- Volume 75, Issue 11, 1994
Volume 75, Issue 11, 1994
- Articles
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- Animal
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Measles Virus Induction of Human Endothelial Cell Tissue Factor Procoagulant Activity in Vitro
Measles virus infection of microvascular endothelium in vivo and ensuing endothelial cell activation may be important in the pathogenesis of subsequent inflammation in target organs. This study investigated the capacity of measles virus to induce procoagulant activity, in vitro, in endothelial cells isolated from human umbilical cord veins. Endothelial cells were infected with a clinical isolate of measles virus propagated in Vero cells. Cells were also incubated with bacterial lipopolysaccharide (10 μg/ml), herpes simplex virus type 1, cytomegalovirus or culture medium alone as positive and negative controls, respectively. Endothelial cell procoagulant activity was measured in a one-stage clotting assay. Measles virus stimulated both a time and dose-dependent endothelial cell procoagulant response by the induction of tissue factor synthesis, confirmed by both immunocytochemistry and its dependence on factor VII for activity. This activity was reduced by u.v.-irradiation of the virus. Infected cells were analysed by double immunofluorescent staining for both tissue factor and measles virus N-protein, and examined using confocal scanning laser microscopy. Cells expressing tissue factor were also positive for the measles virus N-protein. Low levels of interleukin-1 were detected in some viral inocula derived from measles virus-infected Vero cells, however neutralising antibody to interleukin-1 failed to inhibit the endothelial cell procoagulant response to measles virus, whereas it significantly reduced procoagulant activity induced in endothelial cells by recombinant interleukin-1. The capacity of measles virus to induce endothelial tissue factor in vitro, may be relevant to the thrombotic vasculopathy associated with measles virus infection in vivo.
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Extensive Sequence Variation in the Attachment (G) Protein Gene of Avian Pneumovirus: Evidence for Two Distinct Subgroups
More LessThe putative attachment protein of the avian pneumovirus that causes turkey rhinotracheitis is, by analogy with mammalian pneumoviruses, expected to be the major antigenic determinant. We report the nucleotide sequence of the attachment (G) protein genes of five different continental European isolates and compare them with the previously published sequence of the G gene for the focal variant of a U.K. isolate. The nucleotide sequences and the predicted amino acid sequences indicate that there are at least two distinct subgroups, similar to the grouping described for human respiratory syncytial (RS) virus. The U.K. and French isolates form one group and the isolates from Spain, Italy and Hungary form a second. The two subgroups can be easily distinguished on the basis of restriction enzyme digestion of PCR-generated products representing the full-length gene. Within the subgroups the predicted G proteins were highly conserved (98·5 to 99·7% amino acid identity) compared to the levels of identity of RS virus G proteins in the same subgroup (80 to 95%). Between the avian pneumovirus subgroups described here there was an unexpected degree of divergence, the average amino acid identity between members of the two groups being only 38%. This compares with the 53% conservation seen between members of the RS virus subgroups A and B. Comparison of the predicted amino acid sequences showed that the G proteins of members of the two avian pneumovirus subgroups had similar structural features. All proteins had an amino-terminal membrane anchor and the positions of cysteine residues were highly conserved. The potential importance of the high level of variation between the two subgroups in terms of epidemiology of the disease is discussed.
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Dominant Glycoprotein Epitope of Four Corners Hantavirus is Conserved Across a Wide Geographical Area
A newly identified hantavirus, tentatively called Four Corners virus (FCV), was found to be the aetiological agent of a 1993 outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern United States. Immunodominant epitopes of 43 and 31 amino acids were identified in the nucleocapsid protein and G1 glycoprotein, respectively. The G1 genes of different hantaviruses are highly divergent, suggesting that geographically diverse FCVs might fail to cross-react owing to antigenic drift. We now show that the immunodominant epitope of G1 is conserved among 18 FCVs from a broad geographical area, despite extensive nucleotide sequence heterogeneity. Antibodies from all 45 HPS patients, separated by more than 3000 km were shown to be reactive with the dominant G1 epitope. Evidence for limited cross-reactivity between the G1 antigen of a novel hantavirus of the cotton rat and that of FCV is presented.
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In Vivo Interaction of Rabies Virus Phosphoprotein (P) and Nucleoprotein (N): Existence of Two N-binding Sites on P Protein
More LessThe rabies virus phosphoprotein (P) and nucleoprotein (N) are involved in transcription and replication of the viral genome. Interaction between N and P was studied in vivo in transfected cells expressing both proteins. Co-immunoprecipitation assays revealed that the N-P complex is present in cells expressing both proteins as well as in infected cells. Furthermore, immunostaining showed that coexpression of N and P was sufficient to induce the formation of cytoplasmic inclusions similar to those found in infected cells. In addition, deletion mutant analysis of P was performed to identify the regions of P interacting with N. The results indicate that at least two independent N-binding sites exist on P protein: one is located in the carboxy-terminal part of the protein and another between amino acids 69 and 177. The formation of cytoplasmic inclusions seems to require the presence of both N-binding sites on P protein.
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Structural Protein Relationships Among Eastern Equine Encephalitis Viruses
More LessWe have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the E1, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidly-migrating E2-associated protein and a high M r (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The protein profiles of the 33 North American (NA)-serotype viruses examined were remarkably homogeneous, with variation detected only in the E1 protein of two isolates. In contrast, considerable heterogeneity was observed in the migration profiles of both the E1 and E2 glycoproteins of the 13 South American (SA)-type viruses examined. Peptide mapping of individual virion proteins using limited proteolysis with Staphylococcus aureus V8 protease confirmed that, in addition to the homogeneity evident among NA-type viruses and relative heterogeneity among SA-type viruses, the E1 and E2 proteins of NA-and SA-serotype viruses exhibited serotype-specific structural variation. The C protein was highly conserved among isolates of both virus serotypes. Endoglycosidase analyses of intact virions did not reveal substantial glycosylation differences between the glycoproteins of NA- and SA-serotype viruses. Both the HMW protein and the E2 protein (doublet) of EEE virus appeared to contain, at least in part, high-mannose type N-linked oligosaccharides. No evidence of O-linked glycans was found on either the E1 or the E2 glycoprotein. Despite the observed structural differences between proteins of NA- and SA-type viruses, Western blot analyses utilizing polyclonal antibodies indicated that immunoreactive epitopes appeared to be conserved.
