- Volume 78, Issue 11, 1997
Volume 78, Issue 11, 1997
- Articles
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The molecular basis of virulence of the encephalitogenic flaviviruses.
More LessSummary: Studies of flavivirus virulence have focussed on variants with differing neurovirulence or neuroinvasiveness in animal models, with the majority based on determining the contribution of viral structural proteins, in particular the envelope glycoprotein. Specific virulence determinants on protein E have recently been identified by the application of several experimental techniques such as selection and characterization of neutralization escape variants in the presence of anti-E protein MAbs or by site-directed mutagenesis of the E gene of infectious cDNA clones (see Fig. 2, Tables 2 And 3). However, the association of particular amino acid substitutions in protein E with loss of neurovirulence and/or neuroinvasiveness appears to be dependent on the virus system under study and thus the mechanism by which protein E controls these two virulence phenotypes is unclear. Existing data show that neurovirulence and neuroinvasiveness determinants map to identical regions of protein E, including the lateral surface of domain III (the putative receptor binding site) and the base of domain II (thought to be involved in pH-dependent fusion activity). Attenuation of some domain II and III variants is associated with changes in binding to cell membrane proteins or in pH-dependent fusion activity, suggesting that defects in the early events of virus replication are responsible for virulence attenuation. A small number of amino acid changes in domain I are associated with attenuation of virulence, including mutations in the protein E glycosylation site which result in significant reductions in viral glycoprotein expression. The mechanism of attenuation associated with other mutations in domain I is unknown. Several studies have identified putative neurovirulence determinants within other regions of the genome (e.g. non-structural protein genes, 3′ UTR), and in one example the mutation attenuates neurovirulence without alteration in neuroinvasiveness. Further work is necessary to map neurovirulence determinants within these regions. It is likely that significant advances in this field will come from the application of infectious cDNA clone technology.
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Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system.
More LessIn a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289–424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum- free conditions, more than 95 % of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue- 3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.
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Immune responses to the hepatitis C virus NS4A protein are profoundly influenced by the combination of the viral genotype and the host major histocompatibility complex.
More LessThe interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using syntheticfull-length non-structural (NS) 4Aproteins, residues 1658–1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688–1707. In immunized B10 H-2 con- genic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670–1679 and 1683–1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restric- ted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.
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Hepatitis C virus negative strand RNA is not detected in peripheral blood mononuclear cells and viral sequences are identical to those in serum: a case against extrahepatic replication.
More LessPeripheral blood mononuclear cells (PBMCs) from 27 hepatitis C virus (HCV)-infected patients were analysed for the presence of HCV negative strand RNA with strand-specific Tth-based RT-PCR. No negative strand RNA was detected in any sample, and positive strand HCV sequences amplified from PBMCs were identical to those found in serum. These findings suggest that HCV does not replicate in PBMCs, and the presence of HCV sequences at this site is compatible with passive virus adsorption and/or contamination by circulating virus.
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Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.
More LessThe synthesis of long cDNA molecules encoding the complete genome of RNA viruses has recently been demonstrated; this major improvement has numerous practical applications such as construction of infectious cDNA clones or study of sequence variability at the level of a single RNA molecule. Using hepatitis C virus (HCV) as a model, we established an RT-PCR technique for amplification of cDNA fragments with a length of about 5 kb. The RT reaction was carried out with a Moloney murine leukaemia virus reverse transcriptase lacking detectable RNase H activity. For PCR reactions an enzyme mix containing Taq and Pwo DNA polymerases was used. Hot start and addition of 5% DMSO were also important to efficiently achieve long PCR products.
About 106 HCV genome equivalents/ml in serum were needed in order to amplify the HCV genome in only two cDNA fragments covering about 98% of the complete genome. Analysis of the HCV quasispecies is also possible by this method as shown by sequencing of the hypervariable region 1 (HVR1) after cloning of cDNAs. The integrity of the long cDNA clones was proven by (1) restriction analyses, (2) partial sequencing and (3) expression of respective gene products. In vitro transcribed cDNAs were translated in rabbit reticulocyte lysate. Structural and nonstructural HCV proteins were identified by immunoprecipitation using patient serum. These results suggest that the two cDNA clones encode a complete and functional open reading frame of HCV.
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Molecular cloning of a defective hepatitis C virus genome from the ascitic fluid of a patient with hepatocellular carcinoma.
More LessA defective hepatitis C virus (HCV) genome in the ascitic fluid of a patient with hepatocellular carcinoma was cloned and sequenced up to the 3′ poly(U) stretch. When compared with the published Taiwanese HCV sequence, this defective genome contained deletions of single nucleotides at eight sites, double nucleotides at two sites, triple nucleotides at four sites, quadruple nucleotides at one site and replacement of a short stretch of sequence at one site. For comparison, the corresponding regions containing these mutations were also cloned from a serum sample from this patient. Except for deletions of two triple nucleotides in the hypervariable region, the reading frames of all serum-derived clones were intact. The defective HCV genome encoded a truncated core protein with 90 amino acid residues (the last 20 amino acid residues came from a different reading frame), whereas the serum-derived genome encoded a full- length core protein. When expressed in Huh-7 cells, these two proteins were localized to the nucleus and cytoplasm, respectively. Using specific primer-sets, ascites- and serum-derived genomes were each detected alone in ascitic fluid and serum samples, respectively, whereas both sequences were present in ascitic mononuclear cells. The defective sequence thus constituted the major virus population in the ascitic fluid whereas a putative helper genome coexisted with it inside the ascitic mononuclear cells. This sequence is possibly a defective and interfering genome.
