- Volume 84, Issue 4, 2003
Volume 84, Issue 4, 2003
- Review
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Do lipid rafts mediate virus assembly and pseudotyping?
More LessCo-infection of a host cell by two unrelated enveloped viruses can lead to the production of pseudotypes: virions containing the genome of one virus but the envelope proteins of both viruses. The selection of components during virus assembly must therefore be flexible enough to allow the incorporation of unrelated viral membrane proteins, yet specific enough to exclude the bulk of host proteins. This apparent contradiction has been termed the pseudotypic paradox. There is mounting evidence that lipid rafts play a role in the assembly pathway of non-icosahedral, enveloped viruses. Viral components are concentrated initially in localized regions of the plasma membrane via their interaction with lipid raft domains. Lateral interactions of viral structural proteins amplify the changes in local lipid composition which in turn enhance the concentration of viral proteins in the rafts. An affinity for lipid rafts may be the common feature of enveloped virus proteins that leads to the formation of pseudotypes.
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- Animal
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- RNA viruses
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Neutralizing epitopes specific for influenza B virus Yamagata group strains are in the ‘loop’
More LessTo study the neutralizing epitopes of influenza B virus Yamagata group strains, two monoclonal antibodies (mAbs) were used to select escape mutants of the virus. mAbs 5H4 and 3A12 were found to react with B/Yamagata group strains in haemagglutination inhibition and neutralization tests; no reactivity with B/Victoria group strains was observed. Most of the mutants reacted poorly to polyclonal ferret antibody against the 1998 isolate. Analysis of the deduced amino acid sequences identified a single amino acid substitution at residue 141 (Gly→Arg) or 149 (Arg→Gly) in 5H4-escape mutants and 141 (Gly→Arg), 147 (Thr→Ile) or 148 (Ser→Gly) in 3A12-escape mutants. These residues are situated in close proximity in the ‘loop’ of the haemagglutinin molecule. These epitopes have been conserved in B/Yamagata group strains for almost 10 years in Japan but amino acid substitutions in the loop have been observed in clinical isolates only since 1999.
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Oral immunization with recombinant Yersinia enterocolitica expressing a measles virus CD4 T cell epitope protects against measles virus-induced encephalitis
Immunization via the oral route with an attenuated Yersinia enterocolitica strain expressing a fragment of the measles virus nucleocapsid protein (aa 79–161) via its type III protein secretion system induced a T helper type 1 response in immunized C3H mice, which conferred protection against measles virus-induced encephalitis in a time- and dose-dependent manner.
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Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication
More LessNewcastle disease virus (NDV) was examined for its suitability as a vector for the expression and delivery of foreign genes for vaccination and gene therapy. A reporter gene encoding human secreted alkaline phosphatase (SEAP) was inserted as an additional transcription unit at four different positions in the NDV genome, between the NP and P, M and F, and HN and L genes and behind the L gene. Eight infectious recombinant NDV (rNDV) viruses, four in the non-virulent strain NDFL and four in the virulent derivative NDFLtag, were generated by reverse genetics. SEAP expression levels, replication kinetics and virus yield were examined. Replication kinetics of the rNDV viruses in primary chicken embryo fibroblasts showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was inserted between the NP and P genes. With the exception of the strain that carried the SEAP gene behind the L gene, all recombinant strains expressed high levels of SEAP, both in cell culture and in embryonated chicken eggs. In embryonated eggs, the rNDV viruses showed a 2·6- to 5·6-fold (NDFL) or 2·1- to 8·1-fold (NDFLtag) reduction in yield compared with the parent strains. These results show that foreign genes can be inserted at different positions in the NDV genome without severely affecting replication efficiency or virus yield.
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Effects of a point mutation in the 3′ end of the S genome segment of naturally occurring and engineered Bunyamwera viruses
More LessThe genome of Bunyamwera virus (BUN) consists of three segments of single-stranded RNA of negative polarity. The smallest segment, S, encodes the N protein and a nonstructural protein called NSs. We recently described a mutant virus (BUNdelNSs) that does not express NSs but overexpresses N and grows to lower titres than wild-type (wt) BUN. Here we report a BUNdelNSs variant that expresses lower levels of N protein and grows to higher titres. Sequencing of the 3′ and 5′ termini of the BUNdelNSs S RNA segment and analysis using a minireplicon system show that the N overexpressing phenotype results from a single nucleotide substitution at position 16 in the 3′ terminus. This mutation could also be detected in wtBUN populations, and was isolated by plaquing a ‘wt’ variant carrying the mutation. This variant was found to express increased N and NSs levels, and grew to lower titres than wtBUN.