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Persistence of Replicating Coxsackievirus B3 in the Athymic Murine Heart is Associated with Development of Myocarditic Lesions
Coxsackievirus B3 (CVB3)-induced myocarditis was studied in euthymic (nu/+) and athymic (nu/nu) C3H/ HeN (H-2k) mice. Mice were inoculated intra-peritoneally with 106 p.f.u. of CVB3 (Nancy strain) and sacrificed at intervals up to 92 days post-inoculation (p.i.). Viraemia peaked at day 2 to 3 p.i. and ceased at day 5 to 7 p.i. in a synchronized manner in both sets of mice. Very few infectious particles were detected in the blood of nu/nu mice after day 14 p.i. In nu/nu mice, CVB3 persisted in myocardial tissue with constant titres between 2·7 ± 1·9 × 104 and 7·6 ± 5·2 × 104 p.f.u./mg from day 3 to 92 p.i., which were comparable to those of nu/+ mice in the acute phase. In nu/+ mice, the virus was recovered from all animals examined by day 11 p.i. and from three out of 13 mice between days 14 and 21 p.i., yet no virus was recovered from nu/+ mice at day 42 p.i. In nu/nu mice, sense and antisense RNA for CVB3 was detected in the myocardial tissue up to day 42 p.i. by in situ hybridization and up to day 92 p.i. by reverse transcriptase-PCR. Neither sense nor antisense RNA was detected after day 21 p.i. in nu/+ mice with the same techniques. Myocardial tissue damage was analysed morphologically. At day 92 p.i., the area of myocardial injury peaked at 23% of the section in nu/nu mice. In contrast, less than 0·6% of tissue sections contained lesions in nu/+ mice. A neutralizing antibody response to CVB3 was observed in both nu/nu and nu/+ mice. The mean titre of neutralizing antibody was significantly higher at day 21 p.i. in nu/+ mice, but similar at day 42 p.i. with nu/nu and nu/+ mice. Perforin-producing natural killer-like cells, which are considered to play an important role in causing acute myocarditic lesions in immunocompetent mice, were found in the lesions of nu/nu mice persistently infected with CVB3. Prolonged tumour necrosis factor-α mRNA synthesis detected in nu/nu mice appears to reflect the continuous activation of macrophages, which extend phagocytic reactions to virus-infected myocytes. These immunological results suggested that the host immune response devoid of antigen-specific T cell function is not sufficient to terminate CVB3 infection in nu/nu mice. Also, it appears that competent cellular immunity, on the whole, plays a role in curing rather than in aggravating myocarditis in nu/+ mice. In conclusion, our results indicate that the presence of replicating CVB3 is an important factor in the development of myocarditic lesions in C3H/HeN mice.
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Identification of Mengo Virus T Helper Cell Epitopes
More LessTo identify Mengo virus-specific T cell epitopes in mice (the natural host for the virus), lymph node cells were obtained from BALB/c (H-2d ) mice, previously immunized with u.v.-inactivated virus, and stimulated in vitro with each of 116 overlapping peptides (10 to 18 residues long) covering the entire capsid coding region (834 amino acids). T cell epitopes were defined on the basis of specific peptide-induced lymphocyte proliferation. Where proliferation occurred, immunological characterization showed that it was the CD4+ T helper (Th) cell subpopulation that was responsible for the Mengo virus-specific response. Surprisingly, no Mengo virus Th cell epitopes were found in capsid protein VP1 or VP4. Six peptides in VP2 (residues 1 to 15, 99 to 108, 118 to 132, 133 to 147, 227 to 236 and 247 to 256) identified the positions of separate Th cell epitopes, and two overlapping peptides (residues 173 to 182 and 178 to 192) defined an additional Th cell immunogenic sequence. Three individual peptides in VP3 (residues 46 to 58, 136 to 150 and 198 to 212) and two overlapping peptides (residues 1 to 15 and 11 to 20) also represent Th cell epitopes. Similar assays with C57BL/6 (H-2b ) and SJL/J (H-2s ) mice showed that the pattern of recognition of these peptides was H-2 restricted. Each of the previously identified sites of B cell antigenicity in VP2 and VP3 are associated with one Th epitope. Comparison of the experimentally determined Th epitopes with potential T cell epitopes identified by several predictive strategies revealed only a low correlation between authentic and predicted epitopes.
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T cell-stimulatory Fragments of Foot-and-mouth Disease Virus Released by Mild Treatment With Cathepsin D
Cathepsin D and cathepsin B are endosomal/lysosomal proteases that are thought to play a role during in vivo antigen processing, releasing fragments for binding to major histocompatibility complex class II products and subsequent presentation to T cells. Here we treated purified foot-and-mouth disease virus (FMDV) strain A10Holland with both enzymes. Cathepsin D, but not cathepsin B, was shown to release fragments from reduced or non-reduced FMDV under mild conditions in vitro. Twenty-eight predominant cathepsin D-released fragments were purified by HPLC and identified by amino acid composition analysis and sequencing. The unseparated set of fragments produced (the digest) was able to stimulate T cells from eight vaccinated cattle. With respect to the response to intact virus the extent of the response to the digest differed between animals: four animals could be classified as good responders, three as intermediate responders and one as a low responder. Subsequently, we investigated the proliferative T cell response to a large set of synthetic peptides in detail for two animals, one belonging to the group of good responders, the other being the low responder. The peptides covered all 28 cathepsin D-released fragments analysed and also several sequences not recovered from the digest. In this way seven T cell sites could be identified, five of which coincided with cathepsin D-released fragments. The other two T cell sites were VP2[54–72], being a homologue of a T cell site identified for FMDV strain O1K and the N terminus of VP4. Whether the most dominantly recognized T cell site was recovered from the digest or not was shown to be related to the good or low response to the digest. These findings suggest a role for cathepsin D in the release of some but not all T cell-stimulatory fragments from FMDV.
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Transmissible Mink Encephalopathy Species Barrier Effect Between Ferret and Mink: PrP Gene and Protein Analysis
More LessExperimental infection of transmissible mink encephalopathy (TME) in two closely related mustelids, black ferret (Mustela putorius furo) and mink (Mustela visa), revealed differences in their susceptibility to the TME agent. When challenged with the Stetsonville TME agent, a longer incubation period was observed in ferrets (28 to 38 months) than mink (4 months). Western blot analysis of ferret and mink prion proteins (PrP) demonstrated no detectable differences between the proteins. Northern blot analysis of ferret brain RNA indicated that PrP mRNA abundance is similar in infected and uninfected individuals. We amplified the PrP coding region from ferret DNA using the polymerase chain reaction and compared the deduced amino acid sequence of the ferret PrP gene with the mink PrP gene. This comparison revealed six silent base changes and two amino acid changes between mink and ferret: Phe → Lys at codon 179 and Arg → Gin at codon 224, respectively. These changes may indicate the region of PrP that is responsible for the species barrier effect between mink and ferret.
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Localization of Viral protein X in Simian Immunodeficiency Virus Macaque Strain and Analysis of its Packaging Requirements
More LessSimian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) encode the accessory viral protein X (Vpx) known to be incorporated into virions in amounts comparable to those of the Gag proteins. The localization of Vpx within SIVmac-infected HUT-78 cells and SIVmac virions was studied by immunoelectron microscopy. Vpx appeared to be associated with extracellular virions as well as budding viral particles at the surface of infected cells. Immunolabelling of purified viral cores suggested that Vpx was a component of the amorphous material surrounding the core structure. Furthermore, a detergent insoluble fraction containing SIV core proteins was devoid of Vpx. To investigate the protein requirement for packaging of Vpx, BHK-21 cells were co-infected with vaccinia virus recombinants encoding Vpx and other SIV proteins able to assemble into virus-like particles. Analysis by immunoprecipitation of the extracellular particulate material as well as immunoelectron microscopy demonstrated that co-expression of Vpx with the Pr56 gag polyprotein was sufficient for the formation of pseudo-virions containing Vpx. Virus-like particles that appeared upon expression of p16 gag did not contain Vpx. The results suggest that Vpx is packaged into viral particles through its binding to the Gag polyprotein. The precise positioning of Vpx within the space separating the viral envelope from the core structure is postulated to result from the reorganization of viral proteins that occurs upon Gag polyprotein cleavage and budding.