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Sequence variation and phylogenetic analysis of envelope glycoprotein of hepatitis G virus.
More LessA transfusion-transmissible agent provisionally designated hepatitis G virus (HGV) was recently identified. In this study, we examined the variability of the HGV genome by analysing sequences in the putative envelope region from 72 isolates obtained from diverse geographical sources. The 1561 nucleotide sequence of the E1/E2/NS2a region of HGV was determined from 12 isolates, and compared with three published sequences. The most variability was observed in 400 nucleotides at the N terminus of E2. We next analysed this 400 nucleotide envelope variable region (EV) from an additional 60 HGV isolates. This sequence varied considerably among the 75 isolates, with overall identity ranging from 79·3% to 99·5% at the nucleotide level, and from 83·5% to 100% at the amino acid level. However, hypervariable regions were not identified. Phylogenetic analyses indicated that the 75 HGV isolates belong to a single genotype. A single-tier distribution of evolutionary distances was observed among the 15 E1/E2/NS2a sequences and the 75 EV sequences. In contrast, 11 isolates of HCV were analysed and showed a threetiered distribution, representing genotypes, subtypes, and isolates. The 75 isolates of HGV fell into four clusters on the phylogenetic tree. Tight geographical clustering was observed among the HGV isolates from Japan and Korea.
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Inhibition of pestivirus infection in cell culture by envelope proteins E(rns) and E2 of classical swine fever virus: E(rns) and E2 interact with different receptors.
More LessPure preparations of envelope glycoproteins Erns and E2 of classical swine fever virus (CSFV) synthesized in insect cells were used to study infection of porcine and bovine cells with the pestiviruses CSFV and bovine viral diarrhoea virus (BVDV). Almost 100% inhibition of infection of porcine kidney cells with CSFV was produced by 100 μg/ml Erns. After removal of the virus no Erns was needed in the overlay medium (growth medium) to maintain this level of inhibition. In contrast, 100% inhibition of infection of porcine kidney cells with CSFV by 10 ^g/ml E2 was only achieved when E2 was added to the overlay medium. When E2 was omitted, a maximum of 50% inhibition was achieved. This indicated that after the virus and E2 were removed from the cells, infection still occurred, by virus particles which were still bound to the cell surface. Treatment with 100 μg/ml Erns released these particles from the cell surface. Furthermore, Erns bound irreversibly to the surface of cells susceptible or unsusceptible to pestivirus infection and cell-to-cell spread of CSFV was completely inhibited by E2 but not by Erns. These results demonstrated that Erns and E2 interacted with different cell surface receptors. Inhibition of BVDV infection of porcine and bovine cells by CSFV E2 suggested that CSFV E2 and BVDV E2 share an identical receptor. BVDV strain 5250 isolated from pigs was efficiently inhibited by CSFV Erns, whereas several BVDV strains isolated from cattle were not, suggesting that the conformation of Erns plays a role in host tropism.
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Identification and subcellular localization of a 41 kDa, polyprotein 1ab processing product in human coronavirus 229E-infected cells.
More LessThe translation products of the human coronavirus (HCV) 229E open reading frames 1a and 1b, the polyproteins 1a and 1ab, are processed by virus- encoded proteinases. One of the key enzymes in this process is a chymotrypsin-like enzyme, the 3C- like proteinase. In this study we have identified an ORF 1b-encoded, 41 kDa processing product in HCV 229E-infected cells by using a monoclonal antibody with defined specificity. We show that this polypeptide is released from polyprotein 1ab by 3C-like proteinase-mediated cleavage of the peptide bonds Gln-6110/Gly-6111 and Gln-6458/Ser- 6459. Also, we have investigated the subcellular localization of the 41 kDa processing product. Immunofluorescence microscopy revealed a punctate, perinuclear distribution of the 41 kDa polypeptide in infected cells and an identical subcellular localization was observed for three additional pp1ab-derived polypeptides. In contrast, the virus nucleocapsid protein showed a homogeneous cytoplasmic localization.
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Identification of residues critical for the human coronavirus 229E receptor function of human aminopeptidase N.
More LessAminopeptidase N (APN) is the major cell surface receptor for group 1 coronaviruses. In this study, we have isolated and characterized a feline APN cDNA and shown that the transfection of human embryonic kidney cells with this cDNA renders them susceptible to infection with the feline coronavirus feline infectious peritonitis virus, the human coronavirus (HCV) 229E and the porcine coronavirus porcine transmissible gastroenteritis virus. By using chimeric APN genes, assembled from porcine and feline sequences, we have shown that, analogously to the human APN protein, a region within the amino-terminal part of the feline APN protein (encompassing amino acids 132–295) is essential for its HCV 229E receptor function. Furthermore, by comparing the relevant feline, human and porcine APN sequences, we were able to identify a hypervariable stretch of eight amino acids that are more closely related in the feline and human APN proteins than in the porcine APN molecule. Using PCR- directed mutagenesis, we converted this stretch of amino acids within the porcine APN molecule to the corresponding residues of the human APN molecule. These changes were sufficient to convert porcine APN into a functional receptor for HCV 229E and thus define a small number of residues that are critically important for the HCV 229E receptor function of human APN.
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Structural, antigenic and immunogenic relationships between European brown hare syndrome virus and rabbit haemorrhagic disease virus.