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Molecular epidemiology of rabies epizootics in Colombia: evidence for human and dog rabies associated with bats
More LessThree urban rabies outbreaks have been reported in Colombia during the last two decades, one of these is occurring in the Caribbean Region (northern Colombia), while the other two occurred almost simultaneously in Arauca (eastern Colombia) and in the Central Region and ended in 1997. In order to derive phylogenetic relationships between rabies viruses isolated in these three areas, 902 nt cDNA fragments encoding the cytoplasmic domain of protein G and a fragment of protein L were obtained by RT-PCR. These amplicons contained the G–L intergenic region and were sequenced to draw phylogenetic trees. Phylogenetic analysis showed three distinct groups of viruses in the study sample. Colombian genetic variant I viruses were isolated in both Arauca and the Central Region. These viruses are apparently extinct in Colombia. Colombian genetic variant II viruses were isolated in the Caribbean Region and are still being transmitted in that area. The third group of viruses consists of viruses isolated from two insectivorous bats, three domestic dogs and a human. According to sequence analysis, the data here indicate that the isolates in this third group are bat rabies virus variants. This finding is the first that associates bats to rabies in Colombian dogs and humans, showing an unsuspected vector threatening animal and public health.
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Phylogeography of infectious haematopoietic necrosis virus in North America
Infectious hematopoietic necrosis virus (IHNV) is a rhabdoviral pathogen that infects wild and cultured salmonid fish throughout the Pacific Northwest of North America. IHNV causes severe epidemics in young fish and can cause disease or occur asymptomatically in adults. In a broad survey of 323 IHNV field isolates, sequence analysis of a 303 nucleotide variable region within the glycoprotein gene revealed a maximum nucleotide diversity of 8·6 %, indicating low genetic diversity overall for this virus. Phylogenetic analysis revealed three major virus genogroups, designated U, M and L, which varied in topography and geographical range. Intragenogroup genetic diversity measures indicated that the M genogroup had three- to fourfold more diversity than the other genogroups and suggested relatively rapid evolution of the M genogroup and stasis within the U genogroup. We speculate that factors influencing IHNV evolution may have included ocean migration ranges of their salmonid host populations and anthropogenic effects associated with fish culture.
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Amino acids 1–20 of the hepatitis C virus (HCV) core protein specifically inhibit HCV IRES-dependent translation in HepG2 cells, and inhibit both HCV IRES- and cap-dependent translation in HuH7 and CV-1 cells
More LessA self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES- and cap-dependent translation in an in vitro system. This effect was duplicated in a dose-dependent manner with a synthetic peptide representing amino acids 1–20 of the HCV core protein. This peptide was able to bind to the HCV IRES as shown by a mobility shift assay. In contrast, a peptide derived from the hepatitis B virus (HBV) core protein that contained a similar proportion of basic residues was unable to inhibit translation or bind the HCV IRES. A recombinant vaccinia–HCV core virus was used to examine the effect of the HCV core protein on HCV IRES-dependent translation in cells and this was compared with the effects of an HBV core-recombinant vaccinia virus. In CV-1 and HuH7 cells, the HCV core protein inhibited translation directed by the IRES elements of HCV, encephalomyocarditis virus and classical swine fever virus as well as cap-dependent translation, whereas in HepG2 cells, only HCV IRES-dependent translation was affected. Thus, the ability of the HCV core protein to selectively inhibit HCV IRES-dependent translation is cell-specific. N-terminal truncated (aa 1–20) HCV core protein that was expressed from a novel recombinant vaccinia virus in cells abrogated the inhibitory phenotype of the core protein in vivo, consistent with the above in vitro data.
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Sequencing of ‘untypable’ enteroviruses reveals two new types, EV-77 and EV-78, within human enterovirus type B and substitutions in the BC loop of the VP1 protein for known types
More LessThe N-terminal part of VP1 was sequenced for 43 enterovirus isolates that could not initially be neutralized with LBM pools or in-house antisera. Most isolates were found to belong to human enterovirus type A (HEV-A) and HEV-B (18 isolates of each). All HEV-A isolates could be typed by sequencing, with CV (coxsackievirus)-A16 and EV (enterovirus)-71 being dominant (nine and seven isolates, respectively). These types thus seem to have diverged more from their prototypes than the other types. Among the HEV-B isolates, E-18 dominated with five isolates that became typable after filtration. The virus type obtained by molecular typing was verified for 28 of the other patient isolates by neutralization using high-titre monovalent antisera or LBM pools. Twenty-two of the other 30 ‘untypable’ isolates had substitutions in the VP1 protein within or close to the BC loop. Two closely related HEV-B isolates diverged by 19·4 % from E-15, the most similar prototype. Two non-neutralizable HEV-C isolates split off from the CV-A13/CV-A18 branch, from which they diverged by 15·7–18·2 %. Three of the six non-neutralizable isolates, W553-130/99, W543-122/99 and W137-126/99, diverged by >24·2 % from the most similar prototype in the compared region. The complete VP1 was therefore sequenced and found to diverge by >29 % from all prototypes and by >28 % from each other. Strains similar to W553-130/99 that have been identified in the USA are tentatively designated EV-74. The two other isolates fulfil the molecular criterion for being new types. Since strains designated EV-75 and EV-76 have been identified in the USA, we have proposed the tentative designations EV-77 and EV-78 for these two new members of HEV-B.