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Extensive C-terminal Deletion in Human Immunodeficiency Virus Type 1 Env Glycoprotein Arising After Long-term Culture of Chronically Infected Cells
More LessHuman immunodeficiency virus type 1 (HIV-1) chronically infected (Cl) cell lines were established from HIV- lIIIB/LAIinfected MT-4 cells that survived acute infection. The HIV env gene expressed in the two longterm cultured cell lines differed from that of the lines cultured for shorter periods, by coding for a glycoprotein gp160 that had the C terminus deleted. One long-term cultured cell line, Cl-17, was studied in detail. An insertion of a premature stop codon in the env gene caused about 90% of gp160 molecules to be truncated (gp160×), lacking both cytoplasmic and transmembrane domains; these species were secreted into the cell medium, and could form oligomers with other truncated gp160 molecules as well as with their normal counterparts. Cl-17 cells constantly yielded high levels of viral protein and relatively low quantities of infectious virus, without cytopathicity. However, acute infection of fresh MT-4 cells with CI-17-derived virus led to cytopathicity, the rate of which as well as the Env glycoprotein pattern depended on multiplicity: (i) using an infection dose of 10−4 ID50/cell, cells died 7 to 8 days post-infection with normal gp160 synthesis predominating; (ii) with 10−2 ID60, gp160× was produced as early as 48 h postinfection and cell death was delayed. Predominant gp160× formation occurred again when new Cl cell lines were obtained with CI-17-derived virus. Thus, two human immunodeficiency virus variants, a normal and a defective one, are persistently expressed in CI-17 cells. The other long-term cultured Cl cell line also expressed gp160 with a similar (albeit slightly longer) deletion of a C-terminal region in most molecules, but the cell lines that were cultured for shorter periods did not. These results suggest that the emergence of HIV variants with a C-terminal deletion in the Env glycoprotein, which coexist with normal virus, may play a role in maintaining the long-term growth capacity and viability of Cl cells.
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Inhibition of Protein Synthesis by the Human Immunodeficiency Virus Type 1 nef Gene Product
More LessDuring productive infection of human T lymphocytes in cell culture, the expression of human immunodeficiency virus type 1 is temporally regulated by virus-encoded regulatory proteins. Among these Nef, whose function has not been clearly elucidated, is thought to alter CD4+ T cells. We examined the possibility that the nef gene interferes with the translation process in a cell-free system. The results demonstrate that the nef gene product mediates an inhibitory effect on protein synthesis. Conversely, the use of antisense nef mRNA did not affect translation. Further observations suggest that this inhibitory effect is an inherent property of the nef gene product itself and not of its mRNA. The data show that the translational repression directed by Nef is a general phenomenon, acting on its own and on other messengers used as reporter mRNAs. We propose that, as a consequence, Nef can play an important role in the pathogenesis of AIDS.
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Comparative Morphology of Gag Protein Structures Produced by Mutants of the gag Gene of Human Immunodeficiency Virus Type 1
More LessSix mutants that differ in the extent of their carboxy- terminal sequences and two deletion mutants of the gag gene of HIV-1 have been characterized morphologically following their expression in Spodoptera Jrugiperda cells using recombinant baculoviruses. Electron microscopy has revealed distinct morphological forms of the Gag protein that can be classified as either (i) particulate, three-dimensional, spherical or tubular shells or (ii) nonparticulate, two-dimensional, flat, curved or convoluted sheets. Progressive truncation of the carboxy terminus of Gag was accompanied by changes in the morphology and formation of spherical particles from predominantly C-type assembly and budding at the plasma membrane, through B-type intracytoplasmic assembly, to A-type assembly with budding mainly into cytoplasmic vacuoles. Deletions within the Pr24 CA domain of Gag abolished particle formation but retained association of the protein with the plasma membrane. All of the observed morphologies of the mutant Gag proteins could be accommodated within an icosahedral model for the organization of spherical particles and a basic hexagonal arrangement of assembled Gag protein monomers.
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The pk-1 Gene of Autographa Californica Multinucleocapsid Nuclear Polyhedrosis Virus Encodes a Protein Kinase
More LessOpen reading frame (ORF) 9 of the EcoBA I fragment of the baculovirus Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) genome encodes a protein, PK-1, which has strong similarity to serine-threonine protein kinases. The published sequence of the pk-1 gene contains an error that, when corrected, extends the ORF by 228 nucleotides. Transcription of pk-1 was first detectable by primer extension at 12 h post-infection, accumulating to high levels during the very late phase of infection. PK-1 produced in rabbit reticulocyte lysates was able to phosphorylate histone HI. Together, our results suggest that ORF 9 encodes a protein kinase that is expressed during the beginning of the late and throughout the very late phases of AcMNPV infection.
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Microplitis Demolitor Polydnavirus Infects and Expresses in Specific Morphotypes of Pseudoplusia Includens Haemocytes
More LessMicroplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. M. demolitor eggs are never encapsulated by host hae- mocytes when coinfected with its associated polydnavirus (MdPDV) whereas eggs are encapsulated within 36 h when injected into hosts without virus. In this study, infection of specific classes of P. includens haemocytes by MdPDY was examined. Electron microscopic studies indicated that MdPDV entered all haemocyte morphotypes. Northern blot analysis revealed that similar size classes of viral mRNAs were produced in granular cells, plasmatocytes and spherule cells. Expression of a 1·6 kb MdPDV mRNA in haemocytes from parasitized hosts was detectable by in situ hybridization at 2 h post-parasitism (p.p.) and continued through until day 6 p.p. By 12 h p.p., viral expression was detected in greater than 80% of the haemocytes in circulation but thereafter the percentage of haemocytes exhibiting a hybridization signal declined. Similar patterns were observed in haemocytes from larvae injected with calyx fluid or MdPDV plus venom. Granular cells and plasmatocytes from unparasitized larvae were purified on Percoll cushions and maintained in vitro. Both morphotypes were successfully infected with MdPDV and exhibited changes in morphology and adhesiveness very similar to cells from parasitized hosts. Cell-free plasma from parasitized larvae had a variable effect on haemocyte adhesion. Haemocytes cultured in plasma from 1 or 4 day p.p. larvae rapidly spread whereas cells cultured in 7 day p.p. plasma did not. Reciprocally, adhesion of haemocytes from parasitized larvae could not be rescued by cell-free plasma from unparasitized larvae. Together, these data suggest that disruption of the host encapsulation response is mediated primarily by direct infection of granular cells and plasmatocytes by MdPDV.
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The Asialoglycoprotein Receptor Mediates Hepatic Binding and Uptake of Natural Hepatitis B Virus Particles Derived From Viraemic Carriers
As a putative mechanism of hepatitis B virus (HBV) uptake into hepatocytes the interaction between HBV and the hepatic, human-derived asialoglycoprotein receptor (ASGPR) was investigated. Sera from patients with different variations of hepatitis B surface antigen-(HBsAg) positive chronic hepatitis, HBV particles isolated from HBV carriers with high-titre viraemia and commercial HBsAg served as sources of HBV. ASGPR was affinity-purified from human liver. HBV that had bound to isolated ASGPR was either detected by radio-immunoassay using solid-phase bound ASGPR or enzyme immunoassay with biotin-ASGPR bound to immobilized HBV. Furthermore, binding and uptake of purified, 125I-labelled HBV particles into human hepatoma cell lines (HepG2 and HuH7), which constitutively express functional ASGPR molecules, were compared to that of ASGPR-negative COS cells. As a result HBV was found to bind to purified human ASGPR in two different assays. Circulating virus particles from sera with high titre viraemia showed the highest attachment activity to ASGPR. HBV binding to purified ASGPR was saturable and inhibitable by an excess of d-galactose-bearing ligands, by EDTA and anti-receptor immunoglobulin. Lysis of particles by adding detergent abolished immunodetectable HBV binding to purified ASGPR. Commercial HBsAg did not adhere to solid phase-immobilized ASGPR. Monoclonal anti-preS1 antibody (MA18/7) but not anti-preS2 antibody (Q19/10) inhibited virus attachment. Purified and radiolabelled HBV particles showed binding to HepG2 and HuH7 cells but to much lesser degree to COS cells. Cellular binding of HBV was significantly inhibited by blocking of ASGPR function. Both ASGPR ligands and rabbit anti-ASGPR immunoglobulin but not non-immune rabbit serum inhibited uptake of radiolabelled HBV particles into HepG2 cells or HuH7 cells, respectively. This study suggests that HBV virions may enter human hepatocytes via ASGPR molecules by attachment of viral preS1-related envelope binding sites to this receptor.
