- Volume 76, Issue 4, 1995
Volume 76, Issue 4, 1995
- Animal
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A set of African swine fever virus tandem repeats shares similarities with SAR-like sequences
More LessA group of cross-hybridizing DNA segments contained within the EcoRI restriction fragments U′, X and J of a Vero cell-adapted strain (BA71V) of African swine fever virus (ASFV) were mapped and sequenced. Analysis of the nucleotide sequence revealed the presence of a set of long internal repeated sequences composed of five types of tandemly repeat units of about 200 bp. These tandem repeats contain a G-rich core of 10–14 nucleotides surrounded by regions with a high A+T content distributed in oligo(dA).oligo(dT) tracts. Next to the repeated sequences we detected two related open reading frames that are members of a new multigene family (multigene family 300). Comparison of DNA sequences from several virus isolates indicated that this region undergoes frequent rearrangements leading to either duplications or deletions of the repeat units. These ASFV repeated sequences share similarities with chromosomal α satellite DNA, the scaffold-associated region and satellite III of Drosophila. Similar tandemly repeated sequences have not been described in other viruses.
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Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues
High titre replication of human cytomegalovirus (HCMV) in cell culture is restricted to primary human fibroblasts. During acute infection in vivo, HCMV nucleic acids and antigens have been found in various organs. Using only morphological criteria, inconsistent data have been reported about the cell types that can be infected by HCMV. In particular, the role of fibroblasts in organ infections has remained unclear. To define accurately the target cells of HCMV in vivo, tissue sections from lung and gastrointestinal tract of patients suffering from acute HCMV infection were investigated using immunohistochemical double-labelling analyses. Monoclonal antibodies with defined specificity against immediate early (IE), early (E) and late (L) viral antigens and antibodies directed against cell marker proteins were employed to identify infected cells. The results demonstrated that a broad spectrum of cells was infected by HCMV in vivo. Consistent with their susceptibility in culture, fibroblasts formed a major population of HCMV-infected cells. In contrast, haemopoietic cells were only infrequently stained with virus-specific antibodies. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells and macrophages appeared to be permissive for HCMV replication. Contrary to this, polymorphonuclear cells showed only IE gene expression, indicating that these cells were abortively infected. The analysis of the distribution of infected cells in tissue supported the hypothesis that endothelial cells and monocytes/macrophages may play a crucial role in the haematogenous spread of HCMV; in contrast, fibroblasts, smooth muscle cells and epithelial cells may form the cell populations important for the multiplication and spread of the virus in infected tissues.
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cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products
Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras cDNA, together with pΔACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of pΔACMVCAT were also observed in cell lines carrying stably transfected ras oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, β-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21 ras . Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.
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Identification of an epithelial cell differentiation responsive region within the BZLF1 promoter of the Epstein—Barr virus
More LessThe Epstein—Barr virus (EBV) BZLF1 gene product, Zta is able to trigger the viral lytic cycle in latently infected B lymphocytes. Investigations into regulation of the promoter, Zp for the BZLF1 gene in B cells have identified 12-O-tetradecanoylphorbol 13-acetate, anti-immunoglobulin and Zta responsive elements as well as a negative regulatory element within Zp. EBV infects and replicates within squamous epithelial cells and there is evidence to indicate that EBV gene expression is linked to the differentiation status of epithelial cells. We have investigated regulation of Zp in undifferentiated and differentiated cells of the human squamous epithelial cell line SCC12F. Zp was found to be active in SCC12F cells and activity was increased approximately 7-fold upon induction of epithelial terminal differentiation. Sequences responsive to epithelial cell differentiation were contained within the region of Zp from -86 to +12 bp, a region previously shown to contain an AP-1-like binding site, designated the ZII domain. In addition, enhancing sequences were detected in the region -221 to -86 bp. These studies will lead to a greater understanding of differentiation-linked EBV gene expression in human squamous epithelial cells.
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The cytoplasmic C-terminal domain but not the N-terminal domain of latent membrane protein 1 of Epstein—Barr virus is essential for B cell activation
More LessThe Epstein—Barr virus (EBV) latent membrane protein 1 (LMP-1) is essential for EBV-induced immortalization of human primary B cells, transforms rodent fibroblasts and induces the up-regulation of several B cell activation markers when transiently expressed in primary B cells. The biochemical function of LMP-1 is unclear and limited information is available on the involvement of different domains of the protein in B cell activation. The present study describes the characterization of LMP-1 N- and C-terminal deletion mutants in terms of their cell surface distribution and ability to induce activation markers in primary human B cells and in the type I Burkitt’s lymphoma cell line DG75. The C-terminal deletion mutant was detected by immunofluorescence via antibodies targeted against an eight amino acid FLAG epitope substituted for the entire predicted cytoplasmic C-terminal domain. Our findings show that N-terminal deletion mutants of LMP-1 are unable to attain their usual patched distribution on the plasma membrane but retain the ability to activate B cells. In contrast, the C-terminal deletion mutant shows the same patched cell surface distribution as wild-type LMP-1 but is unable to activate B cells. The patched distribution of LMP-1 in the plasma membrane is therefore not sufficient nor necessary for the induction of B cell activation and the results rule out patching as a direct mechanism in LMP-1-induced activation. This is the first study addressing the role of the LMP-1 C-terminal domain in lymphocytes and our results show that this domain is essential for B cell activation and therefore likely to be important for the early events of B cell immortalization by EBV.