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Molecular characterization of M1146, an American isolate of Ljungan virus (LV) reveals the presence of a new LV genotype
Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles in Sweden. This study describes the genetic characterization of a virus, M1146, which was isolated in 1962 from another vole species (Microtus montanus), trapped in Oregon, USA. Based on antigenic properties, M1146 was postulated previously as a putative member of the family Picornaviridae. The near complete genomic sequence verifies that M1146 is a member of the Picornaviridae, most closely related to LVs isolated in Sweden. The strain M1146 possesses typical LV genomic organization, including a cluster of two 2A homologues. There are significant differences throughout the capsid protein region, while the non-structural region of M1146 is closely related to the Swedish LV genomes. Genetic and phylogenetic analyses show that M1146 represents a new genotype within the distinct LV cluster. Isolation of LV from both Swedish and American voles trapped over a period of 30 years suggests a continuous worldwide presence.
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Galactose is needed only for expression of co-receptors used by Theiler's murine encephalomyelitis virus as the virus does not directly bind galactose or use the UDP-galactose transporter as a receptor
More LessTheiler's murine encephalomyelitis virus (TMEV) infects most mammalian cells, but a TMEV receptor has not been identified. Studies have demonstrated that the UDP-galactose transporter (UGT) is critical for TMEV attachment and entry into mammalian cells ( Hertzler et al., Virology 286, 336–344, 2001 ). It was suggested that UGT might function as a TMEV receptor. We have demonstrated that polyclonal rabbit antibodies to human UGT that cross-react with hamster UGT do not block binding to or infection of mammalian cells by either high- or low-neurovirulence TMEV. In addition, incubation of virus with galactose, or blocking galactose on the cell surface with lectins, does not inhibit TMEV binding or infection. Thus, TMEV needs UGT for its transporter activity and galactose for assembly of its co-receptors (attachment factors) but does not bind directly to galactose. Excluding direct involvement of UGT and galactose in TMEV binding and entry provides further insight into how TMEV interacts with the host cell and should facilitate ongoing studies to identify a TMEV receptor.
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Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus
Japanese patients with sporadic acute hepatitis E are infected with polyphyletic strains of hepatitis E virus (HEV). Hepatitis E is considered a zoonotic disease. Thus far in Japan, only three strains of swine HEV have been identified and an antibody study for HEV antibodies has not been done on Japanese pigs. To determine the prevalence of swine HEV infection in Japan and the extent of genetic variation among Japanese swine HEV strains, we tested serum samples obtained from 2500 pigs from 2 to 6 months of age at 25 commercial swine farms in Japan for the presence of IgG antibodies to HEV and swine HEV RNA. Anti-HEV antibodies were detected in 1448 pigs (58 %). One-hundred-and-thirteen (15 %) of the 750 3-month-old pigs and 24 (13 %) of the 180 4-month-old pigs were positive for swine HEV RNA. The nucleotide sequence of a 412 bp region within open reading frame 2 of the 137 swine HEV isolates was determined. Sequence analyses revealed that the 137 isolates shared 76·6–100 % nucleotide sequence identities and were classifiable into genotype III (93 %) or IV (7 %) and that the isolates from the same farm were ⩾97·1 % similar to each other. Phylogenetic analysis showed that the Japanese swine and human HEV isolates segregated into four clusters, with the highest nucleotide identity being 94·4–100 % between swine and human isolates in each cluster. These results indicate that swine HEV is widespread in the Japanese swine population and further support the hypothesis that swine serve as reservoirs for HEV infection.