The capsid protein of a French isolate of the European brown hare syndrome virus (EBHSV) was expressed in the baculovirus system. The recombinant EBHSV (rEBHSV) capsid protein was able to self-assemble into virus-like particles (VLPs). The VLPs were indistinguishable from the infectious EBHSV and displayed morphological characteristics similar to those we have described for the VLPs resulting from the expression of the capsid protein of rabbit haemorrhagic disease virus (RHDV), a closely related calicivirus. Cross-protection experiments showed that vaccination with rEBHSV particles did not protect rabbits against an RHDV challenge. A set of monoclonal antibodies (MAbs) was raised against rEBHSV capsid protein and used together with anti-RHDV and anti-EBHSV MAbs produced against native viruses to study the antigenic relationships between the two calici- viruses. All six anti-EBHSV MAbs delineated discontinuous epitopes; two of them reacted with specific surface epitopes and the remaining four reacted with internal epitopes which were also present in rRHDV. All anti-RHDV MAbs were monospecific; three reacted with surface linear epitope(s), two reacted with internal linear epitope(s) and one with a surface conformational epitope. On the basis of all these results, a classification of RHDV and EBHSV as two serotypes of a single serogroup is proposed.
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Creation of an infectious recombinant Sendai virus expressing the firefly luciferase gene from the 3' proximal first locus.
More LessA genetic engineering approach was made to generate a recombinant non-segmented negative- strand RNA virus, Sendai virus (SeV) of the family Paramyxoviridae, that expresses firefly luciferase. The DNA construct containing the entire open reading frame (ORF) of the luciferase gene followed by the SeV transcription stop and restart signals connected with the conserved intergenic three nucleotides was inserted immediately before the ORF of the viral 3′-proximal nucleocapsid (N) protein gene in a full-length SeV cDNA copy. After intracellular expression of full-length antigenomic transcripts from the engineered cDNA and of the viral nucleocapsid protein and RNA polymerase from the respective plasmids, a recombinant SeV expressing luciferase activity at a high level was recovered, although the tendency of this particular reporter gene product to aggregate in cells made it difficult to estimate the maximum level of expression. The increase in genome length brought about by inserting 1728 nucleotides into the 15 384 nucleotide parental SeV was associated with reduced plaque size, slightly slower replication kinetics and a severalfold decrease in yield of the virus. The inserted luciferase gene was stably maintained after numerous rounds of replication by serial passages in chick embryos. These results indicate the potential utility of SeV as a novel expression vector.
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Differential induction of cytotoxicity and apoptosis by influenza virus strains of differing virulence.
More LessTwo influenza viruses of differing virulence, clone 7a (virulent for humans and ferrets) and A/Fiji (attenuated for both species), induced differential levels of cytotoxicity (as measured by release of lactate dehydrogenase from cells) and apoptosis (as determined by altered nuclear morphology or flow cytometry) in MDCK and U-937 cells. Clone 7a induced more apoptosis and cytotoxicity than A/Fiji, despite the fact that its infectious virus yields were similar to or less than those for A/Fiji. This indicates a greater capacity of clone 7a to induce the two phenomena. Nevertheless the replication process was clearly essential, since UV-irradiated virus induced little or no apoptosis, and apoptosis occurred as a late event post-infection. The possible relevance of these findings to destruction of host cells by influenza virus in vivo is discussed.
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Alteration of the actin-based cytoskeleton by rabies virus.
More LessWe investigated the effect of rabies virus infection on the actin cytoskeleton using various techniques. Confocal microscopic examination of rabies virus- infected neuroblastoma cells at late stages of infection revealed a dramatic decrease in F-actin staining. The results of a fluorimetric assay with pyrenylated actin indicated that purified rabies virus nucleocapsid has no direct action on the kinetics of actin polymerization and only a weak effect on the final extent of polymerization. Videomicroscopy experiments with purified components showed that rabies virus nucleocapsid inhibits the actin-bundling effect induced by dephospho-syn- apsin I, a neuron-specific protein which is known to exert a control on the actin-based cytoskeleton. Thus, the observed decrease in F-actin staining in infected cells might be ascribed to an indirect action of rabies nucleocapsid on the effects of actin- binding proteins such as synapsin I.
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Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia virus, a fish rhabdovirus.
To evaluate the genetic diversity of viral haemorrhagic septicaemia virus (VHSV), the sequence of the glycoprotein genes (G) of 11 North American and European isolates were determined. Comparison with the G protein of representative members of the family Rhabdoviridae suggested that VHSV was a different virus species from infectious haemorrhagic necrosis virus (IHNV) and Hirame rhabdovirus (HIRRV). At a higher taxonomic level, VHSV, IHNV and HIRRV formed a group which was genetically closest to the genus Lyssavirus. Compared with each other, the G genes of VHSV displayed a dissimilar overall genetic diversity which correlated with differences in geographical origin. The multiple sequence alignment of the complete G protein, showed that the divergent positions were not uniformly distributed along the sequence. A central region (amino acid position 245–300) accumulated substitutions and appeared to be highly variable. The genetic heterogeneity within a single isolate was high, with an apparent internal mutation frequency of 1·2 × 10 3 per nucleotide site, attesting the quasispecies nature of the viral population. The phylogeny separated VHSV strains according to the major geographical area of isolation: genotype I for continental Europe, genotype II for the British Isles, and genotype III for North America. Isolates from continental Europe exhibited the highest genetic variability, with sub-groups correlated partially with the serological classification. Neither neutralizing polyclonal sera, nor monoclonal antibodies, were able to discriminate between the genotypes. The overall structure of the phylogenetic tree suggests that VHSV genetic diversity and evolution fit within the model of random change and positive selection operating on quasispecies.