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The role of repetitive DNA sequences in the size variation of Epstein—Barr virus (EBV) nuclear antigens, and the identification of different EBV isolates using RFLP and PCR analysis
The six Epstein—Barr virus (EBV) nuclear antigen proteins (EBNA-1–6) show characteristic size variations between different virus isolates; this is a feature that has been used to identify the source of virus isolates in epidemiological studies (Ebnotyping). We have now studied the correlation between restriction fragment length polymorphisms (RFLPs) within exons coding for the EBNAs and the molecular masses of the respective proteins. The B95-8 EBV strain was used as the prototype virus. The variation in apparent molecular mass of EBNA-1, -3 and -6 correlated positively with the size of RFLP coding for repeat sequences in these polypeptides. For EBNA-2, no correlation between apparent molecular mass and length of the repetitive sequences was found. The EBNA-4 protein showed virtually no variation in apparent molecular mass and RFLP size across the repeat sequence. Based on the strong correlation between apparent molecular mass and RFLP size for EBNA-6, we developed an EBNA-6 PCR assay that discriminated between different isolates of EBV. This assay offers the advantage of EBV characterization using uncultured material (e.g. throat washings, blood or biopsies), thus avoiding the selection against poorly transforming strains that occurs during establishment of lymphoblastoid cell lines required for Ebnotyping at the protein level.
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The cellular RING finger protein PML is not a functional counterpart of the herpes simplex virus type 1 RING finger protein Vmw110
More LessHerpes simplex virus type 1 (HSV-1) immediate early protein Vmw110 (also known as ICP0) is required for the fully efficient expression of viral genes during onset of lytic growth and for normal reactivation from latency. Both Vmw110 and the cellular protein PML are members of the RING finger family of zinc binding domain proteins, a family which includes an increasing number of examples from a wide evolutionary range. The function of the RING finger domain is unknown, and the question arises whether the RING finger (like several other examples of conserved domains) fulfils similar functions in these diverse proteins. Another link between Vmw110 and PML is that at early times of HSV-1 infection Vmw110 migrates to distinct nuclear structures which contain the PML protein. In order to test the possibility that PML and Vmw110, or their RING finger domains, fulfil similar functions, we have constructed recombinant viruses that express either intact PML, or a chimeric Vmw110 protein which contains the PML RING finger in place of its own. The results indicate that the PML and Vmw110 RING fingers are not functionally interchangeable, and that PML is not a cellular functional counterpart of Vmw110.
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Sequence of RNA2 of the Helicoverpa armigera stunt virus (Tetraviridae) and bacterial expression of its genes
More LessThe complete nucleotide sequence of RNA2 of Helicoverpa armigera stunt virus (HaSV), a member of the Tetraviridae, was determined by characterization of cloned cDNA and PCR products and direct sequencing of genomic RNA. The capped, positive sense, single-stranded RNA is 2478 nucleotides in length and has two overlapping open reading frames (ORFs) likely to be cistrons which are situated between terminal non-coding regions of 282 and 168 bases, 5′ and 3′, respectively. Extensive secondary structure of the RNA strand is indicated, including a tRNA-like structure at the 3′ terminus which is the first such structure discerned in an animal virus. The first ORF encodes a 17 kDa PEST protein (p17) of unknown function while the second ORF encodes the 71 kDa coat protein precursor (p71) that is cleaved at an Asn-Phe site into the 64 kDa and 7 kDa coat proteins. The precursor coat protein is 66% identical to that of another tetravirus, the Nudaurelia ω virus, with most of the difference residing in a 165 amino acid region located in the middle of the sequence. Despite the extensive similarity, no serological relationship was observed between the two viruses, suggesting that the dissimilar region is exposed on the capsid exterior. Expression in bacteria of the two RNA2 gene products shows they are likely to be expressed by a leaky scan-through mechanism. Bacterial expression of p71 did not produce virus-like particles while expression of p17 produced large arrays of mostly hollow, hexagonal tube-like structures.
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Characterization of hepatopancreatic parvo-like virus, a second unusual parvovirus pathogenic for penaeid shrimps
More LessThe hepatopancreatic parvo-like virus (HPV) of penaeid shrimp was extracted from infected shrimp tissues, purified and subsequently characterized. The viral particles, icosahedral in shape, are 22 nm in diameter and possess a buoyant density of 1.41 g/ml. They contain ssDNA, of approximately 5 kb in size which encodes a single polypeptide of 54 kDa. On the basis of its general characteristics this pathogenic agent belongs to the Parvoviridae family, but because of two unusual characteristics (capsid protein formed with a single polypeptide and genome structure more closely related to the autonomous parvoviruses rather than the densoviruses), it seems to constitute a novel group in the Parvoviridae family.
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Mutations in the human papillomavirus type 16 E2 protein identify a region of the protein involved in binding to E1 protein
More LessPapillomavirus DNA replication is primarily dependent upon two viral gene products, E1 and E2. Work with bovine papillomavirus has shown that the E2 protein can bind directly to the E1 protein and enhance the binding of E1 to the viral origin of replication. However, little is known about the mechanism of interaction between E1 and E2 proteins. In this study we have analysed in detail the association between human papillomavirus type 16 (HPV-16) E1 and E2 proteins. Using a purified glutathione S-transferase-HPV-16 E1 fusion protein from Escherichia coli and E2 proteins produced by in vitro transcription-translation, we have developed a rapid and simple method for investigating the association between E1 and E2 in vitro. The binding of E2 to E1 was found to be dependent on sequences in the N-terminal activation domain of the E2 protein. Truncated forms of E2, including a putative repressor form of E2 encoding the DNA binding domain, failed to associate with E1 in this assay. The region of E2 required for efficient binding to E1 was then localized using mutants in the activation domain of E2. These results demonstrated that only a short region of E2 was required for association with E1. This region of E2 was found to be highly conserved amongst all papillomaviruses, suggesting a conservation of E2 function and a common mechanism of interaction between these virally encoded proteins.