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Identification and analysis of gp116 and gp64 structural glycoproteins of yellow head nidovirus of Penaeus monodon shrimp
Yellow head virus (YHV) is a major agent of disease in farmed penaeid shrimp. YHV virions purified from infected shrimp contain three major structural proteins of molecular mass 116 kDa (gp116), 64 kDa (gp64) and 20 kDa (p20). Two different staining methods indicated that the gp116 and gp64 proteins are glycosylated. Here we report the complete nucleotide sequence of ORF3, which encodes a polypeptide of 1666 amino acids with a calculated molecular mass of 185 713 Da (pI=6·68). Hydropathy analysis of the deduced ORF3 protein sequence identified six potential transmembrane helices and three ectodomains containing multiple sites for potential N-linked and O-linked glycosylation. N-terminal sequence analysis of mature gp116 and gp64 proteins indicated that each was derived from ORF3 by proteolytic cleavage of the polyprotein between residues Ala228 and Thr229, and Ala1127 and Leu1128, located at the C-terminal side of transmembrane helices 3 and 5, respectively. Comparison with the deduced ORF3 protein sequence of Australian gill-associated virus (GAV) indicated 83 % amino acid identity in gp64 and 71 % identity in gp116, which featured two significant sequence deletions near the N terminus. Database searches revealed no significant homology with other proteins. Recombinant gp64 expressed in E. coli with and without the C-terminal transmembrane region was shown to react with antibody raised against native gp64 purified from virions.
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A cytoplasmic region of the NSP4 enterotoxin of rotavirus is involved in retention in the endoplasmic reticulum
More LessThe rotavirus genome encodes two glycoproteins, one structural (VP7) and one non-structural (NSP4), both of which mature and remain in the endoplasmic reticulum (ER). While three amino acids in the N terminus have been proposed to function as a retention signal for VP7, no information is yet available on how NSP4 remains associated with the ER. In this study, we have investigated the ER retention motif of NSP4 by producing various C-terminal truncations. Deleting the C terminus by 52 amino acids did not change the intracellular distribution of NSP4, but an additional deletion of 38 amino acids diminished the ER retention and resulted in the expression of NSP4 on the cell surface. Brefeldin A treatment prevented NSP4 from reaching the cell surface, suggesting that C-terminal truncated plasma membrane NSP4 is transported through the normal secretory pathway. On the basis of these results, we propose that the region between amino acids 85 and 123 in the cytoplasmic region of NSP4 are involved in ER retention.
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Network analysis of human and simian immunodeficiency virus sequence sets reveals massive recombination resulting in shorter pathways
More LessThe intrinsic recombination rate of human immunodeficiency virus (HIV) exceeds the point mutation rate by a factor of 10. As the majority of infected cells in vivo harbour multiple proviruses, the stage is set for rampant recombination. Therefore, it may be presumed that phylogenic relationships and mutation frequencies will probably be affected by recombination. However, the proportion of homoplasies arising from recombination and mutation is not known. By studying the evolution of the hypervariable regions of the simian immunodeficiency virus envelope gene among four macaques, it is shown that homoplasies arise more from recombination than from point mutation. When recombination is accounted for, the minimum number of substitutions in a sequence set may be reduced by as much as 45 %. In fact, the true number of point mutations in a set of HIV sequences tends to the number of discrete substitutions. Hence, lineages are younger than anticipated previously, although not in proportion to the ratio of the intrinsic recombination/point mutation rate. Recombination also inflates codon polymorphisms.
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p53 facilitates degradation of human T-cell leukaemia virus type I Tax-binding protein through a proteasome-dependent pathway
Human T-cell leukaemia virus type 1 (HTLV-I), the aetiological agent of adult T-cell leukaemia (ATL) and tropical spastic paraparesis (TSP/HAM), transforms human T-cells in vivo and in vitro. The Tax protein of HTLV-I is essential for cellular transformation as well as viral and cellular gene transactivation. The interaction of Tax with cellular proteins is critical for these functions. We previously isolated and characterized a novel Tax-binding protein, TRX (TAX1BP2), by screening a Jurkat T-cell cDNA library. In the present study, we present evidence that the tumour suppressor p53 targets the TRX protein for proteasome degradation. Pulse–chase experiments revealed that p53 enhanced the degradation of TRX protein and reduced the half-life from 2·0 to 0·25 h. p53 mutants R248W and R273H enhance TRX degradation suggesting a transcriptionally independent mechanism. Both HTLV-I Tax and the proteasome-specific inhibitor MG132 inhibited p53-mediated TRX protein degradation. These results suggest that TRX degradation is mediated through activation of the proteasome protein degradation pathway independent of transcriptional function of p53. Our results provide the first experimental evidence that Tax inhibits transciption-dependent and independent functions of p53.
