- Volume 75, Issue 10, 1994
Volume 75, Issue 10, 1994
- Review Article
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- Animal
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A molecular epidemiological study of rabies virus in central Ontario and western Quebec
More LessRabies persists in Ontario wildlife in two predominant species: the red fox (Vulpes vulpes) and the striped skunk (Mephitis mephitis). A protocol applying reverse transcription/polymerase chain reaction (RT/PCR) and restriction endonuclease analysis (REA) to the rabies virus nucleoprotein gene was previously reported by Nadin-Davis et al. (Journal of General Virology 74, 829–837, 1993) to be useful for discrimination of rabies virus variants in Ontario. Four main types, which showed no host species specificity but which did exhibit different geographical distributions, were identified.
Between 1989 and 1992 an area north and west of the city of North Bay experienced unusual and substantial rabies activity. In this report we describe the use of these molecular techniques to investigate the epidemiology of this recent rabies outbreak in central Ontario. It is shown that two of the four previously identified variants had invaded this region from the south and east, but in addition viruses very closely related to arctic isolates of rabies virus were found. The nucleoprotein and glycoprotein genes of this arctic type were sequenced and compared to those of its more southerly neighbours.
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Characterization of the IgA and subclass IgG responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus
In this study, we have investigated the characteristics of secreted IgA and other classes of Ig induced after vaccination of sows with transmissible gastroenteritis virus (TGEV) or the antigenically related porcine respiratory coronavirus (PRCV). Both viruses induced the secretion of neutralizing antibodies of different classes in the sows’ milk, but these protected suckling piglets against TGEV to different degrees. Quantitative differences in the induction of IgA by both viruses were found among the different viral antigenic sites and subsites of glycoprotein S. In TGEV-vaccinated sows, antigenic subsite A was the best inducer of IgA, followed by antigenic site D. After vaccination with PRCV, lower levels of IgA were detected on colostrum and milk, antigenic site D and subsite Ab being the immunodominant sites. This quantitative difference in epitope recognition could explain the differences in newborn piglet protection found using Ig classes purified from the milk of sows immunized with both viruses. Apparently only IgA recognizing at least antigenic sites A and D confers good protection in vivo, whereas any Ig class recognizing only one antigenic site may neutralize the virus in cell culture. These results indicate that the formulation of a subunit vaccine against TGEV has to consider the inclusion of more than one antigenic site involved in virus neutralization.
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Involvement of the vacuolar H+-ATPase in animal virus entry
More LessSemliki Forest virus (SFV) enters cells by receptor- mediated endocytosis, followed by acidification of endosomes by the action of the vacuolar H+-ATPase. Fusion of the viral and the endosomal membrane delivers the viral genome to the cytoplasm. Direct blockade of the vacuolar H+-ATPase by the selective inhibitor bafilomycin A1 (BFLA1) prevented the infection of cells by SFV, if the compound was present during the first minutes of infection. Attachment and penetration of virus particles were not the targets of the antibiotic. BFLA1 and the ionophore monensin potently blocked SFV infection even at low pH, indicating that acidic pH is not sufficient for SFV to deliver its genome to the cytoplasm, but the proper functioning of the H+- ATPase pump is necessary. Other enveloped RNA- containing viruses, such as vesicular stomatitis virus or influenza virus were also blocked by BFLA1, whereas no effect was observed with Sendai virus, which enters into cells by direct fusion with the plasma membrane. Enveloped DNA-containing viruses, such as herpesviruses and vaccinia virus, infected the cells even when the vacuolar H+-ATPase was inhibited by BFLA1; similar behaviour was observed with poliovirus and adenovirus. Animal virus particles promote the internalization of proteins and other macromolecules during entry. BFLA1 blocked co-entry of the toxin α-sarcin when induced by SFV, but not when induced by Sendai virus. The inhibition of the enzyme responsible for acidification of endosomes by means of the potent inhibitor BFLA1 constitutes a selective and powerful tool to analyse the low-pH dependent mechanism(s) during virus entry and will aid in understanding the mechanisms and routes of entry of animal viruses into cells.
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Particle assembly and Vpr expression in human immunodeficiency virus type 1-infected cells demonstrated by immunoelectron microscopy
More LessThe 96 amino acid viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) was detected during virus assembly in intracellular vacuoles and at the plasma membrane on peripheral blood mononuclear cells. In both immature and mature virus particles, Vpr was located immediately beneath the viral envelope, colocalizing with the core structural protein, Gag p24. Vpr was present in intracellular HIV-1 wild-type virions at 50% of the level found in extracellular HIV-1 particles. Cells infected with HIV-1 strains with C-terminal truncations of Vpr manifested a different pattern of Vpr expression. A mutant with an alteration of amino acids 79 to 85 exhibited a 23 % reduction in total levels of Vpr expression, but a marked accumulation of Vpr in intracellular rather than extracellular virions. A mutant with the last 17 amino acids of Vpr deleted expressed only 10% of wild-type levels of Vpr. These observations indicate that Vpr is incorporated into virions from the cytoplasmic aspect of either the vacuolar or plasma membrane. Furthermore, the proportion of Vpr on intracellular compared to extracellular virions is affected by a specific locus within the protein.
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A rodent cell line permissive for entry and reverse transcription of human immunodeficiency virus type 1 has a pre-integration block to productive infection
More LessReplication of human immunodeficiency virus type 1 (HIV-1) is restricted to CD4-expressing primate cells. This tropism may be due partly to the absence from nonprimate cells of a species-specific factor which has an accessory role to CD4 during virus penetration. In this study we describe a rat B lymphocyte cell line in which there is efficient CD4-dependent entry of HIV-1.
However, this cell line has a block to productive infection of HIV-1 at a stage between reverse transcription and integration. Our results demonstrate that the putative accessory factor for HIV-1 penetration is not restricted to primate cells and that there is a novel, uncharacterized cell-virus interaction at a stage between penetration and integration.