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Detection of silencer activity in the long control regions of human papillomavirus type 6 isolated from both benign and malignant lesions
More LessHuman papillomavirus type 6 (HPV-6) DNA is the predominant HPV type found in condyloma acuminata: it is rarely found in carcinomas. We have previously reported cloning and characterizing an HPV-6 from a vulvar condyloma (HPV6-W50) and an HPV-6 from a vulvar carcinoma (HPV6-T70). The E5, E6 and E7 proteins encoded by the two genomes were identical, however, the two genomes differed in the long control region (LCR). Cloning of the entire LCR into the enhancerless plasmid pSVEcat showed that the two LCRs had comparable enhancer activity. Since the major differences between the two LCRs resided in the 5′ end of the LCR, upstream of the L1 polyadenylation signal, we subcloned the two LCRs to analyse more closely their effect on cat gene expression. The data indicated that LCR subclones of the two genomes had comparable chloramphenicol acetyltransferase (CAT) activity. A negative regulatory region was detectable when the test plasmids were transfected into HeLa and C33A cells and in primary keratinocytes. A decrease in CAT activity was also detected when the SV40 early promoter was replaced with the putative HPV-6 E6 promoter. The negative regulatory region functioned in a position- and orientation-independent manner, thus fulfilling the definition of a silencer.
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The human immunodeficiency virus type 1 Nef protein functions as a protein kinase C substrate in vitro
More LessThe human immunodeficiency virus type 1 Nef protein was expressed in Escherichia coli as a C-terminal fusion with glutathione S-transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified GST-Nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. In the presence of [γ32P]ATP, phosphorylation of Nef occurred predominantly on serine residues. Studies with protein kinase inhibitors suggested that protein kinase C (PKC) was involved in Nef phosphorylation. This was supported further by the demonstration that purified PKC was also able to phosphorylate Nef in the absence of cell extract. Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-terminal GST or 6-histidine tag in Spodoptera frugiperda insect cells by recombinant baculoviruses. In extracts from Jurkat T cells and U937 monocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro. Phosphorylation of this Nef-associated protein was inhibited by heparin and is thus likely to be mediated by casein kinase II. The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components.
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Revertants and pseudo-revertants of human immunodeficiency virus type 1 viruses mutated in the long terminal repeat promoter region
More LessThe TAR domain is an RNA secondary structure element within the leader transcript of the human immunodeficiency virus type 1 (HIV-1) virus. TAR RNA forms the binding site for the viral trans-activator protein Tat and cellular co-factors that are involved in induction of the LTR transcriptional promoter. Here, we report that mutations in the single-stranded bulge- and loop-domains of TAR RNA impair the ability of the virus to replicate in T cell lines. Revertant viruses were isolated upon prolonged culturing and analysed through sequencing. The reversion data confirm the importance of both bulge and loop as sequence-specific recognition motifs. We also analysed the replication phenotype of a mutant HIV-1 virus with a substitution in the -19/-3 promoter region. This mutant displayed delayed infection kinetics compared to the wild-type virus, and revertants with increased replication potential could be isolated. Interestingly, all revertants had acquired an additional mutation at position -2. Primer extension analyses revealed that an upstream shift in transcription start site usage was induced by the -19/-3 substitution. This effect was compensated for by the nucleotide substitution near the RNA start site.
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Brefeldin A and monensin arrest cell surface expression of membrane glycoproteins and release of rubella virus
More LessThe maturation of rubella virus (RV) glycoproteins E2 and E1 was examined by using brefeldin A (BFA) and monensin. BFA, which induces the rapid redistribution of Golgi enzymes residing in the Golgi complex into the endoplasmic reticulum (ER), was used to locate the intracellular site for the modification of carbohydrate side-chains on RV E1 and E2 proteins. The monovalent ionophore monensin, which inhibits intracellular transport of proteins through the ER-Golgi complex, was used to block the transport of E1 and E2 glycoproteins through the Golgi complex. BFA and monensin effectively blocked the cell surface expression of RV E2 and E1 proteins, secretion of an anchor-free form of E2 and budding of RV from the plasma membrane. For O-linked glycosylation, addition of N-acetylgalactosamine and galactose to E2 protein was found to take place in the medial to the trans Golgi. A dramatic change in the intracellular distribution of RV structural proteins was observed when transfected COS cells were treated with BFA or monensin, although the proteolytic processing of RV structural protein precursor was not affected. In the presence of BFA or monensin, virus release from infected Vero cells was only 0.1% of the intracellular virus, and the intracellular virus titre decreased as well. Our results suggest that O-linked glycosylation on the E2 protein occurred in the post-ER region and the transport of RV structural proteins to the Golgi complex and post-Golgi compartment may be a rate-limiting step in RV assembly and budding.
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Neurovirulence and neuroinvasiveness of Murray Valley encephalitis virus mutants selected by passage in a monkey kidney cell line
More LessVariants of the prototype Murray Valley encephalitis virus (MVE-1-51) were selected by serial plaque purification and amplification in monkey kidney (Vero) cells. Four clones (C1–C4) at passage levels two and nine (P2 and P9) were examined in 21-day-old Swiss outbred mice for neuroinvasiveness (assessed from LD50 values after intraperitoneal inoculation) and neurovirulence (LD50 values after intracranial inoculation). The growth characteristics of the clones were determined in intracranially inoculated mouse brain and in mouse neuroblastoma, Vero and mosquito (C6/36) cell lines. Genomic RNA of the cloned virus stocks was sequenced through the structural protein genes (E, prM/M and C) and the 5′ untranslated region. Clone C2P2 was of high neuroinvasiveness whereas C2P9 was of low neuroinvasiveness; there were also decreased yields of C2P9 in C6/36 cells compared to C2P2 and MVE-1-51. These changes were associated with the substitution of valine for phenylalanine at amino acid position 141 of the C2P9 E protein. Clone C4P2 was of high neurovirulence and low neuroinvasiveness; C4P9 was of low neurovirulence, a change accompanied by a further reduction in neuroinvasiveness. Concomitantly, C4P9 showed a pronounced reduction in growth rates and yields in 21-day-old Swiss mouse brain, in mouse neuroblastoma cells and in C6/36 cells compared to parental virus. The phenotypic changes in clone 4 appear to be due to mutation(s) within non-structural protein genes.
