- Volume 78, Issue 3, 1997
Volume 78, Issue 3, 1997
- Articles
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Two distinct mechanisms regulate luteovirus transmission efficiency and specificity at the aphid salivary gland.
More LessBarley yellow dwarf luteovirus (BYDV) particles are transmitted by aphids in a species-specific manner. Transmission to plants requires that the virus particles be transported across the basal lamina and plasmalemma of the accessory salivary gland (ASG). To characterize the role of the ASG basal lamina in regulating BYDV transmission, five aphid species were microinjected with purified New York isolates BYDV-PAV or -RPV. Both viruses associated specifically only with the ASG basal lamina. The ability of virions to penetrate the basal lamina was separate from the ability to penetrate the plasmalemma. When the salivary glands of vector, Sitobion avenae, or non-vector, Rhopalosiphum maidis, aphids were incubated in vitro with New York isolate BYDV-MAV, virions only attached to the ASG basal lamina of S. avenae. When anionic and cationic ferritin were microinjected into aphids, only cationic ferritin aggregated on the surface of the ASG basal lamina and at openings of plasmalemma invaginations into the cytoplasm, suggesting that these sites had a net negative charge. In vitro studies of anionic and cationic gold penetration of ASG basal laminae indicated a macromolecular size exclusion limit of approximately 20 nm that depended on charge. Anionic gold particles did not accumulate in the basal lamina as densely as the 25 nm BYDV particles, suggesting that the virus particles have a greater affinity for the ASG basal lamina. These results indicate that both the ASG basal lamina and plasmalemma contain specific components independently involved in the recognition and transmission of luteoviruses.
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Two novel subgenomic RNAs derived from RNA 3 of tomato aspermy cucumovirus.
B Shi, S Ding and R H SymonsTwo abundant subgenomic RNAs, designated RNA 3B and RNA 5, were found to be associated with the V strain of tomato aspermy cucumovirus (V-TAV). Sequence determination showed that the 3'-terminal 323 nucleotides (nt) of RNA 3B was identical to RNA 5, whereas its 5'-terminal 163 nt was a direct repeat (one nt difference) of the 5'-half of RNA 5, and that both RNAs are completely homologous to the 3'-terminal untranslated region of TAV RNA 3. TAV RNAs 3B and 5 were also detected in the infection of a pseudorecombinant virus consisting of TAV RNA 3 and RNAs 1 and 2 from cucumber mosaic virus. Furthermore, only RNA 5, not RNA 3B, was detected in a TAV mutant in which one of the repeats was deleted from RNA 3. These genetic studies clearly show that both RNA species are derived from TAV RNA 3. However, in contrast to TAV RNAs 4 and 4A, which encode coat protein and 2b protein, respectively, RNAs 3B and 5 represent a novel class of subgenomic RNAs from TAV that do not function as mRNAs. Possible functional roles for such a class of viral subgenomic RNAs are discussed.
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Expression and suppression of circulative aphid transmission in pea enation mosaic virus.
More LessPea enation mosaic virus (PEMV) is composed of two autonomously replicating virus RNAs related to the genomic RNAs of viruses in the genera Luteovirus and Umbravirus. The transmission of PEMV resembles that of its luteovirus relatives in utilizing circulative aphid transmission. However, unlike its luteovirus counterparts, PEMV can also be mechanically transmitted. Prolonged mechanical passage of PEMV can lead to the loss of aphid transmissibility, a trait that is mirrored by specific changes in the PEMV virion composition. These changes were used to examine the virus contribution to vector transmission and the mechanisms by which it is regulated. Using a local lesion isolation technique, one aphid transmissible and two aphid non-transmissible isolates of PEMV were compared. Structural analysis of a 54 kDa minor structural subunit unique to the aphid transmissible isolate demonstrated that it was a fusion of the 21 kDa virus coat protein and a 33 kDa protein encoded immediately downstream of the 21 kDa ORF, consistent with the formation of the 54 kDa subunit by translational readthrough. Genetic analyses utilizing exchanges between infectious in vitro transcripts of each isolate demonstrated that although the 33 kDa protein was non-essential for infection, its presence was mandatory for aphid transmission, and that specific changes within the 33 kDa ORF were sufficient to confer or abolish aphid transmission. This study also demonstrates that isolates of PEMV exist as mixtures of aphid transmissible and non-transmissible genotypes, and provides insight into the mechanisms used by this virus to down-regulate aphid transmission in response to a specific selection pressure.
