs 1/17, P<0·001) and L1–GST ELISA (18/18 vs 1/17, P<0·001). In contrast, L1-specific cellular immune responses were detected in a significant proportion of controls but were more prevalent in the VIN patients by proliferation assay (9/17 vs 17/18, P<0·02) and interferon-γ ELISPOT (9/17 vs 13/18, P=not significant). Similar and low numbers of patients and controls were seropositive for E2-specific Ig (2/18 vs 1/17). In spite of previous studies showing the immunogenicity of E2 in eliciting primary T-cell responses in vitro, there was a low prevalence of E2 responses in the VIN patients and controls (2/18 vs 0/17).
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Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells
The E6 protein of the high-risk human papillomavirus type 16 (HPV-16) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We characterized new anti-E6 monoclonal antibodies and used them for precise localization of the E6 oncoprotein within carcinoma cells. Overexpressed E6 protein was predominantly detected in the nucleus of transiently transfected HaCaT cells. While mostly localized at the periphery of condensed chromatin, E6 was also associated with nuclear ribonucleoproteic ultrastructures and with some ribosomal areas in the cytoplasm of SiHa and CaSki cells. The chimeric β-galactosidase–E6 protein expressed in transfected HeLa cells was essentially localized in the nuclear compartment. Together, these data indicate that the E6 sequence of HPV-16 may encode a nuclear localization signal. The preferential nuclear distribution of this viral oncoprotein in HPV-transformed cells correlates with its activities at the transcriptional level.
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Time trends in incidence and prevalence of human papillomavirus type 6, 11 and 16 infections in Finland
Human papillomavirus (HPV) type 16 is the major cause of cervical carcinoma, the incidence of which is decreasing in western countries. In Finland the incidence is, however, increasing in women aged <40 years, but possible underlying changes in HPV-16 epidemiology are unknown. To assess incidence trends of HPV infections, paired sera from a random sample of 8000 women with two pregnancies/sera within 5 years, taken from the serum bank of the Finnish Maternity Cohort (1983–98), were analysed for HPV-6, -11 and -16 antibodies. For 23–31-year-old women, HPV-16 incidence increased over the period 1983–97. HPV-16 seroprevalence increased from 17 % in 1983–85 to 24 % in 1995–97, but HPV-6 and HPV-11 prevalence was stable at 9–12 % throughout the study period. Epidemic spread of the oncogenic HPV-16, but not the non-oncogenic HPV-types, throughout the 1980s and 1990s preceded an increase in the incidence of cervical carcinoma in fertile-aged Finnish women.
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The simian virus 40 minor structural protein Vp3, but not Vp2, is essential for infectious virion formation
More LessThe SV40 capsid is composed of pentameric capsomeres of the major structural protein Vp1. The two minor structural proteins, Vp2 and Vp3, interact with the capsid. Here, the roles of Vp2 and Vp3 were explored during the course of SV40 infection. Start codons of Vp2, Vp3, or both Vp2 and Vp3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40ΔVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40ΔVp3 and SV40 ΔVp2/Vp3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, Vp3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length Vp3 appears to be in virus–cell interactions at post-packaging steps of a permissive infection.
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Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome
Fifteen 8-week-old conventional pigs were selected from a farm where pigs were suffering from postweaning multisystemic wasting syndrome (PMWS). Ten of the animals were diseased pigs showing typical signs of PMWS (wasting and respiratory disorders) and positive for infection with porcine circovirus type 2 (PCV2), and the other five animals selected as controls were pen-mate, apparently healthy pigs. Blood samples and lymphoid tissues were taken from each animal for haematological, serological and histopathological studies. Also, cytokine mRNA expression of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12p40 and IFN-γ from inguinal and bronchial lymph nodes, tonsils, spleen and thymus was determined by semi-quantitative RT-PCR. Pigs suffering from PMWS showed severe alterations of haematological parameters such as anaemia, lymphopenia with decrease of CD8+ and IgM+ cells, monocytosis and neutrophilia. Also, extensive lymphocyte depletion and altered cytokine mRNA expression patterns were seen in most of the examined lymphoid organs. Those cytokine mRNA alterations were characterized by an overexpression of IL-10 mRNA in thymus and IFN-γ mRNA in tonsils, and by decreases in the mRNA expression of several cytokines as IL-2 and IL-12p40 in the spleen, IL-4 in tonsils, and IFN-γ, IL-10, IL-12p40 and IL-4 in inguinal lymph nodes. Also, the IL-10 mRNA overexpression was histologically associated with the thymic depletion and atrophy observed in PMWS pigs. In conclusion, the cytokine mRNA imbalance, specially the increased mRNA levels of IL-10 in the thymus, jointly with the histopathological and haematological disorders, are highly indicative of a T-cell immunosuppression, enhancing the notion that the immune system of PMWS-affected pigs is severely impaired.