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Coronavirus-induced encephalomyelitis: balance between protection and immune pathology depends on the immunization schedule with spike protein S
More LessThe neurotropic mouse hepatitis virus MHV-JHM induces central nervous system (CNS) demyelination in Lewis rats that pathologically resembles CNS lesions in multiple sclerosis. The mechanisms of MHV-JHM-induced demyelination remain unclear and several studies have implicated the role of the immune response in this process. We have shown previously that protective immunity against MHV-JHM-induced encephalomyelitis was induced by immunization with a vaccinia virus (VV) recombinant expressing MHV-JHM S-protein (VV-S). Here, we present evidence that the time of MHV-JHM challenge after immunization with VV-S plays a critical role in protective immunity. The induction of virus-neutralizing S-protein-specific antibodies prior to the MHV-JHM challenge modulates the disease process and a subacute encephalomyelitis based on a persistent virus infection developed. Typical pathological alterations were lesions of inflammatory demyelination. In addition, the results indicate that after seroconversion, CD8+ T cells were no longer essential for virus elimination in contrast to their role in protection during acute encephalomyelitis.
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Analysis of hepatitis C virus quasispecies populations by temperature gradient gel electrophoresis
More LessHepatitis C virus (HCV) forms complex quasispecies populations which consist of a large number of closely related genetic variants. This genetic heterogeneity may cause antigenic variation or drug resistance. We used heteroduplex analysis by temperature gradient gel electrophoresis (TGGE) to characterize genetic variants of HCV. The high resolution of TGGE was proven by comparison of DNA sequence data of different cDNA clones from the HCV 5′NCR with their corresponding migration pattern in TGGE. Using this method we were able to identify virus variants of the HCV 5′NCR even if they only differed from each other by a single base. HCV populations from three patients with chronic hepatitis C were found to consist of genetic variants, although the degree of the heterogeneity varied. In addition, we compared the genetic heterogeneity of the core and E2 regions of the HCV genome in one patient. Our results demonstrate that TGGE is a useful tool for characterization of the genetic heterogeneity of virus populations in vivo.
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All rabbits immunized with type A influenza virions have a serum haemagglutination-inhibition antibody response biased to a single epitope in antigenic site B
More LessNine rabbits were immunized with type A influenza virions and the epitope specificities of the secondary serum haemagglutination-inhibition (HI) antibody response were analysed with a panel of neutralizing monoclonal (MAb) antibody double escape mutants. Each of the latter was made by sequential selection using a MAb directed to an epitope of a discrete antigenic site, site A, site B or site D, of the haemagglutinin (HA). Thus the epitope reactivity of the escape mutants was represented as A+B−D−, A−B+D− and A−B−D+. The HI antibody response of all antisera was biased to the site B epitope. In 9/12 antisera, obtained from seven rabbits immunized with whole virions, the site B epitope was predominant, representing 65–82% of the total HI antibody. The restriction of HI antibody was unaffected by strain of rabbit, route of inoculation (intravenous or subcutaneous), use of Freund’s adjuvant, and up to four immunizing injections. In 3/7 rabbits immunized with whole virus, there was a HI antibody response to the HC2 (site A) or HC10 (site D) epitope, but not both, of equal magnitude to the site B epitope. The HI antibody response in one of the rabbits (#40) became more biased to the site B epitope between the third and fourth immunizing doses. Two further rabbits were immunized with virions which had been partially digested with bromelain and then purified from free HA. Both of these made equal HI antibody responses to the site B epitope and the site D epitope, possibly because their remaining HA spikes were better exposed. Overall, these data demonstrate an unexpected degree of restriction in the production of biologically relevant antibody, such that some rabbits (e.g. #45) mount an HI antibody response which is essentially epitope-specific. Implications for epitope specificity of HI antibody stimulated by human influenza vaccines, and also for the generation of antigenic drift variants are discussed. The reason for the non-responsiveness of the immune system to the many other HI epitopes of the HA is not known.
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Maturation of the dengue-2 virus NS1 protein in insect cells: effects of downstream NS2A sequences on baculovirus-expressed gene constructs
More LessA series of recombinant baculoviruses was constructed in order to study the influence of downstream NS2A sequences on the processing of the dengue virus NS1 glycoprotein in insect cells. NS1 alone was expressed at a high level in its native dimeric form and processed efficiently through the Spodoptera frugiperda (Sf) cell secretory pathway. Recombinant NS1 was found associated with the plasma membrane and was also secreted into the extracellular medium. Although both intra- and extracellular NS1 were processed to an endo H-resistant form in Sf cells, Triton X-114 phase separation analysis further suggested that some modifications in addition to dimerization account for the hydrophobic properties of NS1, and that N-glycosylation was therefore not the only difference between the cell-associated and secreted forms. Cleavage at the NS1-NS2A junction of these recombinants demonstrated that as few as 26 amino acids from the N terminus of NS2A provide a sufficient, but not optimal, recognition sequence for a functional cleavage mediated by a protease present in Sf cells infected with recombinant Autographa californica nuclear polyhedrosis virus expressing NS1.
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Identification of the protease domain in NS3 of hepatitis C virus
More LessNS3 of hepatitis C virus (HCV) is a serine protease that carries out the proteolytic processing of the nonstructural proteins of the HCV polyprotein. Deletion analysis of the N terminus of NS2,3,4 fusion protein revealed that the N-terminal boundary of the active protease resides between amino acids 1050 and 1083. The processing patterns of internal deletion mutants of NS2,3,4 indicated that the C terminus of the enzymically active protease resides between amino acids 1115 and 1218. The N- and C-terminal boundaries of the protease were also confirmed by determining the trans-cleavage activity of internally deleted NS3,4. NS3 protease activity was inhibited by Cu2+ but was slightly enhanced by Zn2+. This report provides a possible approach for development of antiviral agents based on protease inhibitors.