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Biogenesis of the Hepatitis B Viral Middle (M) Surface Protein in a Human Hepatoma Cell Line: Demonstration of an Alternative Secretion Pathway
More LessIn the serum of hepatitis B virus (HBV)-infected patients, two different types of particles, a 42 nm virion and a 22 nm subviral particle, were identified. The envelope of both particles is composed of three proteins, the large (L), middle (M), and major/small (S) surface proteins but the ratio between these components varies in each. The M protein appears in a lesser amount than the S protein in both virion and subviral particles, although it is translated from the same subgenomic RNA, and this is due to its poor initiation context of translation. In addition, only the glycosylated form of M protein is secreted in contrast to both glycosylated and unglycosylated forms of L and S proteins that are secreted. To investigate the biogenesis of M protein, human hepatoma cells transfected with plasmids containing a mutated HBV DNA were used to produce a high amount of M protein. Electron microscopic observation revealed that despite a higher proportion of the M protein being found in the transfected cells, the secreted surface antigen particles possess similar size and density to 22 nm subviral particles. Detailed biochemical analyses showed the following. (1) The unglycosylated M protein was predominantly present in the microsomal fraction but not present in any other subcellular fractions. (2) The M protein formed 22-nm-like particles in the endoplasmic reticulum (ER) and was retained in the post-ER or pre- Golgi regions. (3) In addition to the complex glycosylated form of M protein, a high-mannose form of M protein could be secreted. (4) Normally, no unglycosylated M protein was secreted. However, glycosylation was not essential for M protein secretion since M protein deprived of glycosylation by tunicamycin treatment was detected in the medium. These findings suggest that (i) the M protein was probably translated and co-translocated into the ER and at least one site was glycosylated before leaving the ER resulting in no secretion of unglycosylated M protein, and (ii) the M protein had two secretion pathways, one through the conventional pathway and the other probably directly through the ER.
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Internal Ribosome Entry in the Coding Region of Murine Hepatitis Virus mRNA 5
More LessThe unique region of murine hepatitis virus (MHV) mRNA 5 has two open reading frames, ORF 5a and ORF 5b, that encode small proteins of unknown function. In the experiments described here, we have used the in vitro translation of synthetic mRNAs to examine the expression of these ORFs. Our results show that a synthetic mRNA containing both ORFs is functionally bicistronic. More importantly, the expression of ORF 5b, but not ORF 5a, is maintained in a trieistronic mRNA containing an additional 5′-proximal ORF. Thus, in the context of the MHV mRNA 5 unique region, the initiation of protein synthesis on ORF 5b can occur independently of ribosomes that enter from the 5′ end of the mRNA. We conclude that the translation of ORF 5b is mediated by the internal entry of ribosomes.
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An Element Binding a C/EBP-related Transcription Factor Contributes to Negative Regulation of the Bovine Papillomavirus Type 4 Long Control Region
More LessDeletion of the NR2 element of the long control region (LCR) of bovine papillomavirus type 4 (BPV-4) was observed previously to lead to a fivefold increase in enhancer activity of a subfragment of the LCR. Further characterization of this element indicates that mutations in NR2 lead to increased enhancer activity in both mouse CT3 fibroblasts and in a transformed bovine epithelial cell line derived from an alimentary canal papilloma/m situ carcinoma, but not in primary bovine keratinocytes. Since similar oligonucleotide-nuclear factor complexes were obtained in electrophoretic mobility shift assays (EMSA) for all three cell types, the observed difference in negative activity may result from variation in the NR2-binding factor itself between primary and established/transformed cell lines, or from the involvement of other factors that vary between the lines. Characterization of the NR2-binding factor by heat stability and antibody supershifts in EMSA indicate that the factor is related to the CCAAT/enhancer- binding protein (C/EBP) family, and that one component of the complexes may be C/EBP/β.
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Cervical/vaginal dysplasias of Transgenic Mice Harbouring Human Papillomavirus Type 16 E6-E7 Genes
More LessHalf of the female transgenic mice harbouring human papillomavirus type 16 (HPV-16) E6-E7 genes under control of the mouse mammary tumour virus promoter, developed malignant tumours, including salivary gland carcinomas, lymphomas and skin histiocytomas. Although the E6-E7 genes are aetiological factors for human anogenital carcinoma, the transgenic mice produced no tumours in the anogenital tract. We investigated cytological and histological changes in the anogenital tract of the same transgenic mice. Seventeen (77 %) of 22 transgenic mice developed dysplastic and/or hyperplastic changes in the cervix and vagina. HPV-16 E6-E7 mRNA signals were observed in the genital lesions, while they were not detected in the normal cervices and vaginas of transgenic mice and control mice by RNA in situ hybridization analysis. RNA/PCR analysis using poly(A)+ RNA showed that only a full- length E6-E7 RNA was expressed in three scraped cell samples from dysplasia, whereas full-length and spliced E6-E7 transcripts were in three cell samples from dysplasia/hyperplasia. These results suggest that expression ofboth E6 and E7 genes of HPV-16 is important for inducing dysplastic and hyperplastic changes in the genital epithelium.
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DNA-binding Studies of the Epstein-Barr Virus Nuclear Antigen 2 (EBNA-2): Evidence for Complex Formation by Latent Membrane Protein Gene Promoter-binding Proteins in EBNA-2-positive Cell Lines
The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA- 2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Transactivation of the TP1 promoter and of the BamHl C promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates transactivation. To determine whether EBNA-2 also trans- activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides −135 to − 176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein- protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher Mr in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.
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The Human Cytomegalovirus UL100 Gene Encodes the gC-II glycoproteins Recognized by Group 2 Monoclonal Antibodies
More LessIn human cytomegalovirus (HCMV) the envelope glycoprotein complexes designated gC-II contain two immunologically and biochemically distinct glycoproteins. Monoclonal antibodies (MAbs) recognizing the gC-II glycoproteins have been divided into two groups based on the M r of the glycoproteins they recognize. We have now identified the HCMV UL100 gene as the gene encoding the gC-II glycoprotein recognized by the Group 2 MAbs. To do this, gC-II complexes were immunoaffinity purified and cleaved with cyanogen bromide (CNBr). CNBr peptides were separated by reverse phase high performance liquid chromatography (RPHPLC). Amino acid sequences which matched sequences found in the protein encoded by the HCMV UL100 gene were obtained from three purified peptides. To confirm the assignment we made synthetic peptides using amino acid sequence from the carboxyl terminus of the protein encoded by the UL100 gene. These peptides were used to make murine antibodies. The anti-UL100 antibodies immunoprecipitated gC-II complexes and were reactive with gC-II glycoproteins recognized by Group 2 MAbs in Western blotting. Several overlapping UL100 fusion proteins were expressed in E. coli. Only one of these fusion proteins was recognized by gC-II Group 2 MAbs. None of these UL100 fusion proteins were recognized by gC-II Group 1 MAbs. These data showed that the UL100 gene encoded the gC-II glycoprotein recognized by the Group 2 MAbs and that the epitope recognized by these antibodies was located between amino acids 315 to 372 at the carboxyl terminus.