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A glucocorticoid response element in the LTR U3 region of Friend murine leukaemia virus variant FIS-2 enhances virus production in vitro and is a major determinant for sex differences in susceptibility to FIS-2 infection in vivo
More LessThe nucleotide sequence of the Friend murine leukaemia virus variant FIS-2 LTR has high identity with the closely related Friend murine leukaemia virus (F-MuLV) LTR, except for the deletion of one direct repeat, a few point mutations and the generation of a glucocorticoid response element (GRE) in the U3 region. The GRE can mediate gene induction by glucocorticoids, mineral corticoids, progesterone and androgens, and it has been shown that incorporation of a GRE(s) within the LTR can increase the transcriptional activity of retroviral enhancers. We have previously reported an increased early virus replication in male mice compared with female mice when infected with a virus containing the FIS-2 LTR and have proposed that the GRE might contribute to this sex difference. In the present study, we introduced a single point mutation in the GRE and performed comparative studies in NIH 3T3 cells and in young adult male and female NMRI mice. We found that significantly more virus was produced from NIH 3T3 cells infected with wt FIS-2 than from cells infected with the FIS-2 GRE mutant and that this difference was further augmented by glucocorticoids. The glucocorticoid antagonist RU486 inhibited virus production in a dose-dependent manner. The wt FIS-2 disseminated significantly faster than the FIS-2 GRE mutant in both male and female mice. There was no significant difference in the dissemination rate between male and female mice infected with the FIS-2 GRE mutant. Hence, the GRE in the FIS-2 LTR is one determinant of the significant sex difference in susceptibility to FIS-2 infection.
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- DNA viruses
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Divergence of reiterated sequences in a series of genital isolates of herpes simplex virus type 1 from individual patients
More LessBoth serotypes of herpes simplex virus (HSV), HSV-1 and HSV-2, are aetiological agents of genital herpes, although genital herpes caused by HSV-1 recurs less frequently. The HSV-1 genome contains a number of short, tandemly repeated sequences, and some reiterated sequences can serve as sensitive markers for the differentiation of HSV-1 strains. In the present study, variation in reiterations (assumed to be due to different copy numbers of tandemly repeated sequences) was examined in HSV-1 isolates from genital lesions from the same individual. Six sets (three primary-recurrence sets and three multiple-recurrence sets) of HSV-1 isolates were analysed: the primary-recurrence set consisted of two isolates (one isolated at a primary episode and the other at a recurrent episode) from the same individual; the multiple-recurrence set consisted of plural isolates from different episodes of recurrence in the same individual. Variations in length of the major DNA fragment, containing reiteration I (within the a sequence) and/or reiteration IV (within introns of genes US1 and US12), were detected between isolates of each multiple-recurrence set, but not of the primary-recurrence set. Thus, HSV-1 isolates of multiple-recurrence sets are assumed to have diverged more widely within each set than those of primary-recurrence sets, probably because of more rounds of virus DNA replication. This divergence of reiterations seems to indicate a forward step in the division of HSV-1 from a common ancestor into different lineages.
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RNase L activity does not contribute to host RNA degradation induced by herpes simplex virus infection
More LessIn early herpes simplex virus (HSV) infection, the virion host shutoff (vhs) protein mediates the degradation of mRNA and subsequent shutoff of host protein synthesis. It is unclear whether vhs acts alone or in concert with virus-induced cellular factors for this activity. This paper examines whether RNase L, a virally induced endoribonuclease, contributes to HSV-induced mRNA decay. Results showed that RNA degradation was comparable in wild-type and RNase L−/− cells, demonstrating that HSV-mediated RNA degradation is independent of RNase L activity. Furthermore, the data show that HSV-1 does not significantly induce RNase L activity in murine embryo fibroblasts.
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Stimulation of bovine herpesvirus-1 productive infection by the adenovirus E1A gene and a cell cycle regulatory gene, E2F-4
More LessIdentifying cellular genes that promote bovine herpesvirus-1 (BHV-1) productive infection is important, as BHV-1 is a significant bovine pathogen. Previous studies demonstrated that BHV-1 DNA is not very infectious unless cotransfected with a plasmid expressing bICP0, a viral protein that stimulates expression of all classes of viral promoters. Based on these and other studies, we hypothesize that the ability of bICP0 to interact with and modify the function of cellular proteins stimulates virus transcription. If this prediction is correct, cellular proteins that activate virus transcription could, in part, substitute for bICP0 functions. The adenovirus E1A gene and bICP0 encode proteins that are potent activators of viral gene expression, they do not specifically bind DNA and both proteins interact with chromatin-remodelling enzymes. Because of these functional similarities, E1A was tested initially to see if it could stimulate BHV-1 productive infection. E1A consistently stimulates BHV-1 productive infection, but not as efficiently as bICP0. The ability of E1A to bind Rb family members plays a role in stimulating productive infection, suggesting that E2F family members activate productive infection. E2F-4, but not E2F-1, E2F-2 or E2F-5, activates productive infection with similar efficiency as E1A. Next, E2F family members were examined for their ability to activate the BHV-1 immediate-early (IE) transcription unit 1 (IEtu1) promoter, as it regulates IE expression of bICP0 and bICP4. E2F-1 and E2F-2 strongly activate the IEtu1 promoter, but not a BHV-1 IEtu2 promoter or a herpes simplex virus type 1 ICP0 promoter construct. These studies suggest that E2F family members can stimulate BHV-1 productive infection.