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Positive and negative effects of adeno-associated virus Rep on AAVS1-targeted integration
More LessAdeno-associated virus type 2 integrates preferentially into the AAVS1 locus on chromosome 19 of the human genome. It was reported previously that transfection with two plasmids, one for Rep and the other carrying a transgene flanked by inverted terminal repeats (ITRs), enables preferential integration of the latter into AAVS1. Aiming at increasing the frequency of AAVS1-specific integration, the Rep- to transgene-plasmid ratio necessary to achieve a higher frequency of site-specific integration was examined. 293 cells were co-transfected with the Rep78 plasmid and an ITR-flanked Neo gene at different ratios. G418-resistant clones were selected randomly. Extensive Southern blot analysis showed an optimum range of Rep78 expression. In that range, approximately 20 % of clones harboured the Neo gene at AAVS1. Excess Rep expression, however, resulted in ‘abortive’ integration of the Neo gene, a rearrangement of AAVS1 without transgene integration. Rep78 appeared to cause abortive integration more extensively than Rep68. Deleterious effects of the Rep protein on the AAVS1 locus should be considered to develop an improved AAVS1-targeted system.
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Novel structure of the genome of Rice yellow stunt virus: identification of the gene 6-encoded virion protein
More LessThe genomic region encompassing the L protein gene and a small open reading frame (ORF 6) of Rice yellow stunt virus (RYSV) has been sequenced, thus completing the nucleotide sequence of the RYSV genome. The genome organization of RYSV is unique in the rhabdoviruses because it contains two additional genes when compared to the basic gene order of the family Rhabdoviridae. Phylogenetic analysis revealed that the amino acid sequence of the RYSV L protein is most closely related to that of the L protein of Sonchus yellows net virus, another nucleorhabdovirus. However, the RYSV L protein has a unique acidic N-terminal domain distinct from that of other rhabdoviruses. Moreover, the polypeptide encoded by the ORF 6 was detected by immunoblot analysis in purified RYSV virions. Thus RYSV provides the first example in the family Rhabdoviridae that a small ORF between the G and L genes encodes a virion protein.
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Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus
ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP–MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a ‘double tag’ construct, the GST–PCP–MT cDNA was flanked by the 3′-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST–PCP and MT fragments. MT-His6 was purified on Ni–NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly588/Gly589 bond cleavage. The GST–PCP fragment purified on glutathione S–agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.
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The C-terminal region of the movement protein of Cowpea mosaic virus is involved in binding to the large but not to the small coat protein
More LessCowpea mosaic virus (CPMV) moves from cell to cell as virus particles which are translocated through a plasmodesmata-penetrating transport tubule made up of viral movement protein (MP) copies. To gain further insight into the roles of the viral MP and capsid proteins (CP) in virus movement, full-length and truncated forms of the MP were expressed in insect cells using the baculovirus expression system. Using ELISA and blot overlay assays, affinity purified MP was shown to bind specifically to intact CPMV virions and to the large CP, but not to the small CP. This binding was not observed with a C-terminal deletion mutant of the MP, although this mutant retained the capacity to bind to other MP molecules and to form tubules. These results suggest that the C-terminal 48 amino acids constitute the virion-binding domain of the MP.
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Up-regulation of cathepsin B and cathepsin L activities in scrapie-infected mouse Neuro2a cells
More LessPrion diseases are characterized by the accumulation of an abnormal, proteinase K-resistant isoform of the prion protein, PrPSc, which is generated by a post-translational conversion of the protease-sensitive normal cell-surface glycoprotein PrPc involving major conformational changes. The conversion is thought to occur at the plasma membrane or along the endocytic pathway towards the lysosome. PrPSc aggregates have been found to accumulate in secondary lysosomes. In our study, the activities of two major lysosomal cysteine proteases, cathepsins B and L, were found to be significantly increased in scrapie-infected Neuro2a cells compared with uninfected cells using biochemical and cytochemical methods. We hypothesize that lysosomal proteases may be involved in a ‘second autocatalytic loop’ of PrPSc formation, acting in concert with the well-known autocatalytic enhancement of PrP conversion in the presence of PrPSc.
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Volumes and issues
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Volume 105 (2024)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 36 (1977)
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Volume 34 (1977)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 28 (1975)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)