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Absence of Varicella-zoster Virus (VZV) Glycoprotein V Does not Alter Growth of VZV in vitro or Sensitivity to Heparin
More LessVaricella-zoster virus (VZV) encodes at least five glycoproteins, gpl to gpV. VZV gpV, M r 100K to 110K, is the product of VZV open reading frame (ORF) 14. VZV gpV is homologous to herpes simplex virus gC and pseudorabies virus gill. To determine whether gpV is required for viral replication, we inserted a stop codon after the fifteenth codon of the ORF 14 gene in a cosmid containing the gene. Transfection of human melanoma cells with the cosmid containing the mutant ORF14 gene and three other cosmids resulted in the production of infectious VZV. Immunoprecipitation indicated that the mutant virus did not express gpV. VZV that did not express gpV grew at the same rate as parental virus and was inhibited by heparin to a similar extent. The pattern of inhibition by heparin of the gpV mutant was similar to that reported for a herpes simplex virus mutant that does not contain gC, but different from that described for a pseudorabies virus mutant devoid of gill. These results indicate that VZV gpV is not required for viral replication in vitro.
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Glycoprotein gE-negative Pseudorabies Virus has a Reduced Capability to Infect Second- and third-order Neurons of the Olfactory and Trigeminal Routes in the Porcine Central Nervous System
We investigated the spread of glycoprotein gE (gE)- negative pseudorabies virus (PRV) and its rescued ‘wild- type’ strain into and within the central nervous system (CNS) of 3- and 10-week-old pigs. This is the first study that demonstrates PRV invasion of the porcine CNS via the synaptically linked neurons of the olfactory and trigeminal routes and that demonstrates the role of gE in this invasion. After intranasal inoculation with high doses of virus, gE-negative PRV replicated less efficiently in peripheral tissues. The titres of the gE-negative virus in the oropharyngeal mucosa, olfactory epithelium, draining lymph nodes and trigeminal ganglion were approximately 100-fold lower in 3-week-old pigs and 10fold lower in 10-week-old pigs than titres of the ‘wild- type’ virus. In contrast to the ‘wild-type’ virus, titres of the gE-negative virus were very low or undetectable in the olfactory bulb, brain stem and other tissues of the CNS. Viral antigen of rescued ‘wild-type’ PRV and of gE-negative PRV was detected immunohistochemically in the olfactory epithelium and in neurons of the trigeminal ganglion, and also in the olfactory and trigeminal axons leading towards the CNS. But, in contrast to ‘wild-type’ virus, no viral antigen of the gEnegative virus was detected in second- or third-order neurons in the olfactory bulb or in the brain stem. We conclude that gE-negative PRV can infect first-order neurons of the olfactory and trigeminal routes and is able to spread via their axons towards the CNS. Yet, gEnegative PRV has a greatly reduced capacity to infect second- or third-order neurons. Finally, we report lateral spread of ‘wild-type ’ PRV in the trigeminal ganglion, i.e. nonsynaptic transport from neuron to neuron. Possible mechanisms that could explain the reduced levels of the gE-negative virus in the CNS are discussed.
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The Gene Downstream of the gC Homologue in Feline Herpes Virus Type 1 is Involved in the Expression of Virulence
Feline herpesvirus type 1 (FHV-1) mutants were constructed, carrying a β-galactosidase marker gene integrated into the region downstream of the gene encoding the homologue of glycoprotein C (gC) of herpes simplex virus type 1. In cell culture, no differences in replication were observed between mutants and the parent FHV-1 strain. However, in experimentally infected cats, mutants caused fewer clinical signs after oronasal administration although they replicated to the same extent as the parental strain. Sequence analysis in the region of the UL segment surrounding the insertion site revealed an open reading frame (ORF 2) encoding a putative polypeptide of 2IK. RNA analysis indicated a corresponding transcript of 0·8 kb that was detected late after infection of cells in culture. This particular UL locus downstream of the gC gene has not been thoroughly investigated in any of the herpesviruses. The putative gene product showed only limited evolutionary conservation since similarity could be found only with the assumed homologue of equine herpesvirus type 1. Further characterization of this newly identified FHV-1 gene involved in virulence may provide insight into the development of disease owing to herpesvirus infection.
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Identification and Characterization of the Protein Product of Gene 71 in Equine Herpesvirus 1
More LessEquine herpesvirus 1 (EHV-1) strain Ab4 gene 71 is predicted to encode a primary product with a M r of 80·1K. We have previously constructed a deletion/lacZ insertion mutant, ED71, and demonstrated that gene 71 is dispensable for growth of virus in cell culture. We have now constructed a gene 71 revertant, Re71. To identify and characterize the product of gene 71, we produced a specific antiserum, anti-71, against a β-galactosidase fusion protein containing the carboxy terminus of the gene 71 polypeptide. Using the anti-71 serum, mutant ED71 and the revertant Re71, we have demonstrated that gene 71 encodes a 192K polypeptide. Experiments with glycosylation inhibitors revealed that the protein product of gene 71 is N-glycosylated and heavily O-glycosylated. When the 192K polypeptide is synthesized in the presence of monensin, the M r of the polypeptide is reduced to 80K, the predicted unmodified M r of the gene 71 polypeptide. The gene 71 product is found in virions and L particles in a fully processed form that runs as a diffuse band in electrophoresis, with a M r in excess of 200K. Immunofluorescence and virion surface labelling experiments showed that the polypeptide product of gene 71 is located on cellular membranes and the virion envelope. A time course of infection confirmed that gene 71 is regulated as a leaky late gene in infected cells. Finally, using wild-type EHV-1 Ab4, mutant ED71, revertant Re71 and two antibodies (P19 against EHV-1 glycoprotein gp300, and anti-71) we conclusively demonstrated that gene 71 encodes gp300. This contradicts published results with P19 alone, which indicated gp300 was the product of EHV-1 gene 28.
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Role of the Carboxy Terminus of Herpes Simplex Virus Type 1 DNA Polymerase in its Interaction With UL42
More LessSeveral recent reports implicate sequences at or near the C terminus of the catalytic subunit (POL) of herpes simplex virus type 1 (HSV-1) DNA polymerase in its interaction with the accessory protein UL42. We have investigated further the involvement of this region by three different approaches: anti-idiotype antibodies, a competition ELISA and inhibition of the interaction by peptides. Antibodies raised in rabbits to peptides corresponding to regions of POL all reacted in Western blots with POL. Surprisingly, the sera raised against C-terminal peptides (amino acids 1221 to 1235 and 1224 to 1235) also reacted with UL42. The UL42 reactivity was shown to be due to the presence of anti-idiotype antibodies, providing direct evidence for complementarity of the structure of the extreme C terminus of POL to a region of UL42. To measure the contribution of the C terminus of POL to UL42 binding we developed a competition ELISA using POL, a truncated polymerase lacking the carboxyl-terminal 27 amino acids (POLdl) and UL42. UL42 binding to immobilized POL was inhibited approximately four times more effectively by competition, in solution, with POL than with POLdl, indicating that the C-terminal 27 amino acids of POL are responsible for at least 75 % of the binding energy. A peptide corresponding to these 27 amino acids (residues 1209 to 1235) inhibited both the POL-UL42 interaction and the stimulation of POL by UL42 and did so more effectively than peptides corresponding to amino acids just away from the C terminus (residues 1195 to 1223 and 1177 to 1195).