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Antibody-induced internalization of viral glycoproteins and gE–gI Fc receptor activity protect pseudorabies virus-infected monocytes from efficient complement-mediated lysis
More LessPseudorabies virus (PRV)-infected blood monocytes are able to transport virus throughout the body of vaccination-immune pigs. PRV-infected monocytes express viral glycoproteins in their plasma membrane that can be recognized by virus-specific antibodies. Recently, it has been shown that addition of PRV-specific polyclonal immunoglobulins to PRV-infected monocytes at 37 °C induces internalization of the majority of plasma membrane-expressed viral glycoproteins. This study investigated whether this process may interfere with efficient antibody-dependent complement-mediated lysis (ADCML) of infected monocytes. Therefore, an ADCML assay was set up in vitro. A significant decrease in the percentage of cells lysed by ADCML was observed when antibody-induced internalization of PRV glycoproteins occurred (P<0·005). Furthermore, it is shown (i) that the PRV gE–gI complex, which, like certain other alphaherpesvirus orthologues, possesses IgG-binding capacity, aids in avoiding efficient ADCML of PRV-infected monocytes and (ii) that the efficiency of PRV gE–gI-mediated evasion of ADCML can be decreased by the presence of gE–gI-specific antibodies.
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Heterogeneous Epstein–Barr virus latent gene expression in AIDS-associated lymphomas and in type I Burkitt's lymphoma cell lines
More LessEpstein–Barr virus (EBV) is associated with lymphoma in immunocompromised patients. This study provides evidence that the expression of EBV nuclear antigen-3 genes can be directed from the F promoter in different type I Burkitt's lymphoma cell lines and in some lymphomas from human immunodeficiency virus-infected patients. This expression occurs predominantly after induction of the EBV lytic cycle.
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The BZLF1 promoter of Epstein–Barr virus is controlled by E box-/HI-motif-binding factors during virus latency
More LessThe BZLF1 open reading frame of Epstein–Barr virus (EBV) encodes an important transactivator of replication. During latency, transcription of this gene is switched off. HI motifs have been shown to cause negative regulation of the promoter. Using yeast one-hybrid assays, we isolated the E box-binding protein, E2-2, interacting with these motifs. Electrophoretic mobility shift assays demonstrated that E2-2 binds to HIα, HIβ and HIγ, which contain E box consensus binding sites. Deletion of the HI-associated E boxes and overexpression of E2-2 in transfection assays revealed that these elements act as repressors in lymphoid cells. In contrast, in epithelial cells they contribute to the increased responsiveness of the promoter to transactivation by the BZLF1 protein. The data presented are in accord with an alternative and exclusive binding of different cell type- and differentiation-specific factors, such as E2-2, to the HI-associated E boxes in lymphoid and epithelial cells. This implies a role in cell type-specific virus replication.
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A novel function for the Epstein–Barr virus transcription factor EB1/Zta: induction of transcription of the hIL-10 gene
More LessInterleukin-10 (IL-10) plays a critical role in Epstein–Barr virus (EBV) biology. Indeed, the EBV genome contains a gene (BCRF1) with homology to the human IL-10 (hIL-10) gene. In addition to viral IL-10, which is secreted late in the productive cycle, hIL-10 production is also induced in B cells infected by EBV. The EBV protein LMP-1 and the viral small non-polyadenylated RNAs (EBERs) expressed during latency are involved in hIL-10 induction. In this study, we show that in B cells the viral transcription factor EB1, which is the main inducer of the EBV productive cycle, also activates transcription of the hIL-10 gene and secretion of the hIL-10 protein. Accordingly, EB1 bound directly to specific DNA sequences in the hIL-10 minimal promoter. Moreover, specific disruption of EB1 binding to some of these sites impaired EB1-mediated activation of transcription at the hIL-10 promoter in a transient expression assay. Therefore, an increase in IL-10 production occurs during latency and early and late during the productive cycle. This production of IL-10 might favour the survival of EBV-infected cells in vivo and/or create a microenvironment required for efficient de novo infection of B lymphocytes by EBV virions.
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- Plant
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Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences
More LessRNA interference (RNAi) is a double-stranded (ds)RNA-inducible, sequence-specific RNA-degradation mechanism that operates as a natural antiviral system in plants and animals. Successful virus infection requires evasion or suppression of RNAi. Indeed, RNAi suppressor proteins have been identified in plant and animal viruses, although the molecular mechanism of silencing inhibition is still poorly understood. Because many RNA viruses encode dsRNA-binding proteins (dsRBPs) and as RNAi is triggered by the accumulation of dsRNAs, dsRBPs were examined to see if they inhibit RNAi. Here, it is shown that heterologous dsRBPs suppressed RNAi in plants, indicating that in natural host–virus interactions, pathogen-encoded dsRBPs could inactivate RNAi-mediated host defences.