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Adelaide River virus nucleoprotein gene: analysis of phylogenetic relationships of ephemeroviruses and other rhabdoviruses
More LessThe nucleotide sequence of the Adelaide River virus (ARV) genome was determined from the 3′ terminus to the end of the nucleoprotein (N) gene. The 3′ leader sequence comprises 50 nucleotides and shares a common terminal trinucleotide (3′ UGC-), a conserved U-rich domain and a variable AU-rich domain with other animal rhabdoviruses. The N gene comprises 1355 nucleotides from the transcription start sequence (AACAGG) to the poly(A) sequence [CATG(A)7] and encodes a polypeptide of 429 amino acids. The N protein has a calculated molecular mass of 49429 Da and a pI of 5.4 and, like the bovine ephemeral fever virus (BEFV) N protein, features a highly acidic C-terminal domain. Analysis of amino acid sequence relationships between all available rhabdovirus N proteins indicated that ARV and BEFV are closely related viruses (48.3% similarity) which share higher sequence similarity to vesiculoviruses than to lyssaviruses. Phylogenetic trees based on a multiple sequence alignment of all available rhabdovirus N protein sequences demonstrated clustering of viruses according to genome organization, host range and established taxonomic relationships.
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Influenza virus NS1 protein alters the subnuclear localization of cellular splicing components
More LessIntranuclear structure was studied in influenza virus-infected cells by immunofluorescence microscopy with antibodies specific for fibrillarin, the splicing factor SC35 and the autoantigen p80 coilin. In the course of the infection, an increase in the number of coiled bodies was observed, with a parallel decrease in their size. In addition, the normal speckled pattern of the SC35 factor was altered to generate a more punctate distribution. However, no alteration was observed in the fibrillarin staining pattern. Since an alteration in the splicing of both viral and cellular mRNAs upon expression of influenza virus NS1 protein has been reported previously, the possible effects of NS1 expression on intranuclear structure were assayed. The increase in the coiled body numbers was not specific for the expression of NS1 protein, but alterations in the nuclear location of small ribonucleoprotein particles, as determined by immunofluorescence with an anti-Sm serum or the SC35 splicing factor, were produced by the sole expression of NS1 protein. These results correlate with the previously reported inhibition of splicing induced by NS1 protein expression and suggest an interaction of this influenza virus protein with the cellular splicing machinery.
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Structure of influenza virus ribonucleoprotein particles. II. Purified RNA-free influenza virus ribonucleoprotein forms structures that are indistinguishable from the intact influenza virus ribonucleoprotein particles
More LessInfluenza virus nucleoprotein (NP) was purified from the virus and found to be virtually free from RNA. The morphology of the negatively stained NP was studied using electron microscopy. The monomer protein was found to be a small rod with dimensions of 62 by 35 Å. However, most of the protein was found to exist in polymeric forms ranging from trimers to large structures that were morphologically indistinguishable from the intact viral RNP. Each monomer has two sites for NP–NP contacts at one extremity of the rod. The consequences of this finding for crystallization studies, for in vitro studies on NP–RNA interactions and the possible consequences for in vivo ribonucleoprotein particle assembly are discussed.
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Virus load in chimpanzees infected with human immunodeficiency virus type 1: effect of pre-exposure vaccination
Many reports indicate that a long-term asymptomatic state following human immunodeficiency virus type 1 (HIV-1) infection is associated with a low amount of circulating virus. To evaluate the possible effect of stabilizing a low virus load by non-sterilizing preexposure vaccination, a quantitative virus isolation method was developed and evaluated in four chronically infected chimpanzees infected with a variety of HIV-1 related isolates. This assay was then used to monitor a group of chimpanzees (n = 6) challenged with HIV-1 following vaccination with gp120 or gp160. Data indicated that of the three vaccinated animals which became infected after challenge, the animal with the lowest neutralizing titre at the time of challenge acquired a virus load similar to the control animals, whereas the two other chimpanzees had reduced numbers of virus producing cells in their peripheral circulation. One animal became virus isolation negative, developed an indeterminant PCR signal on lymph node DNA and subsequently became negative for HIV-1 DNA as determined by PCR on PBMC (peripheral blood mononuclear cells) and bone marrow DNA. Recently, the second animal has also become PCR negative. To confirm observations from quantitative virus isolations, quantification of HIV-1 DNA in PBMC and virus RNA in serum was performed by PCR on serially diluted samples at two different time points. Comparison of virus load as determined by these three methods confirmed that there was an effect of vaccination in reducing virus load and demonstrated a correlation between decreased numbers of virus producing cells, HIV-1 DNA containing cells and virus RNA molecules in serum.
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Human T cell leukaemia virus type I env gene-transfected HeLa cells display a decrease in cell fusion ability
More LessThe envelope (env) gene of human T cell leukaemia virus type I (HTLV-I) was inserted into an expression vector, referred to as phMTenv, under the transcriptional control of the human metallothionein IIa gene promoter (hMT-IIa). When this vector was transiently transfected in HeLa cells treated with hMT-IIa inducers, formation of multinucleated cells was observed, indicating the expression of functional surface and transmembrane glycoproteins. Of several HeLa cell clones transfected with phMTenv together with a plasmid carrying the neomycin resistance gene and isolated after selection in G418-containing medium, env mRNA was detected in only two, in the presence of hMT-IIa inducers. Viral glycoproteins were found to be weakly expressed, as detected in immunoprecipitation assays of 125I-surface-labelled cells. These env-transfected HeLa cell clones, although unable to form syncytia when cocultivated with untransfected control HeLa cells, retained the capacity to fuse with HTLV-I-producing C91PL T cells. However, a significant decrease in their fusogenic ability was observed, after treatment with hMT-IIa inducers. Under identical experimental conditions, control HeLa cell clones stably transformed with the same plasmid, but lacking the env gene, were still able to fuse with C91PL cells. These observations suggest that a post-transcriptional step in HTLV-I env expression is impaired, probably leading to the establishment of superinfection interference.