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Genome organization and gene expression of saguaro cactus carmovirus.
More LessThe complete sequence of the single-stranded, (+)-sense RNA genome of saguaro cactus carmovirus (SCV) has been determined. The 3879 nucleotide genome contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 26 kDa protein (p26) and terminates with an amber codon which is readthrough into an in-frame p57 ORF to generate an 86 kDa fusion protein (p86). Two small, centrally located ORFs encode a 6 kDa protein (p6) and a 9 kDa protein (p9), respectively. The 3'-proximal ORF encodes a 37 kDa (p37) capsid protein (CP). Analysis of the nucleotide and predicted amino acid sequences supports the classification of SCV in the genus Carmovirus in the family Tombusviridae. All predicted SCV proteins are expressed in an in vitro translation system. SCV p26 and the readthrough fusion protein p86 are synthesized from the genomic RNA while p6, p9 and p37 CP ORFs at the 3' half of the genome are expressed from two subgenomic (sg) RNAs. The 5' termini of both sg RNAs have been mapped. The large 1614 nucleotide sg RNA contains the p6 and p9 ORFs as the first and the second ORFs respectively from its 5' end. It directs the synthesis of abundant p6 but a small amount of p9. While a synthetic transcript with the p9 ORF at the 5' end is a more efficient messenger for p9, no corresponding sg RNA has been identified in vivo. The smaller 1396 nucleotide sg RNA contains only the p37 ORF and directs the synthesis of SCV CP.
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The beet yellows closterovirus p65 homologue of HSP70 chaperones has ATPase activity associated with its conserved N-terminal domain but does not interact with unfolded protein chains.
More LessThe positive-strand RNA genome of beet yellows closterovirus (BYV) encodes a 65 kDa protein (p65) related to the HSP70 family of cell chaperones. The full-sized BYV p65, and N- and C-terminal fragments, with (His)6 tails, were overexpressed in bacteria and purified by metal-chelate chromatography. Using a polyclonal antiserum raised against the C-terminal fragment of p65, evidence was obtained for expression of the viral protein in planta. Purified recombinant p65 and its N-terminal 40 kDa fragment exhibited Mg2+-dependent ATPase activity in vitro. However, unlike its cellular HSP70 homologues, p65 was unable to bind to denatured protein and its ATPase activity was not stimulated by synthetic peptides which are known to stimulate HSP70 ATPases. Hence, the BYV p65, although being a chaperone-type ATPase, may have a distinct substrate specificity and function in BYV-infected cells.
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Completion of the impatiens necrotic spot virus genome sequence and genetic comparison of the L proteins within the family Bunyaviridae.
More LessThe nucleotide sequence of the large (L) genome segment of impatiens necrotic spot virus (INSV) has been determined, and herewith the complete nucleotide sequence of the whole, tripartite genome of this important tospovirus has been elucidated. The L RNA is 8776 nucleotides long and of negative polarity, containing one large ORF on the viral complementary strand. Comparison of the deduced amino acid sequence of the INSV L RNA primary translation product (330.3 kDa) with those of the L RNAs of other members of the family Bunyaviridae reveals that this protein represents the putative viral RNA-dependent RNA polymerase. A cluster dendrogram of the (putative) RNA polymerases indicates that the genera Tospovirus and Tenuivirus, though both encompassing ambisense plant-infecting viruses, have different affinities to the animal-infecting Bunyaviridae, tospoviruses being most closely related to the genus Bunyavirus, and tenuiviruses to the genus Phlebovirus.