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Sequence determination and phylogenetic analysis of the Akabane bunyavirus S RNA genome segment
More LessThe nucleotide sequence of the small (S) RNA segment of Akabane (AKA) bunyavirus was determined. The segment is 858 nucleotides long and contains two overlapping open reading frames (ORFs), which encode the nucleocapsid (N) and nonstructural (NSs) proteins, consistent with other bunyaviruses. Comparisons with the Aino virus S RNA sequence indicated that there is 73·5% identity in nucleotide sequence. However, the sequence identity of the 5′ non-coding region of the genomic RNA between these two viruses is only 55%. The N ORFs from 20 Japanese and 2 Australian isolates of AKA virus were sequenced and subjected to phylogenetic analysis. This suggested that AKA virus has evolved in multiple lineages. Twenty-three isolates were grouped into three major clusters, and the cluster which includes recent isolates was subdivided into two branches. Thus, phylogenetic analysis of the AKA virus N protein gene gives a greater insight into bunyavirus evolution.
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Variability of the NS(S) protein among Rift Valley fever virus isolates.
More LessEighteen strains of Rift Valley fever (RVF) virus collected over a period of 38 years and isolated from diverse localities in Africa and from various hosts (human, animal and arthropod) were investigated by RT-PCR followed by sequencing of the NSS protein coding region. This region was chosen to analyse variability because, in contrast to the N protein, the NSS protein differs in various phlebo- viruses and there exists an RVF virus (clone 13) in which 70% of the NSS ORF is deleted, suggesting that this sequence is under a weak selective pressure. Sequence data indicated that percentage di-vergence among isolates ranged from 0 to 9·6% at the nucleotide level and from 0 to 9·5% at the amino acid level. Phylogenetic analysis based on the NSS gene revealed two major lineages: Egyptian and sub-Saharan. This led to the establishment of the relatedness between strains and insights into the NSS protein, the function of which is still undetermined. Alignment of the deduced amino acid sequences indicated that the cysteine residues are conserved, as are several motifs representing potential phosphorylation sites.
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Dinucleotide and stop codon frequencies in single-stranded RNA viruses.
More LessTo identify potential selection pressures which lead to RNA sequence conservation, we examined the occurrence rates of dinucleotides in 64 singlestranded RNA virus genomes. These viruses may offer a particular insight into these pressures since their RNA-dependent RNA polymerases lack proofreading capability. This potentiates introduction of mutations into their genomes, yet unidentified selection processes conserve the genomes to a large degree. We report a strong inverse correlation between the C G content and the occurrence of the CpG dinucleotide (r = 0·71) in the RNA virus genomes, in contrast to earlier reports (Karlin et al., 1994, Journal of Virology 68, 2889–2897). We also detected significant suppression of UpA, correlating inversely with genomic U A content. These sup-pressions are coupled with over-representation of the complementary pair of dinucleotides, CpA and UpG. In addition, we highlight the fact that odds ratios for dinucleotides are not independent variables, a situation apparently not widely appreciated in the literature. This led us to view the over-representation of CpA and UpG as a consequential outcome of UpA and CpG suppression in the virus genomes. Potential factors influencing these disturbances are discussed. In addition, higher than random incidence was observed for ‘out-of-frame’ stop codons in the viral RNA genomes, with some preferences for individual codons being exhibited by certain virus groups. The UAG codon appeared more common in the M1 frame, the UGA in the 1 frame.
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Investigation of population diversity of human immunodeficiency virus type 1 in vivo by nucleotide sequencing and length polymorphism analysis of the V1/V2 hypervariable region of env.
More LessIn this study we have analysed variability in the V1 and V2 regions of human immunodeficiency virus type 1 (HIV-1) proviral sequences amplified from lymphoid tissue, brain and other non-lymphoid tissue collected at autopsy from three HIV-1-infected individuals with giant cell encephalitis. We found no evidence for any tissue-specific grouping of variants in the V1/V2 regions, in contrast to previous comparisons of sequences in the V3 region, but consistent with the existence of evolu- tionarily distinct lineages previously observed in these study subjects by sequence comparisons of the p17gag gene. Examination of inferred amino acid sequences from V1 and V2 revealed no correlations between tissue origin with overall charge, length or number of glycosylation sites. Length polymorphism analysis is a rapid method to compare whole populations of HIV-1 variants within a sample, and provides information on the length and diversity of the V1 and V2 hypervariable regions. Based upon a comparison of 42 individuals with CD4 counts ranging from 802 to < 1 at time of death, we found no evidence for changes in the length of V2 with development of AIDS. Using the number of length variants in the V1 and V2 hypervariable region as a marker of the overall degree of variability within HIV populations, we found no evidence for an increase or a decrease in diversity between those with and without AIDS defining illness.
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Cell lines susceptible to infection are permeabilized by cleaved and solubilized outer layer proteins of rotavirus.