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Effect of methyltransferase inhibitors on the regulation of baculovirus protein synthesis
More LessIn the presence of the methyltransferase inhibitor 3-deazaadenosine (3DA-Ado) the production of infectious Autographa californica nuclear polyhedrosis virus (AcMNPV) in tissue culture was only slightly affected, while the synthesis of very late proteins (polyhedrin and p10) was abolished. The synthesis of the influenza virus proteins NS1 and HA, expressed under the polyhedrin promoter, was also abolished by 3DA-Ado. Furthermore, 3DA-Ado interfered with the shut-off of early and late AcMNPV proteins. Most of these results were also obtained with 5-azadeoxycytidine (5A-dCyt). In cells in which NS1 was produced abundantly, at least one specific AcMNPV protein was not synthesized. However, if the production of NS1 was inhibited by 3DA-Ado, or if HA was synthesized instead, this AcMNPV protein showed up normally.
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Differences in the target specificity of the transactivating factors MHBst and HBx of hepatitis B virus
More LessTransient transfections of tissue culture cells with plasmids encoding the transactivating factors MHBst and HBx of hepatitis B virus result in transcriptional stimulation of multiple target genes. Our experiments show that the NF-κB-binding enhancer element of simian virus 40 (SV40) and the AP-1-binding enhancer element of the human metallothionein IIA gene mediate the transactivating function of MHBst and HBx. In contrast, the elements GT(IIC+I) and Sph(II+I) of the SV40 enhancer, that, as a common feature, require binding of transcription factor TEF-1 for activity, efficiently mediate transactivation only by HBx but not by MHBst. This finding suggests that at least one regulatory pathway exists that can only be activated by HBx but not by MHBst.
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Importance of the C terminus of the hepatitis B virus precore protein in secretion of HBe antigen
More LessThe hepatitis B virus (HBV) e antigen (HBeAg) is a 15 kDa soluble antigen derived from a precursor protein (precore protein) by two processing events, cleavage of the N-terminal signal peptide and cleavage of the C-terminal 34 amino acids. So far, the role of the C-terminal sequences in secretion has not been analysed in full. In this study deletion of the last 60 amino acids was found to abrogate HBeAg secretion whereas deletions of the last 10, 25 or 39 amino acids decreased its secretion rate. These data demonstrate that C-terminal precore protein sequences are crucial for HBe secretion and determine its secretion rate.
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Hepatitus B virus: specific binding and internalization of small HBsAg by human hepatocytes
More LessPreviously, we identified human liver endonexin II (EII) present on human hepatocyte plasma membrane as a specific hepatitis B surface antigen (HBsAg) binding protein. We also showed the spontaneous development of anti-idiotypic (anti-HBsAg) antibodies in rabbits immunized with EII and in chicken immunized with the F(ab′)2 fragment of rabbit anti-EII IgG. These findings suggest the existence of a receptor-ligand relationship between EII and HBsAg. In the present study, we demonstrate that small HBsAg conjugated to 10 nm colloidal gold also binds specifically to human hepatocytes. Invagination of the coated pit region at the HBsAg binding sites on the human hepatocyte plasma membrane results in the internalization of the HBsAg-gold particles. The binding and consequently the internalization of HBsAg is inhibited by anti-EII or anti-idiotypic (anti-HBsAg) antibodies. These findings indicate that EII is directly involved in the binding and uptake of hepatitis B envelope proteins.
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Expression of adenovirus type 5 E4 Orf2 protein during lytic infection
More LessThe human adenovirus type 5 E4 transcription unit has the potential to encode at least seven distinct polypeptides from reading frames accessed by differential splicing of a single primary transcript. Only some of these polypeptides have yet been detected during viral infection of cultured cells. Mutational inactivation of the reading frames whose products have not been described has no apparent effect on the growth of virus in standard cultured human cell lines, indicating that these proteins, if they exist, have only a subtle, non-essential role in the replication cycle. We have raised an antiserum to one of these undefined products, E4 Orf2, expressed in bacteria. Using this reagent, it was possible to show that Orf2 was expressed during the lytic cycle in HeLa cells, being a soluble cytoplasmic component appearing with early kinetics. No association of Orf2 protein with other infected cell components was detected.
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The use of general primers GP5 and GP6 elongated at their 3′ ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR
Sequence analysis of human papillomavirus (HPV) general primer GP5/6 mediated PCR products revealed the presence of short highly conserved sequences adjacent to the 3′ ends of both primers. Part of these sequences was used to elongate GP5 and GP6 at their 3′ ends to generate the primers GP5+ and GP6+, respectively. Compared with the GP5/6 PCR, GP5+/6+ specific PCR on 22 cloned mucosotropic HPVs revealed an improved HPV detection, reflected by a 10- to 100-fold higher sensitivity and a markedly increased signal to background ratio, especially at the gel level. As determined on purified DNA, the sensitivity of this GP5+/6+ based assay was at the femtogram level for those HPV genotypes which match strongly with the primers (e.g. HPV-16) and at the picogram level for HPV types (e.g. HPV-39 and -51) having four or more mismatches with one or both primers. Application of both methods on 264 cervical scrapes of a cohort of women participating in a prospective follow-up study revealed an increase of total HPV positivity from 39% (GP5/6 PCR) to 43% (GP5+/6+PCR) of the scrapes. Additional HPV typing by PCR specific for the HPV-6, -11, -16, -18, -31 and -33 revealed that all GP5+/6+ PCR positive cases which were negative by GP5/6 PCR (n = 12) contained HPV types different from these six types. These data indicate that the GP5+/6+ PCR method provides an increased detection level mainly of uncommon, apparently poorly matched HPV types in cervical scrapes and most likely in the enlargement of the spectrum of HPVs detectable by this assay.