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The Lassa fever virus L gene: nucleotide sequence, comparison, and precipitation of a predicted 250 kDa protein with monospecific antiserum.
More LessThe large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya- and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS-lymphocytic choriomeningitis and Tacaribe-Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells.
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Antigenic and genetic analyses of H1N1 influenza A viruses from European pigs.
H1N1 influenza A viruses isolated from pigs in Europe since 1981 were examined both antigenically and genetically and compared with H1N1 viruses from other sources. H1N1 viruses from pigs and birds could be divided into three groups: avian, classical swine and 'avian-like' swine viruses. Low or no reactivity of 'avian-like' swine viruses in HI tests with monoclonal antibodies raised against classical swine viruses was associated with amino acid substitutions within antigenic sites of the haemagglutinin (HA). Phylogenetic analysis of the HA gene revealed that classical swine viruses from European pigs are most similar to each other and are closely related to North American swine strains, whilst the 'avian-like' swine viruses cluster with avian viruses. 'Avian-like' viruses introduced into pigs in the UK in 1992 apparently originated directly from strains in pigs in continental Europe at that time. The HA genes of the swine viruses examined had undergone limited variation in antigenic sites and also contained fewer potential glycosylation sites compared to human H1N1 viruses. The HA exhibited antigenic drift which was more marked in 'avian-like' swine viruses than in classical swine strains. Genetic analyses of two recent 'avian-like' swine viruses indicated that all the RNA segments are related most closely to those of avian influenza A viruses.
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Spontaneous excretion of virus from MDCK cells persistently infected with influenza virus A/PR/8/34.
More LessWhen MDCK cells in a semiconfluent monolayer were infected with 5 p.f.u. per cell of influenza virus A/PR/8/34 (H1N1), a majority of the cells continued to grow stably upon subsequent cultivation with a growth medium containing 50% foetal calf serum. While growing, the cells spontaneously excreted virus, the amount of which declined gradually as the passage number of the cells increased. The extent of virus shedding was significantly increased when the cells were subsequently maintained in a medium containing 0.2% bovine serum albumin. Within the cells, viral messenger RNAs for all eight genes of A/PR/8 were demonstrated by PCR indicating that endogenous viral genes were constitutively transcribed. However, viral proteins as well as viral genes were not demonstrable by radioimmunoprecipitation or ribonuclease protection assays, respectively.
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Inactivation of inhibitors by the receptor-destroying enzyme of influenza C virus.
K Höfling, H D Klenk and G HerrlerThe importance of the receptor-destroying enzyme of influenza C virus for inactivation of inhibitors was analysed. Using three different inhibitors (rat serum, bovine submandibulary mucin and bovine brain gangliosides) inhibition of virus infection was observed only at an inhibitor concentration that was about 100-fold higher than the maximum concentration of inhibitor that could be inactivated by the receptor-destroying enzyme of a given amount of virus. From our data and other observations we conclude that the receptor-destroying enzyme is not required to inactivate inhibitors.
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Nucleotide sequence analysis of the large (L) genes of phocine distemper virus and canine distemper virus (corrected sequence).
More LessThis paper corrects the previously published sequence of the L gene of canine distemper virus (CDV). Errors in the published sequence (M. S. Sidhu et al., 1993, Virology 193, 50-65) led to frame shifts between residues 1021-1032, 1190-1219 and 1645-1650; a deletion of 21 amino acids between residues 1684-1705, and a single residue deletion at residue 1478. Residue 237 is now found to be glycine rather than tryptophan and residue 1626 proline instead of threonine. The sequence of the L gene of phocine distemper virus (PDV) was also determined. Alignment of the morbillivirus L proteins showed that PDV and CDV are more closely related to each other than to rinderpest virus and measles virus. Two regions of low identity are proposed to function as hinge regions between three highly conserved domains (I-III) in the morbillivirus L proteins. New sequence motifs have been identified on the basis of conservation in the morbilliviruses and the Paramyxovirinae.