More LessIt has previously been shown that trypsinized triplelayered particles of rotavirus induce destabilization of liposomes and membrane vesicles in the absence of Ca2 , a condition which leads to solubilization of the outer capsid proteins of the virus. In this work, we have studied the relationship between outer capsid solubilization and permeabilization of membrane vesicles, monitoring particle and vesicle size simultaneously by changes in light scattering. Per- meabilization of intact cells induced by solubilized outer capsid proteins was monitored by following the rate of entry of ethidium bromide into the cells. Solubilized outer capsid proteins separated from double-layered particles induced vesicle permeabi- lization. Solubilization of the outer capsid preceded and was required for vesicle or cell permeabiliza- tion. Membrane damage induced by rotaviral outer proteins was not repaired upon addition of 1 mM Ca2 to the medium. Rotavirus infection and cell permeabilization were correlated in six different cell lines tested. This phenomenon might be related to the mechanism of virus entry into the cell. We propose a new model for rotavirus internalization based on the permeabilizing ability of outer capsid proteins and the cycling of trapped calcium in the endosomal compartment.
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Complete nucleotide sequence of Colorado tick fever virus segments M6, S1 and S2.
More LessThe nucleotide sequences of the tenth (M6), eleventh (S1) and twelfth (S2) dsRNA genomic segments of the Florio strain (N-7180) of Colorado tick fever virus were determined and found to be 675, 998 and 1884 bp, respectively, in length. A nonanucleotide motif and a hexanucleotide motif were found to be highly conserved in the 5′ and 3′ non-coding regions (NCRs), respectively, of the three segments. The first and last three nucleotides of each segment were of inverted complementarity, and segment-specific inverted terminal repeats were detected in the NCRs of the three segments. These findings suggest the occurrence of intracellular panhandle structures for the RNA transcripts. A readthrough phenomenon is suspected in segment M6. The environment surrounding the opal codon (position 1052–1054) of segment M6 conforms to that of leaky opal codons described in the literature.
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Resistance to herpes simplex virus type 1 and its latent infection of human T cell lymphotropic virus type I-transformed T cell lines of rabbits.
More LessFourteen T cell lines of rabbits were infected with herpes simplex virus type 1 (HSV-1) and examined for their susceptibility to lytic infection and ability to support virus replication. T cell lines of CD4 8 phenotype were more vulnerable to lysis and supported higher levels of virus replication than those of other phenotypes. Cell lines of CD4 8 and CD4 8 phenotypes continued to proliferate, while remaining productively infected, for more than a month. These latently infected cell lines could be established by treatment with anti-HSV-1 serum and complement. Viral genes were only partially expressed and the CD8 membrane antigen was down-regulated. Infected cell lines, as well as peripheral blood lymphocytes, were shown to induce meningoencephalitis when inoculated intravenously into syngeneic hosts, suggesting a possible role for infected lymphocytes in HSV-1 transport in vivo.
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Induction of apoptosis by herpes simplex virus type 1.
More LessAlthough herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, in the presence of cycloheximide infection induced apoptosis with characteristic morphological changes as well as endonucleosomal DNA cleavage. The induction of apoptosis without de novo protein synthesis suggests that a structural protein of the HSV-1 virion is responsible for the observed apoptosis.
Apoptosis or programmed cell death (PCD) is a type of animal cell death in which cells die by an active cellular process under the control of a genetically encoded cell suicide programme Apoptosis is characterized morphologically by cell shrinkage, plasma-membrane blebbing, chromatin condensation and nuclear fragmentation and biochemically by degradation of chromosomal DNA into oligonucleosome- sized fragments (Kerr & Harmon, 1991) . Although many animal viruses are known to induce an apoptotic response in infected cells (Clem & Miller, 1994; Shen & Shenk, 1995; White & Gooding, 1994), cells infected with wild-type herpes simplex virus type 1 (HSV-1) do not show apoptotic characteristics . However, based on the finding that mutant virus which lacks the γ34.5 gene induces PCD in infected human cells (Chou & Roizman, 1992; Chou et al. , 1994), the lack of apoptosis in the HSV-1-infected cells has been considered to be an outcome of expression of a viral antiapoptosis gene whose product inhibits one of the steps in the virus-induced signalling leading to PCD . Later, HSV-1 was found to carry, as well as the γ34.5 gene, an antiapoptosis gene which can suppress the cellular apoptotic response induced by hyperthermia (Leopardi & Roizman, 1996) or sorbitol treatment (Koyama & Miwa, 1997) .
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Mutations which alter the DNA binding properties of the herpes simplex virus type 1 transactivating protein Vmw175 also affect its ability to support virus replication.
K E Allen and R D EverettThe crucial role of herpes simplex virus type 1 immediate early protein Vmw175 (ICP4) in the regulation of all classes of viral genes has been established by extensive analysis of temperature- sensitive, insertion and deletion mutants. It has long been known that Vmw175 binds to selected DNA sequences, and recent studies have shown that it interacts with components of the basal transcription machinery. However, the role of DNA binding in its mechanism of action has been controversial. Despite the presence of Vmw175 recognition sites at numerous locations throughout the viral genome, it has proved difficult to establish that these sites are important for the activation of early and late promoters. In this study we have analysed the ability of a large number of insertion and single point mutant derivatives of Vmw175 to bind to DNA and to support virus replication. Vmw175 mutants which were unable to bind to DNA were also incapacitated in terms of their ability to activate gene expression in transfection assays and to complement the growth of a Vmw175 deletion mutant virus. These results strongly support the hypothesis that interaction with DNA is an essential feature of the mechanism of action of Vmw175.
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The product of the US10 gene of herpes simplex virus type 1 is a capsid/tegument-associated phosphoprotein which copurifies with the nuclear matrix.