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Alcelaphine herpesvirus type 1 has a semaphorin-like gene
More LessAlcelaphine herpesvirus type 1, a rhadinovirus causing malignant catarrhal fever of ruminants, has an 1959 nucleotide open reading frame with significant homologies to semaphorin genes. While truncated genes of similar structure have been found in poxviruses, this is the first known example of a semaphorin-like gene in a herpesvirus.
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Nucleotide sequence of the herpes simplex virus type 2 gene encoding the protease and capsid protein ICP35
More LessThe complete nucleotide sequence of the gene encoding the protease and ICP35 capsid protein of herpes simplex virus (HSV) type 2 strain G has been determined. The gene consisits of a 1908 bp open reading frame capable of encoding 636 amino acids. Comparative nucleotide sequence analysis of the gene with published HSV-1 sequences revealed that the HSV-2 nucleic acid segment most likely encodes two distinct proteins. The first protein is the HSV-2 protease, which is required for the assembly of the herpes virus capsid. The second protein has been previously designated as ICP35 in HSV-1, and belongs to the family of herpesvirus capsid proteins.
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The 119 kDa and 124 kDa polyproteins of arabis mosaic nepovirus (isolate S) are encoded by two distinct RNA2 species
More LessArabis mosaic virus (ArMV) is a nepovirus that is serologically distantly related to grapevine fanleaf virus (GFLV). Both ArMV and GFLV induce grapevine degeneration disease. Several ArMV isolates, unlike isolates of GFLV, produce upon in vitro translation of RNA2 a polyprotein (P2) that forms a double band in polyacrylamide-SDS gels. Cloning of full-length copies of RNA2 of an ArMV isolate from grapevine (ArMV-S) revealed that this isolate contained two RNA2s of different length, called RNA2-U and RNA2-L. The two species were not readily separated by electrophoresis of the virion RNA under denaturing gel electrophoresis conditions but could be distinguished by analysis of primer extension and in vitro translation products. The size difference of the two RNA2s is due mostly if not exclusively to differences in their coding regions. The 124 kDa RNA2-U-encoded polyprotein P2′ and the 119 kDa RNA2-L-encoded polyprotein P2″, which comigrate, respectively, with the upper and lower polyprotein bands produced by RNA2 of ArMV-S, were more than 95% identical except in their N-terminal domains. In vitro maturation experiments and sequence comparisons indicate that the N-terminal products of P2′ and P2″ have a molecular mass of 31 kDa and 26 kDa. The genomic organization proposed is similar to that of GFLV RNA2.
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Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo
More LessThe putative 38 kDa movement protein (P38) gene, located on the RNA2 of grapevine fanleaf nepovirus (GFLV), was cloned in Escherichia coli and expressed as a fusion protein fused to six histidines (6HisP38). This protein was purified and used to produce a specific polyclonal antiserum from which immunoglobulins were isolated by immunoaffinity against the recombinant 6HisP38. Western immunoblot analyses on GFLV RNA2 in vitro maturation products showed that the antibodies were specific for P38 protein. This protein was detected as early as 18 h post-inoculation in GFLV-infected Chenopodium quinoa protoplasts and accumulated to very high levels. Tissue-prints and time course experiments on infected C. quinoa plants confirmed that P38 is present at a high level late in infection and is a final maturation product of the GFLV RNA2 polyprotein in vivo. P94 and P66 intermediates of maturation and polyprotein P2 were also detected in vivo but in very low concentrations. No significant difference was observed in the relative amounts of P66 and P94 detected in vivo, contrary to what occurs in vitro. Subcellular fractionation studies showed that P38, although mainly cytosolic, is also found in association with cell wall and membranes. Thought to be the GFLV movement protein, P38 would thus behave in an ‘atypical’ manner.
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Tomato ringspot nepovirus protease: characterization and cleavage site specificity
More LessWe have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have demonstrated that proteins produced from these clones possess a proteolytic activity in trans on the cleavage site between the TomRSV movement and coat proteins. By amino acid homology of the TomRSV 3C-related protease with other nepo- and comovirus proteases, His1283, Glu1331 (or Asp1354) and Cys1433 have been predicted to constitute the catalytic triad. Site-directed mutagenesis of His1283 to Asp abolished the TomRSV protease activity, in vitro and in vivo. The cleavage site between the TomRSV movement and coat proteins has been determined to be Q/G, by direct protein sequencing. Previously, His1451 located in the substrate binding pocket of the TomRSV 3C-related protease has been suggested to be involved in the cleavage site specificity. We show that an inactive TomRSV 3C-related protease is obtained after substitution of His1451 with Leu. These results are discussed in light of the possible relation of the TomRSV 3C-related protease to 3C-related proteases of nepo-, como- and potyviruses.
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The nucleotide sequence of RNA3 and RNA4 of olive latent virus 2
More LessOlive latent virus 2 (OLV2), a virus with particle shapes resembling those of alfalfa mosaic alfamovirus (AMV), has four major RNA species, two of which (RNA3 and RNA4) were completely sequenced. RNA3 was a bicistronic molecule containing two clear-cut ORFs, one of which (ORF1) coded for a 36.5 kDa polypeptide with conserved motifs of the ‘30K superfamily’ movement proteins and the other (ORF2) encoded a 20 kDa polypeptide identified as the viral coat protein. RNA4, which was a little smaller than RNA3 (2078 nt versus 2438 nt), also differed from RNA3 in a few positions, but its in vitro translation did not produce any protein. By contrast, an additional RNA, 1042 nt in size with strong sequence homology with RNA3 and RNA4, was identified in RNA extracts from infected plants. This RNA, which may be a subgenomic form of RNA3 carrying the coat protein cistron, is apparently encapsidated to a very small extent, or not at all. Comparative computer-assisted analysis of virus-coded protein sequences disclosed homologies suggesting that OLV2 may belong to the family Bromoviridae, but as an entity separated from the currently known genera.