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Genetic heterogeneity of hepatitis G virus isolates from different parts of the world.
More LessComparative sequence analysis of a 354 nt fragment of the NS5 region of hepatitis G virus (HGV) isolates was performed to assess two levels of HGV genomic variability: (1) heterogeneity of HGV within an infected individual, and (2) heterogeneity of different HGV isolates. Comparison of nucleotide sequences of DNA clones from two virus isolates demonstrated that in each infected individual HGV is represented by a population of virions with closely related but heterogeneous genomes (quasi-species). Phylogenetic analysis of nucleotide sequences of 42 isolates collected from 14 countries revealed less significant genome variability of HGV as compared to hepatitis C virus. Sequences of all HGV isolates fell into one group of distribution of evolutionary distances. On a phylogenetic tree all HGV sequences segregated into numerous branches. All sequences of isolates from Africa, South and South-East Asia, however, were clustered together and were separated from those of other isolates collected in Europe, North America and Central Asia.
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Long-term evolution of the 5'UTR and a region of NS4 containing a CTL epitope of hepatitis C virus in two haemophilic patients.
More LessHaemophilic patients exposed to unsterilized clotting factor concentrates prior to 1985 have become infected with hepatitis C virus (HCV). We have studied the sequence evolution of the 5'UTR and a region of NS4 over 12 years in one human immunodeficiency virus (HIV) positive haemophilic patient and 14 years for one HIV negative haemophilic patient. One sample each year from the date of HCV infection to 1994 was analysed for genotype, virus load and nucleotide sequence of the two genetic loci. Both patients were infected with HCV genotype 1 throughout the study period. The virus load profiles were similar except that the profile for the HIV infected patient was displaced 4 years earlier relative to the other patient. Mean divergence of the quasispecies at both the 5'UTR and NS4 loci was higher in the HIV coinfected patient. Phylogenetic analysis indicated that evolution of the 5'UTR was host independent, whereas the NS4 region containing a CD8 restricted CTL epitope evolved in a host specific fashion.
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The molecular epidemiology of type 1 poliovirus in Central African Republic.
More LessAn increase in the incidence of acute flaccid paralysis cases associated with wild-type 1 poliovirus occurred in children in the city of Bangui, Central African Republic (CAR), in 1993 and 1994. Genetic relationships of 33 isolates were analysed by restriction fragment length polymorphism and by sequencing the VP1/2A junction region (150 nucleotides) of the viral genome. Two distinct genotypes, A and B, were co-circulating in 1993, while in 1994 only a third genotype, C, was observed. Comparison of the sequences found, with those of the sequences from isolates from neighbouring and other endemic countries revealed that genotype A isolates were related to strains from Egypt (90.7% identity), genotype B isolates to strains from Kenya (96.7% identity), Sudan and Egypt, and genotype C isolates to strains from various countries in western and southern Africa (89.0% identity). Genotypic diversity and genetic linkage with strains from neighbouring countries indicate intense poliovirus circulation and transmission that does not respect national borders. Therefore, eradication of poliomyelitis from CAR can only be achieved by a coordinated multinational strategy that stops poliovirus circulation in the whole of Africa.
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Differential restrictions on antigenic variation among antigenic sites of foot-and-mouth disease virus in the absence of antibody selection.
More LessClonal populations of foot-and-mouth disease virus have been serially passaged in cell culture to analyse variation in the absence of immune selection at different antigenic sites of the virus. Mutant frequencies at the RNA regions encoding two independent antigenic sites (sites C and D) were more than twentyfold lower than for antigenic site A (the G-H loop of VP1). Correspondingly, fixation of amino acid substitutions was very restricted in sites C and D. In spite of such a restriction, neutralization assays using fractionated anti-virus polyclonal antibodies has provided direct evidence of significant antigenic variation in the absence of immune selection at sites unrelated to site A. It is proposed that the degree of tolerance to acceptance of amino acid replacements may modulate the variation at different antigenic epitopes of the same virus.