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Altered expression of a novel cellular gene as a consequence of integration of human T cell lymphotropic virus type 1
By analysing a genomic DNA clone derived from the human T cell lymphotropic virus type 1 (HTLV-1)- infected cell line, TL-Su, we found that an integrated HTLV-1 provirus interrupted the poly(A) signal-containing exon of a novel gene, RY-1. Nucleotide sequence analysis of a cDNA derived from Jurkat cells revealed that the normal RY-1 mRNA could encode a novel protein that has an unique primary structure, suggesting that a nucleic acid binding property was involved. Proviral integration led to an accumulation of aberrant RY-1 mRNA species in the cells. All the aberrant RY-1 cDNAs derived from TL-Su cells terminated at the poly(A) site of the R region of the HTLV-1 long terminal repeat and initiated in the intron, approx. 800 bp upstream from the putative second exon. Furthermore, another intron, downstream from this position, remained unspliced in some of the cDNAs. In addition to the activation by the integrated viral elements of cryptic promoters located upstream, mechanisms involving altered rates of degradation or transport from the nucleus to the cytoplasm of intron-containing RNA were suggested.
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Reactivity of primate sera to foamy virus Gag and Bet proteins
In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent Mr and the cytoplasmic 60K Mr Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52 %) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.
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Cruciform structure of a DNA motif of parvovirus minute virus of mice (prototype strain) involved in the attenuation of gene expression
More LessIt has previously been reported that the region between nucleotides 259 and 383 immediately downstream from the P4 early promoter of parvovirus minute virus of mice, prototype strain (MVMp) is responsible for transcriptional attenuation. Attenuation results from the premature pausing of RNA polymerase II within this sequence (designated to as att) and seems to depend on potential RNA secondary structure. To assess the attenuation capacity of att under near physiological conditions, the early transcription unit of MVMp was replaced by the chloramphenicol acetyltransferase reporter gene under control of the early P4 promoter, in the presence or absence of att. The resulting recombinant vectors were encapsidated in parvovirus particles and replicated in cells after co-infection with the wild-type virus. The att fragment reduced the rate of expression of the reporter gene by approximately threefold, confirming previously reported data from transfection experiments performed in the same cellular system. This attenuation factor is unexpectedly high, considering that the ‘readthrough’ fold of the nascent viral transcript is thermodynamically more stable than the ‘attenuation’ configuration. In an attempt to elucidate this point, we sought for the presence of secondary structures in the template DNA molecule. In vitro nuclease probing of viral dsDNA revealed that the att fragment had a cruciform configuration with both complementary strands folding into the computer- predicted stem-loop ‘attenuation’ structure. These observations lead us to propose that the secondary structure of the DNA template may prompt the formation of the ‘attenuation’ stem-loop in nascent mRNAs by bringing corresponding self-complementary sequences into close proximity.
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Adeno-associated virus type 2 interferes with early development of mouse embryos
More LessThe human helper-dependent adeno-associated virus type 2 (AAV-2) has been shown to induce differentiation in various cell types in culture including pluripotent embryonic cells, in the absence of helper virus. To assess whether induction of differentiation may influence developmental processes we analysed the effect of AAV- 2 on developing mouse embryos. In vitro infection of fertilized eggs induced arrest of development at the twocell stage. Moreover, micro injection of AAV-2 DNA (comprising either the complete AAV-2 genome or a fragment containing the P5 promoter region) into onecell embryos, blocked development at the morula stage. In vivo, AAV-2 infection of pregnant mice led to fetal death and early abortion. These results demonstrate that the human adeno-associated virus, which is thought to be non-pathogenic, can perturb embryonic development in mice. This may provide a suitable animal model system to further elucidate the biological significance of the recent detection of adeno-associated virus DNA in human abortion material.
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Mechanism of translation of the bicistronic mRNA encoding human papillomavirus type 16 E6–E7 genes
More LessThe transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are expressed from a bicistronic mRNA with a characteristic spacing of 3 to 6 bp between the termination codon of E6 and the initiation codon of E7. Plasmid pSP64E6E7 which contains the reading frames of both E6 and E7 was constructed in order to study the expression of both proteins in a coupled transcription/rabbit reticulocyte translation system. Both E6 and E7 proteins were expressed simultaneously. This translation could be interfered with by antisense oligonucleotides corresponding to various regions of the transcript. Antisense oligonucleotides targeted at sequences flanking either side of the translation initiation codon of the E6 open reading frame were effective in inhibiting the synthesis of both proteins, whereas oligonucleotides complementary to the coding regions downstream of the first start codon showed either a considerably reduced effect or none at all. In particular, there was limited inhibition of E7 translation by antisense oligonucleotides flanking the translation start region of the E7 gene. In the presence of RNase H, it was possible to selectively inhibit the synthesis of either E6 or E7 by several gene-internal antisense oligonucleotides. We conclude that HPV16 E6-E7 bicistronic mRNA is fully functional and that both proteins are translated with equal efficiency via the scanning mechanisms with reinitiation at the second open reading frame. In addition, both AE6 and AE7 may have therapeutical potential as they are capable of inhibiting the proliferation of CaSki cells which contain the HPV16 genome.
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Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter
More LessIt has been demonstrated that the hepatocyte nuclear factor 1 (HNF1) binding site is critical for the majority of the hepatitis B virus (HBV) large surface antigen promoter activity in differentiated hepatoma cell lines. Examination of a series of clustered point mutations in the minimal large surface antigen promoter demonstrated that the HNF1 and TATA box binding sites are the major regulatory elements required for transcription from this promoter. Synthetic promoter constructs containing the large surface antigen promoter HNF1 binding site and TATA box element upstream of the luciferase open reading frame were tested for their transcriptional activities in HepG2.1 cells in the absence or presence of an HNF1 expression vector. These synthetic promoter constructs displayed a similar level of transcriptional activity and induction by HNF1 in comparison with the full-length large surface antigen promoter, suggesting that additional HBV sequences are dispensable for full transcriptional activity. The distance between the HNF1 binding site and TATA box element in the synthetic promoter constructs appeared to influence the transcriptional activity modestly and in a periodic manner.