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Nucleotide Sequence of the Polyhedrin Gene Region of Helicoverpa zea Single Nucleocapsid Nuclear Polyhedrosis Virus: Placement of the Virus in Lepidopteran Nuclear Polyhedrosis Virus Group II
More LessThe polyhedrin gene (polh) of Helicoverpa zea single nucleocapsid nuclear polyhedrosis virus (HzSNPV) was identified and shown by sequence analysis of the iscoRI I genomic fragment to encode a 246 amino acid polypeptide that has greater than 80 % sequence identity to known polyhedrins. It is preceded by an AT-rich region containing the conserved late promoter motif TAAG, which was identified as a transcription start point. Downstream ofpolh there were several similarities in genome arrangement to other nuclear polyhedrosis viruses (NPVs). These include open reading frame (ORF) 8, immediately downstream of polh, encoding a 412 amino acid protein with multiple tandem proline residues, which is homologous to ORF8 (ORF1629) of Autographa califomica multiple nucleocapsid NPV. Phylogenetic analysis of the polh gene region shows that HzSNPV is a member of the previously described lepidopteran NPV group II and that it is most closely related to polh of the NPVs of Malacosoma nuestria, Spodoptera littoralis, Orgyia pseudotsugata (single nucleo- capsid-type virus) and Buzura supressaria.
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DNA Sequence and Transcriptional Analysis of the Simian Varicella Virus Glycoprotein B Gene
More LessThe varicella-zoster virus (VZV) glycoprotein B (gB) is a major viral antigen which elicits immunity and neutralizing antibodies. In this study, the genomic map position and DNA sequence of a simian varicella virus (SW) homologue of the VZV gB gene was identified and the transcript analysed. A 32P-labelled VZV gB DNA probe hybridized to a subclone of the SW BamHl B restriction endonuclease fragment indicating the fine map position of SW DNA sequences homologous to the VZV gB gene. The SW gB DNA sequence was determined and analysis revealed a 2751 base pair open reading frame (ORF) with 71·1% identity to the VZV gB gene and 53·8% identity to the herpes simplex type 1 gB gene. The SW gB ORF encodes a 916 amino acid polypeptide with a predicted molecular mass of 104K. The deduced SW and VZV gB polypeptides share 78·9 % amino acid identity and predicted TV-linked glycosylation sites, cleavage sites and transmembrane regions. 32P-labelled SW gB DNA and RNA probes hybridized to a 3·5 kilobase SW polyadenylated transcript. Primer extension experiments identified transcript start sites for the SW and VZV gB genes and permitted a comparison of the sequences upstream of the SW and VZV gB ORFs. The SW and VZV gB promoter elements are similar in content and align closely. The VZV gB transcript start site suggests a gB polypeptide initiation site which is inconsistent with the previously reported ATG start codon.
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Delineation of a Sequence Required for Nuclear Localization of the Protein Encoded by Varicella-zoster Virus Gene 61
More LessAll characterized alphaherpesviruses encode a protein whose N-terminal region contains a novel zinc-binding motif, the C3HC4 domain. Homology between the different proteins is in general limited to key residues in this domain. In order to identify a separate landmark site in the C3HC4 protein encoded by varicella-zoster virus gene 61, namely the region required for nuclear localization, we have analysed a range of mutants in transient expression and immunofluorescence experiments. A basic region (RGAKRR) at residues 387 to 392 was found to be required for nuclear localization, and residues 390 and 391 were critical.
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Identification, Sequencing and Expression of the Glycoprotein L Gene of Murine Cytomegalovirus
DNA sequence analysis of the genome of the Smith strain of murine cytomegalovirus (MCMV) revealed an open reading frame (ORF) with amino acid sequence identity to glycoprotein L (gL) of other herpesviruses. The ORF is 822 bp in size and has the capacity to encode a protein of 274 amino acids. It has significant identity with the gL genes of human CMV and human herpesvirus 6. The coding sequence of the gL gene of MCMV strain K181 was also determined, and expressed in Escherichia coli as a fusion protein with glutathione S- transferase using the pGEX expression system. Two antibody-binding regions were identified on the basis of the reactivity of a series of truncated gL constructs with anti-MCMV antibodies. One was mapped to residues 1 to 38 and the other between residues 230 and 274. Polyclonal antibodies specific to gL were raised against the lull-length gL fusion protein. The antisera were shown to react with a 46K protein present in purified virions by Western blotting. Treatment of purified virions with endoglycosidase-H or -F resulted in reductions in M r of the 46K species to 42K and 3IK, respectively. The antisera did not exhibit any neutralizing activity in a plaque reduction assay.
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Expression of the Epstein–Barr Virus Envelope Fusion Glycoprotein gp85 Gene by a Recombinant Baculovirus
More LessThe gp85 envelope glycoprotein of Epstein-Barr virus (EBV) has a role in the molecular mechanism of infection, enabling fusion between the viral and host cell envelopes, a role in common with the homologous gH glycoproteins in other herpesviruses. A glutathione S- transferase bacterial fusion protein (GST85N-S) was generated, containing 178 amino acids from the C terminus of gp85 and including a known gp85 linear epitope. A panel of EBV-positive human antisera contained no antibodies to linear epitopes presented on the purified GST85N-S protein, indicating that primary protein structure in this region of gp85 is not a B cell target. This bacterial fusion protein was used to raise a rabbit monospecific polyclonal antiserum capable of detecting gp85 in a Western blot. The majority of recombinant baculovirus-expressed gp85 obtained from cell extracts prepared with SDS appeared on Western blots as heterogeneous high M r protein aggregates and consistently included 84K, 81K and 70K bands. Recombinant gp85 aggregation was increased by boiling the sample prior to gel electrophoresis. The 84K and 81K proteins were completely sensitive to endoglycosidase H treatment, indicating that these glycosylated species did not undergo further post-translational processing. Immunofluorescence studies revealed that recombinant gp85 was not transported to the insect cell surface. It reacted only with antibodies recognizing denatured gp85 and not with antibody to native gp85. Therefore expression of the gene encoding gp85, BXLF2, alone in the baculovirus expression system is insufficient for the synthesis of a correctly transported, processed, folded and antigenically native form of recombinant gp85.
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Molecular Determinants of the V3 Loop of Human Immunodeficiency Virus Type 1 Glycoprotein gp120 Responsible for Controlling Cell Tropism
More LessWe and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gpl20. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal β-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gpl20 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.
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Inhibition of Human T Cell Leukaemia Virus Type I Long Terminal Repeat Expression by DNA Methylation: Implications for Latency
More LessHuman T cell leukaemia virus type I (HTLV-I) provirus DNA was found to be methylated in patients with adult T cell leukaemia. We have therefore examined the possibility that DNA methylation might contribute to HTLV-I latency. In vitro methylation of HTLV-I long terminal repeat (LTR)-chloramphenicol acetyltransfer- ase or LTR-Luciferase constructs at eight HpaII sites, a subset of the eukaryotic methylation site CpG, resulted in a three- to fourfold inhibition of transcription in transfected cells. Inhibition of transcription by methylation of all CpG methylation sites using Sxsl methylase was much more pronounced (50- to 80-fold). As partial methylation of the LTR showed, methylation of the promoter region was responsible for most of the effect. Whereas cellular stimulation by a combination of phorbol 12-myristate 13-acetate and Tax was able to reverse the HpaII methylation effect, the inhibition by SsjI methylation was not suppressible under these conditions. The results are in line with a possible function of DNA methylation in HTLV-I latency.