We have identified the herpes simplex virus type 1 (HSV-1) US10 gene product using rabbit polyclonal antisera raised against a recombinant 6xHis-US10 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with 34 and 36 kDa proteins in HSV-1 KOS-infected cells as shown by Western blotting and immunoprecipitation experiments. The 36 kDa protein was immunoprecipi- tated with the US10 antiserum from 32P-labelled lysates of Vero cells infected with HSV-1 KOS, demonstrating that the US10 protein was phos- phorylated. Indirect immunofluorescence studies localized the US10 protein mainly to nuclei as large discrete particles at later times post-infection (p.i.), and nuclear fractionation studies revealed that the protein was tightly associated with the nuclear matrix. Moreover, analysis of isolated intracellular capsids showed that both phosphorylated and unphosphorylated forms of the US10 product were also associated with the capsid/tegument. These results indicate that the US10 gene of HSV-1 encodes a capsid/tegument-associated phospho- protein which copurifies with the nuclear matrix.
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Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection.
More LessTo facilitate the evaluation of vaccines against bovine herpesvirus type 1 (BHV-1), a cotton rat model of intranasal (i.n.) BHV-1 infection was established. Cotton rat lung cells were similar to bovine cells in their ability to support BHV-1 replication in vitro. Furthermore, i.n. inoculation of cotton rats with BHV-1 resulted in pulmonary lesions comparable to BHV-1 infection in cattle. Using this model, the potential of i.n. and gastrointestinal (g.i.) immunization was examined with recombinant human adenoviruses expressing glycoprotein D (gD) of BHV-1 to induce protective immunity against BHV-1.The replication-competent virus (gD-dE3) was more efficient than the replication-defective virus (gD-dE1E3) in inducing gD- specific antibody in the serum and in the respiratory tract. Furthermore, i.n. immunization with gD-dE3 stimulated antigen-specific antibody-secreting cells in the lung 12 weeks following immunization. Protection against BHV-1 challenge correlated with gD-specific antibody levels such that i.n. immunization with gD-dE3 conferred complete protection, while g.i. immunization conferred only partial protection of the lungs of most animals against BHV-1 challenge. In comparison, immunization with gD-dE1E3 by either route resulted in only a partial reduction of BHV-1 titre in the respiratory tract. The results obtained demonstrate that mucosal immunization with replication-competent recombinant adenovirus expressing gD of BHV-1 can induce immunity and protection against BHV-1 challenge.
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Characterization of the assembly and processing of infectious laryngotracheitis virus glycoprotein B.
More LessInfectious laryngotracheitis virus (ILTV) is an alpha- herpesvirus that causes severe upper respiratory infections in chickens. Although ten putative ILTV glycoprotein genes have been identified by sequence analysis, no ILTV glycoprotein has been extensively characterized. In order to delineate the synthesis and processing pathway of ILTV glycoprotein B (gB), rabbit polyclonal antibodies were raised against a Cro-gB- β-galactosidase fusion protein. Through immunoprecipitation analysis of ILTV-infected chicken embryo liver cells it was determined that ILTV gB is initially synthesized as a 110 kDa monomeric precursor protein which rapidly assembles into homodimers composed of 100 kDa subunits. The dimer form of ILTV gB is rapidly cleaved to form two disulphide-linked species of 58 kDa. The apparent reduction in mass (from 110 to 100 kDa) of the mature form of gB during processing in the Golgi apparatus appears to be a common feature of avian herpesvirus gB proteins.
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The human cytomegalovirus UL98 gene encodes the conserved herpesvirus alkaline nuclease
More LessThe human cytomegalovirus (HCMV) UL98 gene is predicted to encode a homologue of the conserved herpesvirus alkaline nuclease. To determine if the HCMV UL98 gene product is functionally homologous to other herpesvirus alkaline nucleases, the HCMV UL98 protein was purified and its activity characterized in vitro. Extracts of HCMV-infected cells were fractionated using Q Sepharose, phos- phocellulose and native DNA cellulose chromatography. UL98 immunoreactivity copurified with alkaline pH-dependent nuclease activity. The purified protein migrated at its predicted size of approximately 65 kDa in denaturing polyacrylamide gels, and displayed nuclease activity in an activity gel assay. Enzyme activity was characterized by a high pH optimum, an absolute requirement for divalent cation, salt sensitivity, and 5′ to 3′ exonuclease activity. DNA digestion resulted in 5′ monophos- phoryl mono- and oligodeoxyribonucleotides. Kinetic analyses revealed a turnover rate of greater than 200 per min, and similar apparent affinity and rate constants on single- and double-stranded DNA. These results indicate that a functional alkaline nuclease activity is conserved among distant members of the herpesvirus family, and are consistent with a highly conserved role in the virus life cycle.
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Cloning and functional characterization of the origin of lytic-phase DNA replication of rat cytomegalovirus
More LessA cis-acting sequence within the rat cytomegalovirus (RCMV) genome (or/Lyt) that directs initiation of lytic-phase DNA replication is identified in this report. RCMV or/Lyt was localized within a 4 3 kb NcoI fragment that is situated immediately upstream of the gene encoding the major DNA-binding protein. The activity of or/Lyt was investigated in a transient replication assay, in which the ability of plasmid constructs to promote DNA replication was tested. Replication of or/Lyt-containing plasmids was autonomous and resulted in the generation of high-molecular-mass concatemers of head-to-tail- linked plasmid oligomers. or/Lyt-mediated replication was found to depend on viral DNA polymerase activity supplied by RCMV infection. The sequence required for or/Lyt function was found to reside within a 3·3 kb HincII-NcoI fragment. The RCMV or/Lyt sequence is highly complex, containing 23 direct repeats (DRs) and 16 inverted repeats (IRs) of lengths greater than 10 bp. Two of the DRs (DR21 and DR22) are exceptionally large, being 80 and 88 bp in length, respectively. In addition, two sequence elements (of 127 and 120 bp) with dyad symmetry were identified within or/Lyt. Although the sequence similarity of RCMV or/Lyt with its human cytomegalovirus counterpart is limited, there is a striking resemblance in the overall organization of several IRs and DRs within both sequences.