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In vitro infection of human hepatoma cells (HepG2) with hepatitis B virus (HBV): spontaneous selection of a stable HBV surface antigen-producing HepG2 cell line containing integrated HBV DNA sequences
More LessThe degree of susceptibility of human hepatoma (HepG2) cells to direct hepatitis B virus (HBV) infection remains unknown. We previously observed a low level of Dane particle production and viral DNA replication after in vitro infection of HepG2 cells with serum-derived HBV. However, this culture system appeared to be affected by variations as human hepatocyte cultures. In the present study, HBV infection of HepG2 cells led to a significant increase in the secretion of three envelope antigens (HBsAg, preS2Ag and preSl Ag) at 4 days post-infection, and Northern blot analysis revealed the presence of both preSl (2·6 kb) and preS2/S (2·2 kb) transcripts. Expression of preSlAg and the corresponding viral RNA became undetectable on 21 days post-infection whereas the 2·2 kb RNA species persisted and was associated with secretion of subviral HBs particles expressing preS2-epitopes and banding between 30 and 35% sucrose. At 35 days post-infection (fifth passage), a sudden high level production of HBsAg and preSlAg was observed, followed by a massive cell death (90 %). A stable HBsAg-producing HepG2 cell line, designated HepG2-BV3, grew out of the surviving cells. HepG2- BV3 cells could integrate HBV DNA sequences and produce the three HBV surface antigens. Treatment with dexamethasone increased the HBsAg and preSlAg secretion. Such a HBsAg-producing HepG2 cell line obtained by in vitro HBV infection seems to mimick events that occur in the naturally occurring persistent chronic infection, and therefore may be an efficient in vitro model for studying the contribution of viral integration in the dysregulation of HBV and liver- specific genes expression.
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Analysis by RNA-PCR of latency and reactivation of herpes simplex virus in multiple neuronal tissues
More LessFollowing intracameral inoculation with herpes simplex virus type 1 (HSV-1), BALB/c mice develop acute necrotizing chorioretinitis and infectious virus is detected in the eyes, trigeminal ganglia, brain, spinal cord and adrenal glands during acute infection. In this study, we analysed the latent phase of this experimental animal system. In mice which survived the acute infection, latent HSV-1 was recovered from the trigeminal ganglia, brain and adrenal glands by cocultivation with Vero cells. In these tissues, both the unspliced latency-associated transcript (LAT) and the spliced LAT were detected by RNA-PCR. Following in vivo administration of cyclophosphamide and dexa- methasone to induce viral reactivation, ICPO mRNA became detectable in the multiple neural tissues, and the spliced LAT disappeared whereas the unspliced LAT remained detectable by RNA-PCR. Sequence analysis of the RNA-PCR products revealed that the GC-AG splicing signal previously reported for LATs from trigeminal ganglia was also detected in LATs from the brain and adrenal glands, suggesting that the splicing of LATs might be associated with the maintenance of and/or reactivation from latency. The generalized latent infection of HSV-1 described in this study might serve as an experimental model of possible viral reactivation from organs that do not innervate the primary port of entry.
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The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein
More LessThe products of herpes simplex virus type 1 (HSV-1) genes UL5, UL8 and UL52 form a complex in virus- infected cells that exhibits both DNA helicase and DNA primase activities. UL8 protein was purified from insect cells infected with a recombinant baculovirus and used to generate monoclonal antibodies (MAbs). MAb 0811 was shown to recognize the UL8 protein in both Western blots and immunoprecipitation assays and to co-precipitate the other two proteins in the complex from insect cells triply infected with recombinants expressing the UL5, UL8 and UL52 polypeptides. Experiments performed using extracts from doubly infected cells indicated that UL8 could interact separately with both the UL5 and UL52 proteins. Similar experiments using a recombinant virus that expressed the HSV-1 origin-binding protein (OBP), UL9, demonstrated a direct physical interaction between the helicase-primase complex and OBP which involved the UL8 subunit. The C-terminal DNA-binding domain of OBP is dispensable for this interaction, as evidenced by the ability of MAb 0811 to co-precipitate a truncated UL9 protein, containing only the N-terminal 535 amino acids, with UL8.
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Comparative studies of the structural proteins and glycoproteins of equine herpesviruses 2 and 5
More LessThe structural proteins of equine herpesvirus 2 (EHV-2) and EHV-5, recently shown to be gammaherpesviruses, were identified and compared. Labelled proteins and glycoproteins were separated by SDS-PAGE and although EHV-2 and EHV-5 had similar protein profiles, bands in some positions were virus-specific. Six glycoproteins, with distinct profiles, were identified for both EHV-2 and EHV-5. Rabbit antisera to EHV-2 and EHV- 5 and horse antiserum to EHV-2 were used in radio- immunoprecipitations, Western blot analysis and ELISA to investigate the immunogenicity and cross-reactivity of virus proteins. These analyses revealed that while EHV-2 and EHV-5 proteins share many common epitopes, they also possess type-specific epitopes. A 0·71 kb region of the EHV-2 glycoprotein B (gB) gene was expressed as a fusion protein in Escherichia coli. Antiserum raised in a rabbit to the EHV-2 fusion protein was used to identify a 64K EHV-2 protein as EHV-2 gB. Antiserum to EHV-2 gB was used to identify a 66K EHV-5 protein as EHV-5 gB. These proteins, which may represent subunits of gB rather than the entire molecule, appear the most immunodominant of the structural virion proteins as identified by Western blot.