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Assembly of single-shelled cores and double-shelled virus-like particles after baculovirus expression of major structural proteins P3, P7 and P8 of Rice dwarf virus
More LessExpression of the core capsid protein P3 of Rice dwarf virus in a baculovirus system resulted in the formation of single-shelled core-like particles in insect cells in the absence of any other capsid proteins. Double-shelled virus-like particles were also observed upon mixing or co-expression of P3 and the major outer capsid protein P8, suggesting that P3 and P8 have the ability to form double-shelled particles both in vivo and in vitro. Core protein P7 expressed in a similar manner was incorporated into the virus-like particles.
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Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata
The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP–TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP–TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.
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The optimal temperature for RNA replication in cells infected by Soil-borne wheat mosaic virus is 17 °C
More LessSystemic infection of wheat plants with Soil-borne wheat mosaic virus (SBWMV) requires temperatures below 20 °C. Here we examine the cause of the temperature sensitivity by inoculating infectious in vitro transcripts of SBWMV RNA1 and RNA2 to barley mesophyll protoplasts. After RNA inoculation, protoplasts were incubated at temperatures between 15 and 25 °C for up to 48 h. Western blot analysis showed that the capsid protein accumulated most abundantly at 17 °C but was not detectable at 25 °C. Northern blot analysis showed that the wild-type RNA1 and RNA2 and their subgenomic RNAs accumulated most abundantly at 17 °C but were barely detectable at 25 °C. An RNA1 mutant in which the p152 and p211 replicase genes were placed between the 5′- and 3′-untranslated regions also replicated most efficiently at 17 °C but not at 25 °C. Thus, the requirement for temperatures lower than 20 °C for SBWMV infection is primarily determined by replication of RNA1, which encodes the viral RNA replicase.
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Potato mop-top virus: the coat protein-encoding RNA and the gene for cysteine-rich protein are dispensable for systemic virus movement in Nicotiana benthamiana
Full-length genomic cDNA clones of the Swedish isolate of Potato mop-top virus (PMTV) were transcribed in vitro using T7 RNA polymerase. The combination of RNA 1, 2 and 3 synthesized in the presence of m7GpppG cap analogue was infectious when inoculated onto Nicotiana benthamiana plants. Also, the combination of RNA 1 (encodes the viral replicase) with RNA 3 [encodes the triple gene block proteins and a small cysteine-rich protein (CRP)] was infectious and both RNAs moved systemically in N. benthamiana plants in the absence of RNA 2, which encodes the coat protein (CP). However, the yellow mosaic symptoms that typically developed following PMTV infection with all three RNAs were not observed in plants infected with RNA 1+RNA 3. Site-directed mutagenesis experiments revealed that expression of the putative CRP was not required for systemic infection and symptom induction in N. benthamiana. These data show that PMTV represents an example of a multipartite virus capable of establishing systemic infection without the CP-encoding RNA, and also without the putative CRP.
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Nucleotide sequence and genome organization of Cucumber yellows virus, a member of the genus Crinivirus
More LessThe genome of Cucumber yellows virus (CuYV), isolated in Japan from cucumber (Cucumis sativus L.), was completely sequenced and shown to be bipartite. CuYV RNA1 consisted of 7889 nucleotides and encompassed seven open reading frames (ORFs), which is typical of the Closteroviridae, including a heat-shock protein 70 homologue, a coat protein and a diverged coat protein (CPd). CuYV RNA2 consisted of 7607 nucleotides and included two ORFs: ORF1a potentially encoded a polyprotein containing putative papain-like protease, methyltransferase and helicase domains, and ORF 1b potentially encoded an RNA-dependent RNA polymerase, which is probably expressed via a +1 ribosomal frameshift. The size and organization of the CuYV genome are similar to those of Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, indicating that CuYV is a member of that genus, although CuYV differed in several points from LIYV.