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Cooperative interaction between Bcl-2 and Epstein-Barr virus latent membrane protein 1 in the growth transformation of human epithelial cells
More LessThe Epstein-Barr virus latent membrane protein 1 (LMP-1) is essential for virus-induced B cell immortalization and can protect B lymphoma cell lines from apoptosis signals in vitro via induction of cellular Bcl-2 expression. However, we have reported that high-level expression of LMP-1 in epithelial cells (RHEK-1 cells) itself induces apoptosis. This apoptotic event occurs in the absence of detectable Bcl-2 expression in the LMP-1- transfected epithelial cells. In this study, we transfected the bcl-2 gene into the LMP-1-containing cells and examined the effect of Bcl-2 upon LMP-1- mediated apoptosis, and upon the growth phenotype of the transfected cells. The results show that ectopic expression of Bcl-2 specifically blocks apop- totic death induced by LMP-1. This was observed from cell culture viability and from gel electrophoresis and flow cytometric assays of the degree of DNA fragmentation in cultured cells. Furthermore, co-expression of LMP-1 and Bcl-2 in RHEK-1 cells enabled the cells to grow under low-serum conditions and to form colonies in semi-soft agar medium. These results suggest, therefore, that these two proteins play important complementary roles in the process of EBV-associated epithelial cell transformation. It appears significant, therefore, that LMP-1 and Bcl-2 are frequently co-expressed in the malignant cells of an EBV-positive epithelial tumour, nasopharyngeal carcinoma.
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BHRF1, a viral homologue of the Bcl-2 oncogene, is conserved at both the sequence and functional level in different Epstein-Barr virus isolates
More LessBHRF1, a component of the restricted early antigen (EA) complex of the Epstein-Barr virus (EBV) lytic cycle, encodes a 17 kDa putative transmembrane protein with both sequence and functional homology to the Bcl-2 proto-oncogene. To determine whether there was any sequence variation over the BHRF1 open reading frame (ORF), 15 EBV isolates from different geographical regions and from both healthy donors and patients with EBV-associated diseases were sequenced. A small number of base changes which resulted in amino acid substitutions in the BHRF1 protein were found relative to the prototype B95.8 EBV sequence and these were predominantly clustered near the amino terminus of the BHRF1 protein outside conserved domains identified in the Bcl-2 homologues. In transient transfection assays none of the mutations in the BHRF1 ORF from eight different EBV isolates had a
significant effect on BHRF1 protein localization compared to the B95.8 BHRF1 protein. However, transient expression of the adenovirus 12 19K protein or Bcl-2 resulted in localization patterns distinct from that observed with BHRF1 protein. Whilst all eight EBV isolates and E1B-19K gave comparable levels of protection to the DNA-damaging agent c/s-platin, Bcl-2 did not afford significant protection. Thus, despite several amino acid changes in the BHRF1 ORF of some of the EBV isolates studied, the ability of the protein to protect against cis-platin induced apoptosis is conserved. The highly conserved nature of BHRF1 amongst different EBV isolates at both the sequence and functional level supports the proposed important role of BHRF1 in delaying cell death, thereby maximizing the production of progeny virus and facilitating the establishment of virus persistence.
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The role of exogenous p53 and E6 oncoproteins in in vitro transformation by bovine papillomavirus type 4 (BPV-4): significance of the absence of an E6 ORF in the BPV-4 genome
More LessBovine papillomavirus type 4 (BPV-4) does not possess an E6 ORF. The E6 oncoprotein of human papillomavirus (HPV) binds and degrades the tumour suppressor protein p53, thus contributing to tumour progression. Since BPV-4 lacks E6, it is unknown how the virus evades the tumour suppressor properties of p53 in the induction of tumours of the gastrointestinal tract. Mutations in the p53 gene have been detected both in papillomas and carcinomas, suggesting that p53 dysfunction plays a part in these neoplasias. BPV-4 can transform primary foetal bovine cells (PalFs) in cooperation with an activated ras gene, but the transformed cells are neither immortal nor tumori- genic. Co-transfection with the HPV-16 E6 (16E6) ORF confers immortality but not tumorigenicity. To investigate the role of p53 in BPV-4 cell transformation in vitro, we transfected PalFs and p53- null mouse fibroblasts with BPV-4 DNAin combinations with ras, 16E6 ORF and mutant (V143A) p53 cDNA. Transfection of PalFs with BPV-4 DNA, ras and mutant p53 led to cell immortalization, indicating that 16E6 and mutant p53 are functionally equivalent in conferring immortality. However, cotransfection of PalFs with BPV-4 DNA, ras, and both mutant p53 cDNA and 16E6 ORF resulted in cells which were fully transformed to tumorigenicity. In p53-null mouse fibroblasts, BPV-4 DNA induced transformation by itself, but the transformed cells were incapable of suspension growth. The cotransfection of BPV-4 DNA with 16E6ORF produced many more transformed colonies and the cells were capable of growing in suspension. In this system, therefore, 16E6 confers anchorage-independence to BPV-4-transformed cells in a p53-independent fashion.