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Polyvalent and monoclonal antibodies identify major immunogenic proteins specific for human herpesvirus 7-infected cells and have weak cross-reactivity with human herpesvirus 6
More LessHyperimmune rabbit and mouse sera raised to human herpesvirus 7 (HHV-7)-infected cells and an immune human serum identified 20 [35S]methionine-[35S]cysteine- labelled proteins specific for HHV-7-infected cord blood mononuclear cells, ranging in apparent M r from 136K to 30K. The major proteins had apparent M r values of 121K, 100K, 87K, 85K, 60K, 51K, 46K, 42K, 40K and 36K. The human serum also identified seven [3H]glucosamine-labelled glycoproteins, with apparent M r values of 100K, 89K, 82K, 67K, 63K, 53K and 41K. Four monoclonal antibodies (MAbs) specific for HHV- 7-infected cells were derived. Two reacted with a family of five antigenically related polypeptides (87K, 85K, 70K, 61K and 57K in apparent M r), designated asthep85 complex. Two reacted with 121K and 51K M r proteins designated as pl21 and p51, respectively. Human sera react with high frequency with the p85 complex and to a lesser extent with pl21; hence these two proteins appear to be immunodominant for both humans and laboratory animals. The hyperimmune mouse serum and some of the MAbs showed some cross-reactivity with HHV-6A(U1102)- and 6B(Z29)-infected cells. The implications of cross-reactivity with respect to the human immune response to HHV-6 and -7 infections and prevalence analyses are discussed.
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Sequence variation in the Epstein–Barr virus latent membrane protein 1
More LessThe sequence of the latent membrane protein 1 (LMP- 1) gene was analysed in Epstein-Barr virus (EBV) isolates from specific regions representing both type 1 and type 2 EBV. A predominant strain marked by an XhoI restriction enzyme polymorphism (REP) within the LMP-1 gene has been identified in type 1 EBV in nasopharyngeal carcinoma (NPC) from Southern China. This polymorphism was also present in type 2 EBV in NPC from Alaska. In this study, the sequence of the LMP-1 gene was determined in these samples representing type 1 and type 2 EBV and was compared with the prototype lymphoid strains. Consistent nucleotide variation in the amino terminus of LMP-1 was identified in strains marked by the XhoI REP. These changes were present in both EBV type 1 and type 2 strains. Three types of sequence variation were detected in the carboxy terminus of LMP-1. The LMP-1 sequences differed in the number of an 11 amino acid repeat element. In the prototype EBV type 1 (B95-8) sequence and in the type 1 Raji and type 2 HR-1 strains, the third repeat element contained an insertion of 5 amino acids that were also the first five unique amino acids after the last partial repeat element. The third variation was a deletion of amino acids 343 to 352 of the B95-8 LMP-1. This deletion was detected in the type 1 Chinese EBV strains, but was not detected in the type 2 Alaskan strains although the Chinese and Alaskan strains have nearly identical amino acid changes at the amino terminus. Numerous other amino acid changes were detected in the carboxy terminus which did not cosegregate with either EBV type, amino acid changes in the amino terminus, or specific geographic regions. These data indicate that EBV strains can be distinguished by sequence differences within LMP-1 and that unlike the divergence between type 1 and type 2 EBV in Epstein-Barr nuclear antigen sequences, different EBV types are nearly identical in LMP-1 sequence.
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Epstein-Barr virus types 1 and 2 have nearly identical LMP-1 transforming genes
More LessAlthough variation in the gene encoding the Epstein- Barr virus (EBV) nuclear protein 2 accounts for much of the difference in the transforming activities of the two EBV strain types, divergence in the principal lymphocyte growth-altering gene, LMP-1, has not been previously evaluated. We have now determined the nucleotide sequence of the LMP-1 gene of a type 2 isolate of EBV, AG876. Surprisingly, the AG876 LMP-1 protein is 93 % identical to the prototype type 1 EBV strain B95–8, and this is well within the range of variability previously noted among type 1 EBV LMP-1 genes.
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Characterization of proteins encoded by the short unique region of herpesvirus of turkeys by in vitro expression
More LessNine open reading frames mapping in the short unique (Us) region of the genome of herpesvirus of turkeys (HVT) were expressed by in vitro transcription and translation. The observed Mr s of US10, SORF3 and US2 were as predicted from the sequence but there were discrepancies between the observed and predicted Mr s of US1, protein kinase, gl, gD and gE. These could be accounted for in most cases by post-translational and co-translational processing. Analysis of the synthesized products at different time points provided evidence for post-translational modification of HVT protein kinase. Translation in the presence of microsomal membranes resulted in co-translational processing of HVT gD, gl and gE by glycosylation and signal peptide cleavage.
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Replication of human cytomegalovirus in cells deficient in β 2-microglobulin gene expression
More LessTo study the roles of β 2-microglobulin (β2-m) and major histocompatibility complex (MHC) class I expression in human cytomegalovirus (HCMV) infection, the ability of HCMV strain AD-169 to infect and replicate in a human melanoma cell line (FO-1), which is β,-m- deficient and cannot express MHC class I on its cell surface, was examined. Susceptibility of FO-1 cells was compared with human foreskin fibroblasts (HFF) and FO-1H cells (FO-1 cells that have been transfected with the human β2-m gene, restoring MHC I expression on the cell surface). As judged by the HCMV immediate early 1 (IE-1) antigen expression, HCMV was able to infect FO-1 cells, although somewhat less efficiently than HFF. However, the expression of HCMV late (L) antigen and the production of virus was significantly less for FO-1 cells than for HFF. Analysis of the FO-1H transfectants revealed that expression of IE-1 and L HCMV antigens was comparable to FO-1 cells, which lack MHC I. Treatment of FO-1 and FO-1H cells with sodium butyrate prior to inoculation did not alter the expression of MHC I in either cell type, but did increase susceptibility of both cell types to HCMV infection, as well as the expression of L antigens and production of virus. These studies indicate that HCMV infection of FO-1 cells is independent of β 2-m and MHC class I expression.