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Reovirus λ1 Protein: Affinity for Double-stranded Nucleic Acids by a Small Amino-terminal Region of the Protein Independent From the Zinc Finger Motif
More LessThe reovirus λ1 protein, a major component of the inner capsid, has been shown to exhibit an affinity for dsRNA in a ‘Northwestern’ filter-binding assay. In the present study it was demonstrated that the protein can bind dsDNA as well as dsRNA. A bacterial expression system was used to study the protein region able to bind to nucleic acids. The amino-terminal 187 amino acids of λ1 were fused to the bacterial maltose-binding protein and shown to be sufficient for binding to nucleic acids. The putative zinc finger present on λ1 is not encompassed in this fragment of the protein. Site-directed mutagenesis also indicated that this zinc finger motif is unrelated to binding. In contrast, mutations introduced in a previously suggested nucleotide-binding motif almost completely prevented the binding. These data indicate that the amino-terminal end of λ1, encompassing its nucleotide-binding motif, is involved in the affinity of this protein for nucleic acids.
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Rotavirus Causes Selective Vimentin Reorganization in Monkey Kidney CV-1 Cells
More LessThe effect of rotavirus infection on cytoskeletal organization was examined in cultured African green monkey kidney (CV-1) cells. Rhesus rotavirus caused significant and selective changes in the organization of the vimentin filament network without having any effect on microtubules or actin. Double-immunofluorescence studies showed that at 6 h post-infection, and in the absence of cytopathic effect, the normal arrays of vimentin fibres radiating from multiple sites around the nucleus were lost. Vimentin fibres became irregularly distributed in the cytoplasm and were totally disrupted in the later stages of infection. Vimentin reorganization occurred independent of extracellular Ca2+ levels.
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Association of Serine in Position 1124 of Hantaan Virus Glycoprotein With Virulence in Mice
More LessHantaan virus (HV) of the genus Hantavirus causes a fatal disease in suckling mice following intraperitoneal or intracerebral infection. HV cl-1, which was obtained from the 76–118 strain of HV by growth in Vero E6 cells, exhibited high mortality rates in mice whereas mice infected with HV cl-2 survived without any clinical signs. To determine the molecular basis for the marked difference in virulence, we compared the nucleotide sequences of the large (L), medium (M) and small (S) segments of HV cl-1 genome with those of HV cl-2 and found that there was only one predicted amino acid substitution. This amino acid substitution was in position 1124 of the glycoprotein encoded by the M genome segment, in which serine in HV cl-1 was replaced by glycine in HV cl-2. Although there were several nucleotide and amino acid differences between the parental 76–118 strain and HV cl-1, the serine in position 1124 of the glycoprotein was common to the pathogenic parent and the pathogenic mutant. These results suggest that this substitution may be responsible for the virulence of this hantavirus.
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Full-length Sequence of the Genome of Hepatitis C Virus Type 3a: Comparative Study With Different Genotypes
Hepatitis C virus (HCV) type K3a (type 3a), which represents a minor genotype in Europe, the U.S.A. and Asia, appears to be significantly distributed throughout Australia and Brazil. We amplified the HCV-K3a/650 genome by reverse transcription polymerase chain reaction in ten overlapping fragments and determined the nucleotide sequences. The total sequence was 9454 bases in length and contained an open reading frame of 3021 amino acids, which is 10 or 11 amino acids longer than in HCV type 1 and 12 amino acids shorter than the sequence of type 2. These differences were due to the different lengths of both the putative envelope protein E2 and the NS5A regions, whose nucleotide lengths differ between types 1 and 2 also. Phylogenetic analysis of the putative core region and a portion of NS5B encoding the Gly-Asp-Asp motif indicated that HCV- K3a closely matched the corresponding type 3a group. The deletion and addition of amino acids in both E2 and NS5A may be associated with their pathobiological features.
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Variation in Biological Properties of Cauliflower Mosaic Virus Clones
More LessInfectous clones were prepared from virion DNA of three cauliflower mosaic virus (CaMV) isolates, 11/3, Xinjiang (XJ), and Aust, to investigate pathogenic variation in virus populations. Of 10 infectious clones obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed from those of the 11/3 wild-type. Virus from two clonal groups of 11/3 was transmissible by aphids whereas that from two others was not. Of the five infectious clones obtained from isolate XJ, two groups were identified, one of which differed symptomatically from the wild- type. Only one infectious clone was obtained from isolate Aust and this had properties similar to the wild- type. Restriction enzyme polymorphisms were found in some clonal groups and these correlated with symptoms. Other groups with different pathogenic properties could not be distinguished apart by restriction site polymorphisms. Further variation was observed in the nucleotide sequences of gene II (coding for aphid transmission factor) from these viruses as compared with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg mutation in gene II similar to that of other non-deleted non-transmissible CaMV isolates. The second had a 322 bp deletion at the site of a small direct repeat similar to that of isolate CM4-184 although occurring in a different position. The gene II deletion of isolate 11/3 produced a frame-shift that separated genes II and III by 60 bp. Most CaMV clones studied remained biologically stable producing similar symptoms during subsequent passages. However, one clone (11/3–7) produced two new biotypes during its first passage suggesting that it was relatively unstable. Our results show that wild-type populations of CaMV contain a range of infectious genome variants with contrasting biological properties and differing stability. We suggest that a variety of significant viral phenotypic changes can occur during each infection cycle resulting from relatively small genome changes.
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Complete Nucleotide Sequence of Peanut Clump Virus RNA 1 and Relationships With Other Fungus-transmitted Rod-shaped Viruses
More LessThe complete nucleotide sequence of RNA 1 of the tentative furovirus peanut clump virus (PCV) has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 5897 nucleotides in length and contains three long open reading frames (ORFs). The 5′ -terminal proximal ORF has the potential to encode a polypeptide of M r 130942 (P131) containing methyltransferase and RNA helicase homologous domains and displaying homology with large nonstructural proteins of alpha-like viruses, which are known or thought to be involved in virus replication. The PI31 ORF is followed in-frame by a second ORF which is probably expressed by partial readthrough of the UGA termination codon of the P131 ORF to produce a polypeptide of M r 191044 (P191). The readthrough region of PI91 contains the characteristic ‘core’ RNA polymerase motif, indicating that the PCV replicase proteins are expressed as a pair of overlapping proteins as in the tobamoviruses, tobraviruses and the furovirus soil-borne wheat mosaic virus (SBWMV). Sequence comparisons indicate that PI31 and P191 are most closely related to the replicase proteins of SBWMV and the hordeivirus barley stripe mosaic virus (BSMV) but are only distantly related to the replicase of the furovirus beet necrotic yellow vein virus (BNYW). The 3′ -terminal proximal ORF can encode a putative polypeptide of M r 14556 (PI5) which displays homology to small cysteine-rich proteins of hordeiviruses and SBWMV. We have corrected four errors in the sequence of PCV RNA 2 published previously by Manohar et al. (Virology 195, 33–41, 1993). One of these changes causes two small ORFs near the 3′ terminus of RNA 2 to be fused together to create an ORF for a putative polypeptide of M r 16833 (PI7) which displays extensive homology with the third protein of the triple gene block of BSMV RNA β.