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Inhibition of feline immunodeficiency virus infection by CD9 antibody operates after virus entry and is independent of virus tropism.
More LessA monoclonal antibody which blocks infection with feline immunodeficiency virus (FIV) was found previously to react with the cell surface molecule CD9, implicating CD9 in the process of virus entry. We report here that inhibition by anti-CD9 antibody does not operate at the level of virus entry but at a subsequent stage in the virus life-cycle. Moreover, inhibition of infection is independent of the passage history of the virus or the virus subtype. Inhibition of FIV infection by anti-CD9 antibody does not operate in 3201 cells, which do not express this surface antigen. However, ectopic expression of CD9 on 3201 cells enhances infection with FIV, suggesting that the role of CD9 may be direct rather than via cellular signalling pathways. These results suggest a novel control point in the lentivirus life-cycle which might be susceptible to modulation by natural antagonists.
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Rapid degradation of CD4 in cells expressing human immunodeficiency virus type 1 Env and Vpu is blocked by proteasome inhibitors.
More LessHuman immunodeficiency virus (HIV) type 1 encodes three genes, Vpu, Env and Nef, that decrease cellular CD4. Vpu and Env act cooperatively to accelerate degradation of CD4 in the endoplasmic reticulum. Here we report that Vpu/Env-induced CD4 degradation is inhibited by lactacystin, a specific inhibitor of the proteasome, and by other proteasome inhibitors, but not by non-proteasome protease inhibitors. We also note that Vpu has amino acid sequence homology with a segment of IkappaB known to be involved in proteasome-mediated degradation, suggesting that HIV-1 could have transduced cellular sequences to enhance down-regulation of CD4.
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Analysis of vif-defective human immunodeficiency virus type 1 (HIV-1) virions synthesized in 'non-permissive' T lymphoid cells stably infected with selectable HIV-1.
C Ochsenbauer, T Wilk and V BoschIn order to facilitate analyses of the molecular function of the human immunodeficiency virus type 1 (HIV-1) Vif protein, we have developed a cell culture model-system which allows permanent production of genotypically and phenotypically vif-defective HIV-1 virions in 'non-permissive' H9 cells. Using recombinant, replication-competent HIV-1 proviruses coding for a selectable marker gene (gpt) instead of nef, two stably infected H9 subclones named M2 (vif-mutant) and WX (wild-type), respectively, were generated. Virions released from cell line M2--displaying the expected vif-defective phenotype--are produced permanently, and in an at least 50 times higher amount than virus particles from acutely vif-negative HIV-1-infected H9 cells. Analysis of viral protein composition and the electron-microscopic morphology of vif-mutant virions did not reveal any detectable differences in comparison to wild-type virions.
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The major homology region of bovine leukaemia virus p24gag is required for virus infectivity in vivo.
More LessIn order to gain insight into the role of the major homology region (MHR) in the infectious potential of bovine leukaemia virus (BLV), mutations were introduced into the capsid gene of an infectious molecular clone. A provirus that was designed to contain only a slightly modified version of the MHR (substitution of phenylalanine 147 with a tyrosine) was still infectious in vivo. Furthermore, the provirus loads were not significantly different from those obtained with a wild-type virus. A second mutant was designed to analyse a mild modification of the MHR at the level of arginine 150. The substitution of this residue with a lysine completely destroyed the infectious potential of the recombinant virus. Finally, a third mutant that was deleted in the MHR region was unable to infect the host. Thus it appears that the integrity of the MHR domain is essential for BLV infectivity in vivo.
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The inhibitory effects of antisense RNA on hepatitis B virus surface antigen synthesis.