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The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis
More LessSequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease. Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease. Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95 %. These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.
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Short and long term dissemination of deletion mutants of adenovirus in permissive (cotton rat) and non-permissive (mouse) species
W. Oualikene, P. Gonin and M. EloitAs a first step in the evaluation of the safety of replication-defective adenoviruses used as gene transfer vectors, the dissemination of wild-type (wt) adenovirus (Ad) type 5, Ad-dl327 (deleted for the E3 gene) and Ad- gp50 (deleted for E3 and E1A and therefore replication defective) was studied in mice and cotton rats. Of each virus, 10s p.f.u. was inoculated, either by the intranasal or the intramuscular route, and virus isolation was undertaken after sacrifice of the animals 3 or 31 days after inoculation. E3 deletion had no significant effect on viral spread, whereas El A deletion reduced it significantly. After intramuscular inoculation of the E3 — /E1A— virus, it could be isolated only from the local lymph node. Intranasal inoculation led to a wider distribution including lungs, liver, kidneys and lymph nodes. The pattern of dissemination of the E3 —/E1A— virus was the same in mice and cotton rats, providing indirect evidence of the lack of replication of this virus in this species permissive for the wt virus. However, in the case of infection with a wt strain in E3 —/E1A— adenovirus-inoculated recipients, both viruses were found in lymph nodes. In this situation the risk of phenotypic complementation of the E1A gene remains to be studied further.
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Analysis of p53 status in tonsillar carcinomas associated with human papillomavirus
Tonsillar squamous cell carcinomas (a total of 14) were examined both for the presence of human papillomavirus (HPV) DNA and for p53 alterations. General primer- mediated HPV polymerase chain reaction (GP-PCR) revealed the presence of HPV DNA in 12/14 cases. Subsequent typing by HPV type-specific PCR and sequence or hybridization analysis of GP-PCR products revealed DNA from HPV 16 in seven cases, from HPV 33 in two cases, and from HPV 7, HPV 16/33 and HPV 33/59 each in a single case. p53 immunohistochemistry performed on nine HPV containing tonsillar carcinomas using polyclonal serum CM-1 showed elevated p53 levels in four cases. These included 3/5 HPV 16 containing carcinomas and the HPV 33/59 containing carcinoma. Analysis of p53 mutations using denaturing gradient gel electrophoresis (DGGE) of GC-clamped PCR products of exons 5 to 8 showed p53 gene alterations in 3/13 cases, incuding 2/11 HPV positive cases and 1/2 HPV negative cases. The alterations included a silent point mutation within exon 8 of an HPV 16 containing carcinoma, a 1 bp deletion within exon 8 of an HPV 33 containing carcinoma, and a missense mutation within exon 7 of one of the HPV negative carcinomas. There was evident discrepancy between p53 immunohistochemistry and gene analysis. Four HPV containing cases showing elevated p53 levels did not reveal the presence of exon 5 to 8 alterations affecting the amino acid code, suggesting the presence of mutations occurring in other exons or non-mutational p53 stabilization. The data indicate that HPV and elevated p53 can coexist in tonsillar carcinomas and that despite the low frequency of p53 mutations the presence of HPV is not exclusively related to the absence of mutated p53.
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Origin and evolution of the c-src-transducing avian sarcoma virus PR2257
Avian sarcoma virus PR2257 transduced de novo the c- src gene and about 900 bp of 3‣ non-coding sequences belonging to the src locus. This virus contains only one mutation in the c-src coding sequence causing a reading frame shift after Pro-525. The molecular clone studied was derived from a cell line of transformed quail fibroblasts, Cl. It contains endogenous virus (ev) derived sequences in the U5 and 3‣ non-coding regions, indicating that multiple recombination occurred with endogenous virus. Here we investigated the possible evolution of PR2257 when the original tumour was repeatedly passaged in vivo. After 16 passages a new virus, designated PR2257/16, appeared with a tenfold higher titre. The sequence ofPR2257/16 was determined and showed that PR2257/16 resulted from recombination of PR2257 with the env gene of the helper virus (td daPR-C) This recombination expanded the env gene content in PR2257/16 and, in addition, five point mutations occurred in its genome. Because we thought that an endogenous virus might be involved in the mechanism of c-src transduction, we also reinvestigated the presence of ev sequences in PR2257 proviruses from several early passages of the original tumour. We found that in contrast with the first isolate from the C7 cell line, the provirus in these tumours did not contain such sequences. These results do not support the hypothesis that endogenous sequences were involved in the transduction process.
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Inhibition of feline immunodeficiency virus gene expression and replication by alphaherpesvirus ICP4 homologues
We investigated the effects of pseudorabies virus (PRV), herpes simplex virus type 1 (HSV-1), and equine- herpesvirus type 1 (EHV-1) ICP4 homologues on feline immunodeficiency virus (FIV) long terminal repeat (LTR)-directed gene expression. This was done by using the transient expression chloramphenicol acetyltrans- ferase (CAT) assay in Crandell feline kidney (CRFK) and Felis catus whole fetus 4 cells transfected with a chimeric FIV FTR-CAT reporter construct in combination with effector plasmids expressing the PRV, HSV-1 or EHV-1 ICP4 homologue. The experiments demonstrated that the ICP4 homologues could significantly inhibit FIV FTR-directed gene expression. Moreover, the ICP4 homologues also exhibited a marked inhibitory effect on FIV replication in CRFK cells cotransfected with an infectious molecular clone of FIV.