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Evidence for an Internal Ribosome Entry Site Within the 5′ Non-translated Region of Turnip Mosaic Potyvirus RNA
More LessThe genomic RNA of potyvirases has a characteristic 5′ non-translated region (5′NTR) to which a viral protein, VPg, is covalently attached. This suggests that the viral RNA lacks a conventional cap structure and thus its translation may not proceed in the same way as most cellular mRNAs. To investigate the role of the 5′NTR during translation, various derivatives of the turnip mosaic potyvirus (TuMV) leader were fused to the reporter gene β-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit reticulocyte lysate and in planta following microprojectile DNA delivery into tobacco cell suspensions. GUS transcripts fused with the TuMV 5′NTR, whether they were capped or not, were efficiently translated, whereas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in excess over functional transcripts, translation was inhibited in a dose-dependent manner. Similarly, transcripts synthesized from the reverse complement of the 5′NTR inhibited translation to the same extent as the wild-type sequence, indicating that cap independence was not conferred by a specific sequence within the viral leader. A stable hairpin loop was placed in front or after the viral sequence. This hairpin loop normally prevented translation of control GUS transcripts but when the viral leader was positioned after it a significant level of GUS activity was measured, whether the transcripts were capped or not. On the other hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes bound to an internal site within the TuMV 5′NTR and then presumably scanned the sequence for the initiator AUG.
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The NTP-binding Motif in Cowpea Mosaic Virus B Polyprotein is Essential for Viral Replication
We have assessed the functional importance of the NTP-binding motif (NTBM) in the cowpea mosaic virus (CPMV) B-RNA-encoded 58K domain by changing two conserved amino acids within the consensus A and B sites (GKSRTGK500S and MDD545, respectively). Both Lys-500 to Thr and Asp-545 to Pro substitutions are lethal as mutant B-RNAs were no longer replicated in cowpea protoplasts. Transiently produced mutant proteins were not able to support trans-replication of CPMV M-RNA in cowpea protoplasts in contrast to transiently produced wild-type B proteins. Therefore loss of viral RNA synthesis was a result of a protein defect rather than an RNA template defect. Mutant B polyproteins were correctly processed in vitro and in vivo and the regulatory function of the 32K protein on processing of B proteins was not affected by these mutations. Since regulation of processing by the 32K protein depends on interaction with the 58K domain, the mutations in the NTBM apparently do not interfere with this interaction. The Asp-545 to Pro substitution left intact the binding properties of the 84K precursor of the 58K protein, with respect to ATP-agarose, whereas the Lys-500 to Thr substitution decreased the binding capacity of the 84K protein, suggesting that the Lys-500 residue is directly involved in ATP binding. The Lys-500 to Thr substitution in the 58K domain resulted in an altered distribution of viral proteins, which failed to aggregate into large cytopathic structures as observed in protoplasts infected with wild-type B-RNA. However viral proteins containing the Asp-545 to Pro substitution showed a normal distribution in protoplasts.
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Localization of Functional Regions of the Cucumber Mosaic Virus RNA Replicase Using Monoclonal and Polyclonal Antibodies
More LessMonoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV la and 2a proteins, as immunogens. Five out of eight monoclonal antibodies, which bound to the la and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro. Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the la protein containing putative helicase and methyltransferase domains respectively. Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps. Prior interaction of the latter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase. Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also produced using peptides corresponding to conserved helicase and polymerase motifs in the la and 2a proteins. The greatest inhibition was shown by antibodies to a peptide containing the GDD motif. These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.
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Mapping Local and Systemic Symptom Determinants of Cucumber Mosaic Cucumovirus in Tobacco
More LessCucumber mosaic cucumovirus (CMV) can be divided into two subgroups, I and II. LS-CMV and most other subgroup II strains cause a mild, systemic mottle on tobacco and can induce a necrotic etching (necrotic rings) symptom on inoculated tobacco leaves. In contrast, Fny-CMY and most other subgroup I strains cause severe, systemic mosaic symptoms on tobacco, but do not induce the necrotic etching symptom. Full- length cDNA clones of all three genomic RNAs of LS- CMV were constructed and infectious RNAs were generated from these clones. Using pseudorecombinants constructed from the infectious transcripts of LS-CMV and Fny-CMV, we found that both RNAs 1 and 2 of Fny-CMV are involved in determining the severity of systemic symptom on tobacco, and that LS-CMV RNA 3 contains the determinant for the necrotic etching symptom. Chimeras formed between Fny- and LS-CMV RNA 3 were used to demonstrate that the inducer of the necrotic etching symptoms mapped to the 5′ 618 nucleotides of LS-CMV RNA 3, and required sequences in both the 5′ non-translated region, as well as the 3a gene of CMV.
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The 3a Protein from Cucumber Mosaic Virus Increases the Gating Capacity of Plasmodesmata in Transgenic Tobacco Plants
More LessThe 3a protein, encoded by RNA 3 of cucumber mosaic virus (CMV), is the putative movement protein of viral progeny in infected plants. An analysis of transgenic tobacco plants constitutively expressing the CMV 3a protein showed that the protein is accumulated in leaves at every stage of development. In fully expanded leaves the protein is immunodetectable mostly in a cell-wall- enriched fraction. Dye-coupling experiments using fluor- escent-dextran probes were performed on fully expanded leaves to study the modifying effect of CMV 3a protein on the gating capacity of plasmodesmata. Movement of fluorescein-isothiocyanate-labelled dex- tran with a mean molecular mass of 10000 Da, and an approximate Stokes’ radius of 2·3 nm, was detected between cells of the 3a protein transgenic plants, but not in the control plants. These results are consistent with the idea that the CMV 3 a protein is involved in the modification of plasmodesmata and, therefore, in the cell-to-cell spread of the virus.
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Mapping of the RNA-binding Domain of the Alfalfa Mosaic Virus Movement Protein
In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by site- directed mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding.
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Lethal Mutations Within the Conserved Stem-loop of African Cassava Mosaic Virus DNA are Rapidly Corrected by Genomic Recombination
More LessThe nonanucleotide motif TAATATTAC occurs in the intergenic region of all geminiviruses that have been examined to date. The motif is invariably located within the loop of a potential stem-loop structure that has been implicated in viral DNA replication. To investigate the contribution of these sequences to virus proliferation, African cassava mosaic virus (ACMV) DNA B mutants have been screened for their ability to infect Nicotiana benthamiana when co-inoculated with DNA A. Mutants in which the putative stem structure was altered by the introduction of single nucleotide mismatches remained as infectious as the wild-type virus and the mutations were retained in the progeny. Mutants containing nucleotide substitutions within the loop sequences were similarly infectious but analysis of progeny showed that in most cases wild-type sequences were restored by recombination with DNA A. Stem-loop deletion mutants of both genomic components were not infectious when co-inoculated, although they were once again efficiently rescued by recombination when inoculated with the wild-type components. Co-inoculation of genomic components containing the motif TAGTAT- TAC did not result in a systemic infection while mutants containing the motif TAATACTAC were infectious and the mutation was stable. The results demonstrate that ACMV will tolerate some modification to this highly conserved region of the genome that might allow more precise mapping of the position at which the viral DNA is nicked during replication.
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Volumes and issues
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Volume 105 (2024)
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Volume 71 (1990)
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Volume 70 (1989)
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Volume 69 (1988)
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Volume 68 (1987)
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Volume 67 (1986)
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Volume 66 (1985)
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Volume 65 (1984)
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Volume 64 (1983)
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Volume 63 (1982)
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Volume 62 (1982)
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Volume 61 (1982)
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Volume 60 (1982)
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Volume 59 (1982)
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Volume 58 (1982)
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Volume 57 (1981)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)