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Disruption of the human papillomavirus type 16 E2 gene protects cervical carcinoma cells from E2F-induced apoptosis
More LessHuman papillomavirus type 16 (HPV-16) is a DNA tumour virus that has been implicated in the development of cervical cancer. In non-transformed HPV-infected cells, the HPV E2 protein regulates transcription of the viral E6 and E7 oncogenes. Malignant transformation is usually accompanied by disruption of the E2 gene and consequent deregulated expression of E6 and E7. Here we show that re-introduction of the HPV-16 E2 protein into an HPV-16-transformed cervical carcinoma cell line results in a decrease in growth rate and, in the absence of serum growth factors, cell death via apoptosis. E2 expression increases E6/E7 mRNA levels. This brings about an increase in E7 protein levels, which in turn leads to an increase in free E2F, a condition that has previously been shown to induce apoptotic cell death. Despite the increase in E6 mRNA there is no detectable E6 protein in these cells and E2 expression does not reduce the activity of a p53-responsive promoter. Our data suggest that disruption of the E2 gene produces HPV- transformed cells that are less liable to undergo apoptosis and, therefore, more likely to form cervical tumours.
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Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting
More LessRecombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.
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Characterization of the hepatitis B virus major surface antigen promoter hepatocyte nuclear factor 3 binding site
More LessTranscription of the HBV 2·1 kb RNAs is regulated by the major surface antigen promoter. Previously, transient transfection analysis identified regulatory sequence elements in this promoter located between 189 and 1 which govern the level of transcription from this promoter and appear to bind only ubiquitous transcription factors including NF1, Sp1 and NF-Y. However, in vivo transcription analysis in transgenic mice has demonstrated that the expression of the HBV 2·1 kb RNAs is largely restricted to hepatocytes. In this study, the presence of a functional HNF3 transcription factor binding site located between 231 and 240 in the major surface antigen promoter suggests that the in vivo liver-restricted expression of the 2·1 kb RNAs may be governed by this liver-enriched transcription factor. The identification of a functional HNF3 binding site upstream of the DNA polymerase open reading frame also supports the contention that transient transfection analysis may fail to detect all of the cis-acting regulatory sequence elements involved in modulating the level of transcription from the viral promoters.
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Presence of integrated DNA sequences of adeno-associated virus type 2 in four cell lines of human embryonic origin
The human helper virus-dependent parvovirus adeno-associated virus (AAV) has been found in human female genital tissues including material from first trimester miscarriage. In the latter case, AAV type 2 (AAV-2) DNA and viral proteins were detected mainly in the trophoblast cell layer of placenta. In this report, we present evidence that AAV DNA is also present in established human trophoblast cell lines (JEG-3, JAr, BeWo) and in the human amnion cell line FL. In cells of these lines, AAV-2 DNA could be detected both by PCR and Southern blot analysis. Restriction enzyme analysis indicated that AAV DNA was integrated into the host cell genome. Although the cell lines supported AAV replication when infected with AAV-2 and adenovirus type 2 (Ad2) as a helper virus, superinfection with Ad2 alone did not induce replication of AAV DNA, i.e. it failed to rescue AAV from its integrated state. This is probably due to rearrangements within the integrated AAV genome. The presence of AAV DNA in cells derived from human embryonic tissue corroborates the suggestion that human embryonic tissue may be one of the targets of AAV infection.
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The 3′ untranslated region of alfalfa mosaic virus RNA3 contains a core promoter for minus-strand RNA synthesis and an enhancer element
More LessThe 3′ untranslated regions (UTRs) of the three genomic RNAs of alfalfa mosaic virus consist of a 3′ homologous sequence of 145 nt and upstream unique sequences 18–34 nt in length. Mutations were made in the 3′ UTR of a cDNA clone of RNA3. Point mutations in five AUGC motifs which interfere with specific binding of coat protein to the 3′ UTR had no effect on template activity of RNA3 for minus-strand RNA synthesis in vitro by purified viral RNA-dependent RNA polymerase (RdRp). Deletion analysis showed that the 3′ homologous sequence of 145 nt was sufficient for a low level of template activity in the in vitro RdRp assay and a similarly low level of RNA3 accumulation in plants. The presence of an additional sequence of nucleotides 145–165 from the 3′ end of RNA3 enhanced template recognition by RdRp in vitro and accumulation of RNA3 in vivo to wild-type levels.
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Expression of the coat protein of potato virus X from a dicistronic mRNA in transgenic potato plants
More LessTransgenic potato plants were generated which express a dicistronic mRNA corresponding to the 8 kDa protein and coat protein (CP) genes of potato virus X (PVX). Northern blot analysis of RNA isolated from these plants indicated that both the 8 kDa protein and the CP are translated from this dicistronic mRNA. Expression of both of these proteins in transgenic plants was demonstrated by Western blot analysis, and suggests that translation of CP takes place either by initiation by internal entry of ribosomes or by a termination/reinitiation mechanism. CP was detected in protoplasts electroporated with RNA transcripts of the dicistronic construct in the presence or absence of a stable hairpin structure at the 5′ terminus, suggesting that the former model is more likely. Similar results were obtained when a DNA fragment containing the PVX CP leader sequence was placed between two reporter genes in the transient assay system. These results suggest that potexvirus CP expression may take place from the genomic RNA as well as from the subgenomic RNA.
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