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Defective response to T cell mitogens in mice injected with human immunodeficiency virus type 1-infected U937 cells
Swiss mice were injected intraperitoneally with uninfected or human immunodeficiency virus type 1 (HIV-1) infected human U937 cells. At 6 days, no residual human cells were detected in mouse tissues as determined by PCR analysis of DNAs from injected mice using primers and probes for the human HLA-DQ alpha gene. At 6 to 12 months, approximately 60% of the HIV-1-injected mice had antibodies to HIV-1 gp 120 andgp41 proteins. Fifteen percent of the animals showed evidence of HIV-1 infection as determined by PCR analyses of DNA from peripheral blood leukocytes and by in situ hybridization for detection of HIV-1 mRNA in peritoneal cells. In this set of experiments, spleen cells from mice sacrificed at different times after injection were cultured for 48 h in the presence or absence of mitogens [i.e.: concanavalin (Con A) or anti-CD3 antibody] and then tested for lymphocyte proliferation. At 10 to 12 months, splenocytes from approximately 80 % of Swiss mice injected with HIV-1-infected U937 cells exhibited a marked defect in their proliferative response to Con A or anti-CD3 antibody as compared with spleen cells from both uninjected or U937 cell-injected mice. Similar results were obtained at 12 months in C3H/HeJ mice. Non-responding spleen cells from HIV-l-injected Swiss mice did not proliferate in response to anti-CD3 antibody even in the presence of co-stimulatory molecules such as phorbol myristate acetate or anti-CD28 antibody. Splenocytes from these mice also exhibited an impaired capacity to produce interferon-λ and interleukin-4 after mitogen stimulation. No T cell defects were observed in control-injected mice. Immunofluorescence analyses revealed a significant decrease in the percentage of both CD4+ and CD8+ spleen cells in HIV-l-injected mice. These data indicate that immunocompetent mice can be used to investigate some HIV-1- related immune dysfunctions in vivo.
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Interleukin 4 and human immunodeficiency virus stimulate LFA-1-ICAM-1-mediated aggregation of monocytes and subsequent giant cell formation
More LessThe effects of recombinant interleukin 4 (IL-4) on cell cluster and multinucleated giant cell (MGC) formation from human immunodeficiency virus (HlV)-infected and uninfected monocytes were examined. Human blood monocytes were isolated by centrifugal elutriation and monoclonal antibody-complement-dependent lysis of residual T cells, and infected with low passage HIV strains. Monocytes were exposed to recombinant IL-4 (1 to 20 ng/ml), continuously after inoculation with HIV. Monocyte expression of ICAM-1 but not LFA-1 was significantly enhanced by IL-4 although substrate adherence was a more potent stimulus. Monocyte cluster and MGC formation was quantified after fixation and staining with Giemsa. Clusters of HIV-infected and uninfected monocytes were consistently and significantly increased at 4 to 7 days after IL-4 stimulation. The combination of HIV and IL-4 was more stimulatory than either treatment alone. In two out of five uninfected and three out of seven HIV-infected monocyte cultures, MGC formation was also markedly increased at 10 to 14 days after stimulation. Incubation with anti-LFA-1 (anti- CD 11 a, anti-CD 18) and anti-ICAM-1 (anti-CD54) monoclonal antibodies reduced IL-4-stimulated aggregation in HIV-infected and uninfected monocytes and subsequently reduced MGC formation. Anti-ICAM-1 was not as effective as anti-CD 11a or anti-CD 18 in inhibiting aggregation of HIV-infected monocytes and in these cultures anti-ICAM-2 was also inhibitory. Extracellular HIV antigen concentrations were not consistently reduced by anti-CDlla or anti-ICAM-1. Hence IL-4 markedly enhanced monocyte aggregation in both HIV-infected and uninfected monocytes, probably through enhanced LFA-l-ICAM-1 interactions in all cultures and LFA-l-ICAM-2 interactions in infected monocytes, leading subsequently to MGC formation in some cultures.
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Nucleotide sequence of Nilaparvata lugens reovirus genome segment S8 coding for the major outer capsid protein
More LessThe complete nucleotide sequence of genome segment 8 (S8) of Nilaparvata lugens reovirus (NLRV) was determined. It consisted of 1802 nucleotides containing a long open reading frame (562 amino acids), which was expressed in Escherichia coli as a fusion protein. The expressed S8 product, a 62K protein, was detected by Western blotting using IgG directed against intact NLRV particles. This result indicates that S8 encodes the major outer capsid protein of NLRV. The protein exhibited 18·6 % amino acid sequence identity with the predicted translation product of S10 of rice black- streaked dwarf virus.
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A definition of bovine rotavirus virulence
More LessRotaviruses are accepted as enteric pathogens of calves but many natural infections are subclinical. In the present paper, the outcome of inoculation of gnotobiotic calves of three ages (the second day of life, the second week of life and calves aged 6 weeks and over) with doses of 105·0 to 106·5 TCID50 was compared for three bovine rotavirus isolates (C3–160, 17/4 and 39/58). The clinical outcome of infection was dependent on both calf age and rotavirus isolate. Age-dependent resistance to infection was not found. By contrast, age-dependent resistance to disease was found with rotavirus isolates C3–160 and 17/4 but not with 39/58. All three isolates caused disease in calves inoculated on the second day of life but only one, 39/58, caused disease in the two older groups. Peak levels and duration of virus excretion were similar in clinically normal (106·7 ± 1·08 TCID50 per g of faeces for 4·6 ± 1·2 days) and diseased (107·45 ± 0·94 TCID50 per g of faeces for 5·3 ±0·98 days) calves of all ages, but the onset of virus excretion occurred sooner in clinically affected calves (1·6 ± 0·63 days in clinically affected compared with 3·7 ±1·5 days in clinically normal calves, P < 0·01). The present study confirmed the findings of an earlier study (Bridger & Pocock, 1986) which showed that bovine rotaviruses differ in virulence for calves in the second week of life and that older calves are susceptible to rotavirus infection and disease. In addition, the present study demonstrated for the first time, that differences in rotavirus virulence are not apparent with calves inoculated on the second day of life, an age which has been used commonly to assess rotavirus virulence. It is suggested that rotaviruses that cause disease in calves only on the second day of life should be described as of low virulence whereas those that cause disease in all ages should be described as virulent.