J Wu and M A GerberAntisense RNA-mediated inhibition of gene expression has the potential for gene therapy of virus infections. We studied the inhibitory effect of antisense RNA directed against the hepatitis B virus (HBV) genome on the expression of the HBV surface antigen (HBsAg). Three prokaryotic antisense RNA expression constructs were produced which expressed antisense RNA complementary to the entire coding region (1.4 kb) and to 1.0 kb and 582 bp of the 5' region of HBsAg mRNA, respectively. In an in vitro translation system, all three antisense RNAs showed concentration-dependent inhibitory effects on translation of HBsAg mRNA. In a coupled in vitro transcription and translation system, concentration-dependent inhibition of HBsAg synthesis was observed for all above mentioned antisense RNAs. Three mammalian antisense RNA expression vectors were then constructed, expressing the same antisense RNAs as used above. Transfection of the vectors into Hep3B cells (an HBsAg secreting cell line) resulted in almost complete blockage of HBsAg production, whereas control vector transfected cells secreted high levels of HBsAg. The inhibitory effect lasted for more than 10 months post-transfection. To examine the possible mechanism of the antisense RNA effect in the cell line, we measured HBV mRNA levels in the transfected cells and found that the mRNA levels in the antisense RNA expressing cells were much lower than those in the control cells. Therefore, in Hep3B cells, the antisense RNAs inhibited HBsAg synthesis, at least partially, through the reduction of HBV mRNA levels.
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Comparison of hepatitis B virus core promoter sequences in peripheral blood mononuclear cells and serum from patients with hepatitis B.
More LessIt has been reported that hepatitis B virus (HBV) DNA is present in peripheral blood mononuclear cells (PBMCs), although it is unclear whether it actually replicates there or is adsorbed from serum. HBV DNA sequences from the core promoter and precore regions were amplified from PBMCs and serum taken from 13 patients with hepatitis B infection. Analysis by single strand conformation polymorphism, direct sequencing and cloning revealed identical HBV DNA sequences in both PBMCs and serum from five patients with acute hepatitis and in five out of eight patients with chronic hepatitis. However, in the remaining three chronic hepatitis cases, HBV DNA sequences in both PBMCs and serum were different: two patients harboured HBV DNA sequences from their PBMCs with deletions/insertions in the core promoter region and one patient harboured HBV DNA sequences from their PBMCs with two nucleotide substitutions. These findings point to a possible presence of independent HBV DNA replication in PBMCs.
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Detection and characterization of human papillomavirus type 45 DNA in the cervical carcinoma cell line MS751.
J Geisbill, U Osmers and M DürstThe cervical carcinoma-derived cell line MS751 was examined for human papillomavirus (HPV) DNA and RNA. A genomic fragment containing both viral and cellular sequences was cloned. Sequence analysis showed that MS751 cells contain a partially deleted HPV-45 genome integrated at a single chromosomal site. HPV sequences from the E6-E7 region are expressed as poly(A)+ RNA.
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JC virus regulatory region rearrangements and genotypes in progressive multifocal leukoencephalopathy: two independent aspects of virus variation.
More LessJC virus (JCV) causes the central demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV strains excreted in the urine are distinguishable from those in PML tissue by the configuration of their regulatory region to the right of ori: the archetypal regulatory region, 267 nucleotides long, is rearranged in PML tissue by deletion and duplication. Within the coding region JCV shows variations as a result of virus evolution. Four major genotypes are distinguishable of which Type 1 is based in Europe and Type 2 in Asia. Here, the regulatory region rearrangements and the viral genotypes of 29 JCV strains from PML brain were determined. Rearrangement patterns and genotypes were not associated. In general, deletions occurred before duplications, but exceptions to this rule exist. Each configuration of the 29 rearranged regulatory regions was unique and could be derived directly from the non-rearranged, archetypal form.
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Suppression of high mobility group protein T160 expression impairs mouse cytomegalovirus replication.