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Sequence polymorphism in the 5′NTR and in the P1 coding region of potato virus Y genomic RNA
Potato virus Y (PVY) the type member of the genus Potyvirus, occurs world-wide as isolates which differ in host range and the type of symptoms caused. The sequences of a 5′ segment of viral RNA overlapping the 5′ non-translated region (5′NTR) alone (ten isolates) or the 5′NTR and the adjacent P1 coding region (eight isolates) were established. These data were used to quantify the polymorphism in the 5′-terminal part of the PVY genome. Nucleotide sequence identity between isolates ranged from 66–100% in the 5′NTR and from 70–100% in the P1 coding region. The lowest amino acid sequence similarity between PVY P1 was 77%, illustrating the high variability of this protein in the PVY species. Phylogenetic trees based on either 5′NTR or P1 sequence analyses resulted in the same clustering of the studied isolates into three groups. Group I comprises potato isolates all inducing ‘tobacco veinal necrosis’ symptoms. Group II contains isolates inducing either ‘tobacco veinal necrosis’ or mosaic symptoms in tobacco. Group III contains mainly pepper or tomato isolates inducing mosaic symptoms in tobacco and shows a geographical clustering of the Tunisian isolates. This clustering into three groups is discussed in comparison with phylogenetic trees previously obtained from capsid gene or 3′NTR sequence analysis in the PVY species. Multiple sequence alignment indicated conserved motifs potentially involved in viral functions.
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Processing of the plum pox virus polyprotein at the P3−6K1 junction is not required for virus viability
More LessProteolytic processing of the potyvirus polyprotein is mainly performed by the virus-encoded NIa protease, whose cleavage sites are characterized by conserved heptapeptide sequences. Partial processing at the cleavage site present between the P3 and 6K1 cistrons by the plum pox potyvirus (PPV) NIa protease has been previously shown to occur in vitro. We have now studied the role of polyprotein processing at the P3−6K1 junction in vivo, using a full-length PPV cDNA clone. PPV mutant transcripts containing a histidine for glutamine substitution in the cleavage site sequence (a change that abolishes in vitro processability) are able to infect Nicotiana clevelandii plants, indicating that normal processing at the P3−6K1 junction is not required for virus viability. However, disease symptoms were not detected and virus accumulation occurred after a second site mutation was introduced into the 6K1 cistron during replication. This additional change did not restore the in vitro processability of the mutant heptapeptide. Changes at other positions in the heptapeptide (that only slightly altered the in vitro processability of this NIa site) were also engineered and it was found that these mutations affected the time course and severity of the symptom induction process. A possible regulatory effect on the function of the potyvirus P3+6K1 protein by processing at the P3−6K1 junction is discussed in light of our present results with PPV.
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The complete nucleotide sequence of RNA 3 of citrus leaf rugose and citrus variegation ilarviruses
S. W. Scott and Xin GeComplete sequence data for the RNA 3 of both citrus leaf rugose (CiLRV) and citrus variegation (CVV) ilarviruses have been determined. The RNAs are 2289 nt (CiLRV) and 2309 nt (CVV) in length and both contain the typical Bromoviridae arrangement of two open reading frames (ORFs) which, when translated, code for proteins that correspond to the M r 32 000 (32K) putative movement proteins (ORF 1) and the coat proteins (ORF 2) of the respective viruses. The 3′ termini of both viruses can be folded to form a secondary structure similar to those reported for other ilarviruses. These are the first complete nucleotide sequences for RNA 3 of members of subgroup 2 of the ilarviruses. The two viruses share substantial homology in nucleic acid sequence, code for identically sized coat proteins and share high levels of identity in the translated products of both ORFs. Although related, these viruses differ sufficiently to be considered distinct. The RNA 3s of CiLRV and CVV appear to be distinct from those of other ilarviruses for which comparable sequence data are available and also from the closely related alfalfa mosaic virus.
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Vectors based on maize streak virus can replicate to high copy numbers in maize plants
More LessThe genome of maize streak virus (MSV) consists of one molecule of circular, single-stranded DNA of 2.7 kb. A reporter gene (bar) coding for phosphinothricin acetyltransferase was inserted into the small non-coding region of the MSV genome. The recombinant bar-containing MSV vectors were introduced into maize seedlings via agroinfection. The chimeric viral DNA was found to replicate to high copy numbers in maize leaves resistant to the application of the herbicide Basta. This establishes the usefulness of MSV as an efficient replicating vector in cells of maize plants.
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Mixed-subunit capsids can be assembled in vitro with coat protein subunits from two cucumoviruses
More LessVirus particles were reassembled in vitro from tomato aspermy virus strain V (V-TAV) RNA and a mixture of subunits prepared from V-TAV and 35S-labelled cucumber mosaic virus strain T (T-CMV). Immunodiffusion tests showed that the reassembled particles reacted with polyclonal antisera raised against both V-TAV and T-CMV. Radioactivity was found in the precipitin line formed between the reassembled particles and antiserum raised against T-CMV as well as in the precipitin line formed between the reassembled particles and antiserum raised against V-TAV. This shows that 35S-labelled T-CMV protein subunits were incorporated with V-TAV protein subunits into the same particles. Thus, coat proteins of V-TAV and T-CMV can coassemble and form mixed-subunit capsids in vitro.
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Rice ragged stunt Oryzavirus genome segment 9 encodes a 38 600 M r structural protein
The complete nucleotide sequence of rice ragged stunt virus genome segment 9 (S9) was determined. The S9 segment is 1132 nucleotides long and has a long open reading frame starting from the first AUG codon at nucleotide position 14–16 and terminating at a UAG codon located at 1028–1030, which could encode a polypeptide with an M r of 38 600 (P9). The encoded polypeptide has no sequence homology to polypeptides of any other plant reoviruses published previously. An immunological study demonstrated that P9 was the smallest of the structural proteins. The P9 polypeptide was expressed as a fusion protein with maltose binding protein in Escherichia coli. Antisera to purified virions and to the fusion protein reacted with both the bacterially expressed polypeptide and the smallest polypeptide of the purified virus in immunoblotting analyses.
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