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Biological activity, binding site and affinity of monoclonal antibodies to the fusion protein of respiratory syncytial virus
The neutralizing activity and fusion-inhibition activity per unit weight of immunoglobulin were determined for each of a panel of 20 monoclonal antibodies (MAbs) to the fusion (F) protein of respiratory syncytial (RS) virus. Neutralization did not correlate with fusion-inhibiting activity, suggesting that the F protein plays at least two independent, antibody-sensitive roles in viral infection. Antibodies with the highest biological activity against A2, a subgroup A strain of RS virus, neutralized a subgroup B strain (8/60) poorly, suggesting a degree of antigenic variation that may be important in human infection.
All but one fusion-inhibiting MAb bound to protein blots and binding was mapped to two areas on overlapping F protein fragments. One MAb with relatively poor fusion-inhibiting activity bound only to fragments C-terminal of amino acid 384, the remainder bound only to fragments containing residues 253 to 289. MAbs directed to the latter site were heterogeneous in neutralizing activity, subgroup specificity and fusion- inhibiting activity. These variations between MAbs could not be accounted for by differences in their binding avidities. We suggest that this binding site is not the complete antibody epitope which probably includes conformation-dependent elements.
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Correlation of proteolytic cleavage of F protein precursors in paramyxoviruses with expression of the fur, PACE4 and PC6 genes in mammalian cells
More LessThe fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains and human parainfluenza virus type 3 (HPIV3) has a multibasic amino acid sequence at the cleavage site, and intracellular cleavage activation occurs in a variety of cells. The host protease responsible for the cleavage has been proposed to be a subtilisin-like protease (subtilase) such as furin (the product of the fur gene). We found that the lymphocyte cell lines MOLT-4, Ramos and Daudi, in addition to NALM6, lacked the ability to fully cleave the F protein precursor of virulent NDV. In contrast, MT4 as well as the non-lymphocyte cell lines HeLa and Hep2 cleaved the F protein precursor efficiently. To investigate the role of subtilases in proteolytic processing, we examined the gene expression of candidate subtilases, furin, PACE4 and PC6 in these cleavage-competent and -incompetent cells. Considerable expression of the fur gene was observed in the cleavage-competent cells, but little or no expression was detected in the cleavage- incompetent cells. PACE4 and PC6 gene expression was observed in some of the cleavage-competent cells but not in the cleavage-incompetent cells. These results suggest that furin is the protease responsible for cleavage activation of the F protein of virulent NDV strains in cultured mammalian cells and the possibility is raised that PACE4 and PC6 also participate in processing in some of the cells. On the other hand, the HPIV3 F protein was cleaved efficiently in lymphocyte cells deficient in subtilases, suggesting that an unknown protease other than furin, PACE4 or PC6 may be involved in the processing.
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Comparative analysis of the gene encoding the nucleocapsid protein of dolphin morbillivirus reveals its distant evolutionary relationship to measles virus and ruminant morbilliviruses
More LessA morbillivirus of uncertain origin recently killed hundreds of Mediterranean dolphins. This is the first report of the nucleotide and deduced amino acid sequence of a dolphin morbillivirus (DMV) gene. The sequence of the nucleocapsid (N) gene including boundaries was determined. When the DMV N gene coding region was compared with the corresponding sequences of other morbilliviruses a distant evolutionary relationship between these viruses and DMV was apparent. Phylogenetic analysis of the sequence data provided further evidence that DMV is not closely related to any known morbillivirus, whereas phocine distemper virus exhibits a relatively close relationship to canine distemper virus.
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Role of surface glycoproteins in influenza virus pyrogenicity
More LessEleven H3N2, seven H1N1 and three H3N1 influenza virus reassortants of the pyrogenic A/Puerto Rico/8/34- A/England/939/69 clone 7a (H3N2) (A/7A) and poorly pyrogenic A/Fiji/15899/83 (H1N1) (A/Fiji) parents were analysed genetically for the parental origin of their genes and for their pyrogenicity in ferrets. All H3N2 reassortants were pyrogenic and produced significantly more fever than A/Fiji but differences in pyrogenicity between them could not be correlated with either single or constellations of genes. All H1N1 reassortants were poorly pyrogenic compared with A/7A but one (Am29) produced significantly more fever than A/Fiji. No correlation of the increased fever with inserted A/7A genes was evident in Am29 and, while mutations were detected in the HI haemagglutinin of this reassortant using monoclonal antibodies, similar mutations were present in the other H1N1 reassortants which showed no increase in fever production. The three H3N1 reassortants were intermediate in their pyrogenicity, being more pyrogenic than A/Fiji and less pyrogenic than A/7A. Overall, these results support previous conclusions that the haemagglutinin and/or neuraminidase play a major role in the pyrogenicity of influenza virus.