M Gariglio, P Foresta, C Sacchi, M Lembo, L Hertel and S LandolfoThe high mobility group (HMG-1) box proteins bind both non-B-DNA conformations and specific nucleotide sequences. They have been implicated in a wide variety of cellular functions involving DNA, such as transcription, replication and recombination. To determine whether HMG-1 box protein T160 plays a role in virus replication, we employed an antisense strategy to inhibit its expression in NIH 3T3 cells. The two T160 clones that expressed levels of T160 50% lower than those expressed by clones transfected with the empty vector (Neo+ clones) were investigated with respect to their permissiveness to the growth of viruses representing three families: Rhabdoviridae, vesicular stomatitis virus (VSV); Picornaviridae, encephalomyocarditis virus (EMCV), and Alpha- and Betaherpesviridae, herpes simplex virus 1 (HSV-1) and mouse cytomegalovirus (MCMV), respectively. They displayed a high degree of resistance to MCMV replication, but were fully permissive to the other viruses. Competitive PCR and probing IE-1 products by Western blot analysis showed that this resistance was not due to depressed levels of virus adsorption during the early phases of infection. We therefore conclude that T160 is involved in replication of the betaherpesvirus MCMV.
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Processing and intracellular localization of the herpes simplex virus type 1 proteinase.
More LessThe herpes simplex virus type 1 (HSV-1) capsid protein VP24 (encoded by UL26) was expressed as a GST-fusion protein and used to prepare a group of monoclonal antibodies. These were used to characterize the protein in capsids and virus infected cells and demonstrated that it exists as two polypeptide species. The nature of the relationship between these two species was investigated and found to be associated with disulphide bonding. Under non-reducing conditions a species corresponding to dimers of VP24 was identified in preparations of B capsids, the site of action of the proteinase. Biochemical subcellular fractionation studies suggested that only cleaved forms of UL26 and UL26.5 gene products could be detected in the nucleus of the infected cell at early times post-infection.
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Vaccinia virus serpin B13R (SPI-2) inhibits interleukin-1beta-converting enzyme and protects virus-infected cells from TNF- and Fas-mediated apoptosis, but does not prevent IL-1beta-induced fever.
More LessThe vaccinia virus (VV) strain Western Reserve B13R gene encodes a 38.5 kDa intracellular polypeptide that is non-essential for virus replication in vitro and does not affect virus virulence in a murine intranasal model. The protein has 92% amino acid identity with the cowpox virus cytokine response modifier A (crmA) protein which inhibits the interleukin (IL)-1beta converting enzyme (ICE). Here, we show that extracts from THP-1 cells infected with VV strains expressing B13R prevent the cleavage of in vitro transcribed and translated pro-IL-1beta into mature IL-1beta. Similarly, THP-1 cells infected with VVs expressing B13R process pro-IL-1beta into mature IL-1beta inefficiently in situ. Despite its inhibition of ICE, B13R does not prevent fever in infected mice, a systemic effect mediated by IL-1beta. Instead, fever is controlled by the VV IL-1beta receptor, encoded by gene B15R, and deletion of both the B13R and B15R genes did not increase the febrile response compared to deletion of B15R alone. The B13R protein does, however, block apoptosis mediated by anti-Fas antibodies or by tumour necrosis factor (TNF) and cycloheximide. Using DNA fragmentation, chromium release and microscopic analyses it was shown that cells infected with wild-type VV strain WR, or a revertant virus in which the B13R gene had been re-inserted into the B13R deletion mutant, are more resistant than uninfected cells or deletion mutant-infected cells to apoptosis mediated by anti-Fas and TNF.
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Translational properties of the untranslated regions of the p10 messenger RNA of Autographa californica multicapsid nucleopolyhedrovirus.
More LessProtein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the insect-cell lysates was measured. The translational properties of the p10 transcripts were very similar in lysates made from either uninfected or baculovirus-infected insect cells. Determination of optimal salt conditions for either uncapped or capped transcripts showed that the p10 5'UTR was used very efficiently for translation initiation in vitro, even in the absence of a cap-structure at its 5' end. Addition of cap-analogue to the in vitro translation assays did not inhibit p10 5'UTR-driven translation, while translation of a cap-dependent mRNA was severely inhibited. These data suggest that the very late mRNAs of baculovirus are translated in a cap-independent manner.
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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)