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In vitro cell-free conversion of bacterial recombinant PrP to PrPres as a model for conversion
More LessPrion diseases are associated with the conversion of the normal cellular prion protein, PrPC, to the abnormal disease-associated protein, PrPSc. This conversion can be mimicked in vitro using PrPSc isolated from the brains of scrapie-infected animals to induce conversion of recombinant PrPC into a proteinase K-resistant isoform, PrPres. Traditionally, the ‘cell-free’ conversion assay has used, as substrate, recombinant PrPC purified from mammalian tissue culture cells or, more recently, from baculovirus-infected insect cells. The cell-free conversion assay has been modified by replacing the tissue culture-derived PrPC with recombinant PrP purified from bacteria. Bacterial expression and chromatographic purification give high yields of recombinant radiolabelled untagged protein, eliminates artefacts that may be due to cellular factors or antibody fragments normally present in labelled PrP preparations and allows accurate and rapid variation of protein sequence using standard molecular biological techniques. In addition, these cell-free conversion assays were carried out under more physiological conditions, giving more relevance to the assay as a model for conversion. To validate its use in this assay, this bacterial recombinant PrP has been shown to have the conversion properties of mammalian PrPC: (i) it converts to a proteinase K-resistant isoform in the presence of PrPSc; (ii) the efficiency of this conversion by PrPSc of different strains and species parallels that found in vivo; and (iii) its cell-free conversion is inhibited by Congo Red analogues in a structure-dependent manner similar to that seen in in vivo and in vitro cell assays.
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Studies of the transmissibility of the agent of bovine spongiform encephalopathy to pigs
Studies to test the transmissibility of the bovine spongiform encephalopathy (BSE) agent to pigs began in 1989. Parenteral inoculation of the agent by three routes simultaneously (intracranially, intravenously and intraperitoneally) produced disease with an incubation period range of 69–150 weeks. Pre-clinical pathological changes were detected in two pigs killed electively at 105 and 106 weeks post-inoculation. Infectivity was detected by bioassay in inbred mice in the CNS of those pigs that developed spongiform encephalopathy. Infectivity was also found in the stomach, jejunum, distal ileum and pancreas of terminally affected pigs. These findings show that pigs are susceptible to BSE. In contrast, disease failed to occur in pigs retained for 7 years after exposure by feeding BSE-affected brain on three separate days, at 1–2 week intervals. The amounts fed each day were equivalent to the maximum daily intake of meat and bone meal in rations for pigs aged 8 weeks. No infectivity was found in tissues assayed from the pigs exposed orally. This included tissues of the alimentary tract. It is suggested that these pigs did not become infected. The relatively high oral exposure used in these experiments compared with feed-borne exposure in the field may explain the absence of an epidemic of spongiform encephalopathy in domestic pigs concurrent with the BSE epidemic in the UK.
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Cell-associated variants of disease-specific prion protein immunolabelling are found in different sources of sheep transmissible spongiform encephalopathy
More LessScrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSEs) or prion diseases affecting domestic and exotic ruminants. In previous immunohistochemical studies, we have shown that different sheep TSE sources may be distinguished by both the proportion of disease-specific prion protein (PrPd) accumulation relative to different cell types in the brain (the ‘PrPd profile’) and by different labelling patterns for PrP peptide sequences within phagocytic cells. In the present study, we have further characterized the intracellular accumulation patterns of PrPd in the lymphoreticular system (LRS) and in the brain of sheep clinically affected with scrapie or BSE. BSE-infected PrPARQ/ARQ sheep of different breeds were compared with scrapie-infected sheep of different PrP genotypes. Cases of BSE infection could be distinguished from scrapie cases by a marked reduction in labelling of PrPd containing the 84–105 amino acid residues in phagocytic cells of the LRS and in neurones and glia of the brain. These results therefore indicate that TSE agent-dependent processing of PrP in specific cell types within the brain and LRS can be used to distinguish between BSE in PrPARQ/ARQ sheep and scrapie in sheep of several PrP genotypes. Three different N-terminal peptide antibody labelling patterns were recognized for different cell types in different tissues of BSE-infected sheep, suggesting that different truncated forms of PrPd are formed following infections with this agent strain. These variations in the cleavage sites of BSE PrPd may be due to cell-specific variation in endosomal–lysosomal digestion or to cell- and tissue-specific differences in BSE PrPd conformation.
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Molecular analysis of iatrogenic scrapie in Italy
An accidental intra- and interspecies transmission of scrapie occurred in Italy in 1997 and 1998 following exposure to a vaccine against Mycoplasma agalactiae. PrPSc in affected sheep and goats, collected from a single flock exposed to vaccination 2 years earlier, was molecularly typed. In five animals with iatrogenic scrapie, a PrPSc type with a 20 kDa core fragment was found in all areas of the brain investigated. In three sheep and one goat, this isoform co-occurred with a fully glycosylated isoform that had a protease-resistant backbone of 17 kDa, whereas in two sheep and four goats, the two PrPSc types were detected in different regions of the brain. In sheep with natural field scrapie, a PrPSc type with physico-chemical properties indistinguishable from the 20 kDa isoform was found. The present results suggest the co-presence of two prion strains in mammary gland and brain homogenates used for vaccination.
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