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Endosymbiotic bacteria associated with circulative transmission of potato leafroll virus by Myzus persicae
More LessIn order to understand the molecular mechanisms underlying circulative transmission of potato leafroll virus (PLRV) by aphids, we screened Myzus persicae proteins as putative PLRV binding molecules using a virus overlay assay of protein blots. In this way, we found that purified PLRV particles exhibited affinity for five aphid proteins. The one most readily detected has an M r of 63K, and was identified as symbionin. This is the predominant protein synthesized by the bacterial endo- symbiont of the aphid and is released into the hae- molymph. Since further studies clearly showed that PLRV particles also bind to native symbionin, it was envisaged that virus particles when acquired into the haemocoel of an aphid interact with symbionin. Inhibition of prokaryotic protein synthesis by feeding M. persicae nymphs on an antibiotic-containing artificial diet prior to PLRV acquisition reduced virus transmission by more than 70%. The major coat protein of the virus was found to be degraded in the antibiotic- treated aphids; this would obviously have resulted in an increased exposure of viral RNA to enzymic breakdown and concomitant loss ofinfectivity. For these reasons we conclude that endosymbiotic bacteria play a crucial role in determining the persistent nature of PLRV in the aphid haemolymph and that symbionin is probably the key protein in this interaction.
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Nucleic acid-binding properties of the P1 protein of turnip mosaic potyvirus produced in Escherichia coli
More LessThe N-terminal P1 protein of turnip mosaic potyvirus (TuMY) polyprotein was overexpressed in Escherichia coli, purified by metal chelation chromatography under denaturing conditions and renatured. U.v. cross-linking experiments indicated that the recombinant protein interacted with RNA, and gel retardation electrophoresis demonstrated that more than one molecule of PI bound one molecule of RNA. Formation of the protein-RNA complexes was dependent on the conformational state of PI and was stable at relatively high concentrations of NaCl. P1 had the ability to bind ssRNA and ssDNA, with similar affinity, but was not able to bind to dsDNA. The TuMV protein had the additional characteristic of binding dsRNA with affinity similar to that observed with single-stranded nucleic acids.
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The nucleotide sequence of citrus leaf rugose ilarvirus RNA-2
Xin Ge and S. W. ScottThe nucleotide sequence of citrus leaf rugose ilarvirus (CiLRV) RNA-2 consists of 2990 nucleotides and contains one open reading frame (ORF) which encodes a deduced translation product of 832 amino acids with a calculated Mr of 95501 (95K). The 5‣ terminus of the RNA has a m7Gppp cap. Both the nucleotide sequence of CiLRV RNA-2 and its translated polypeptide share homologies with the nucleotide sequence and translated polypeptide, respectively, of RNA-2 of alfalfa mosaic virus (A1MV). The homology occurs in the central region of both the nucleic acid sequence and the polypeptide. Homologies between either CiLRV or A1MV and other Bromoviridae (cucumber mosaic virus - CMV, brome mosaic virus - BMV and cowpea chlorotic mottle virus - CCMV) were far less. Alignment of the 104 amino acid region (polymerase signature) of the 95K protein against 10 other ‘alpha-like’ plant viral polymerase signatures showed that CiLRV and A1MV are more closely related to each other than to CMV, BMV or CCMV. This is the first report of the full-length sequence of RNA-2 of an ilarvirus.
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The nucleotide sequence of apple mosaic virus coat protein gene has no similarity with other Bromoviridae coat protein genes
A double-stranded cDNA was synthesized from in vitro polyadenylated apple mosaic virus (ApMV) RNA 3 using oligo(dT) and sequence-specific primers, and was cloned into plasmid vectors. A set of overlapping cDNA clones was used to determine the nucleotide sequence of RNA 4. ApMV RNA 4 was found to contain an open reading frame (ORF) of 666 nucleotides, which was Hanked by 5′ and 3′ non-translated sequences of 55 and 264 nucleotides, respectively. The ORF encoding the coat protein was identified by comparing the predicted amino acid sequence with that obtained from direct protein microsequencing of the native viral coat protein. The ORF encodes a protein with an M r of 25056. The nucleotide sequence of the ApMV coat protein gene showed no similarity to those of alfalfa mosaic virus, tobacco streak virus (TSV), brome mosaic virus or cucumber mosaic virus. The predicted amino acid sequence of the amino-terminal region of the ApMV coat protein is basic, rich in cysteine residues and may contain a zinc finger motif similar to that found in TSV.
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Similarities between the secondary structure of satellite tobacco mosaic virus and tobamovirus RNAs
More LessThe secondary structure of satellite tobacco mosaic virus (STMV) RNA was predicted using computer simulations of RNA folding. The analogies of structural elements in the 3′ end untranslated regions (3′ -UTR) of tobamoviral RNAs were analysed. In addition to the tRNA-like structure and pseudoknot stalk, which are found in all known RNAs of tobamoviruses and STMV, another region of stable consecutive pseudoknots was predicted in the 3′ -UTR of STMV RNA. A similar pattern of repeated structural units, containing pseudoknot stalks and parts of the tRNA-like structure, was also found in odontoglossum ringspot virus (ORSV) RNA 3′-UTR. The predictions on the structure are supported by sequence comparisons which point to an important functional role of 3′ terminal pseudoknots in STMV RNA as well as in other tobamoviral RNAs. The possible participation of pseudoknotted structures in the interactions with coat protein in STMV is discussed.
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The infectivity of dimeric potato yellow mosaic geminivirus clones in different hosts
More LessHead-to-tail dimeric clones of both DNA A and DNA B of potato yellow mosaic geminivirus (PYMV) were constructed. These constructs were infectious when inoculated onto Nicotiana benthamiana plants either as DNA or by agroinoculation and were also infectious for tomato plants by agroinoculation. The dimers were not infectious for potato plants following inoculation by either method. Symptom induction required both DNA A and DNA B but agroinoculation with DNA A alone resulted in virus spread in 30% of the inoculated N. benthamiana plants. Leaf disc explants of N. benthamiana, tomato and potato could all be infected by agroinoculation indicating that the method of delivery of the DNA to intact potato plants was unsuitable for successful inoculation rather than an inherent inability of the virus to replicate/spread in potato per se. Neither whole plants nor leaf discs of sugar beet supported the replication of PYMY DNA.
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