- Volume 71, Issue 4, 1990
Volume 71, Issue 4, 1990
- Review Article
-
- Animal
-
-
-
The pH independence of mammalian retrovirus infection
More LessThe pH dependence of early steps in the infection of human and other cells by mammalian retroviruses and retroviral pseudotype particles of vesicular stomatitis virus (VSV) was investigated for 10 strains of retrovirus, including C-type and D-type oncoviruses and human lentiviruses. When cells were treated with weak bases (NH4C1 and amantadine) to raise the pH of endocytic vesicles, only ecotropic murine leukaemia virus (MLV-E) and VSV showed pH-dependent entry. Pretreatment of retrovirus stocks in media below pH 5·0 did not reduce their titres but inactivated VSV to <10−8 of the initial titre. VSV(MLV-E) pseudotype infection in five out of six mouse and rat cell lines was inhibited by NH4Cl, indicating that infection proceeds via receptor-mediated endocytosis. In contrast, NH4Cl treatment has no effect on the infection of XC cells in which MLV-E induces syncytia. It is postulated that the pH-independent entry and cell fusion of XC cells by MLV-E may result from the activity of a cell surface proteinase that cleaves viral gp70 at neutral pH.
-
-
-
-
Host cell proteins required for measles virus reproduction
More LessWe have developed a cell-free system derived from measles virus-infected cells that supported the transcription and replication of measles virus RNA in vitro. The data suggest that tubulin may be required for these reactions, since an anti-β-tubulin monoclonal antibody inhibited viral RNA synthesis and the addition of purified tubulin stimulated measles virus RNA synthesis in vitro. Tubulin may be a subunit of the viral RNA polymerase, since two different anti-tubulin antibodies, one specific for the β- and another specific for the α-subunit of tubulin, coimmunoprecipitated the measles virus L protein as well as tubulin from extracts of measles virus-infected cells. Other experiments further implicated actin in the budding process during virus maturation, as there appeared to be a specific association of actin in vitro only with nucleocapsids that have terminated RNA synthesis, which is presumably a prerequisite to budding.
-
-
-
Cytotoxic T lymphocytes do not control lymphocytic choriomeningitis virus infection of BB diabetes-prone rats
BioBreeding Worcester diabetes-prone (BBdp) rats develop insulin-dependent autoimmune-driven diabetes mellitus spontaneously and intravenous administration of 1 × 107 p.f.u. of lymphocytic choriomeningitis virus (LCMV) to young adult mice prevents disease. The virus is lymphotropic, binding to and replicating in such cells. BBdp rats fail to generate virus-specific major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) responses when challenged with this dose or other doses of LCMV, Pichinde virus or vaccinia virus. Yet such rats clear virus effectively and show no evidence of persistent infection. Associated with this clearance of virus and establishment of immunity is the production of neutralizing antibodies.
In contrast, diabetes-resistant (BBdr) rats generate virus-specific CTL responses. Furthermore LCMV binds to fewer lymphoid cells of BBdr rats (in comparison to those of BBdp rats) and replicates in fewer lymphocytes (by one order of magnitude) from these rats. Thus, unlike mice in which CTLs play a dominant role in the control of LCMV infection, BBdp rats do not overcome this infection via CTLs. In addition, both the BBdp rats and their BBdr counterpart may provide useful models for determining whether or how individual lymphocyte subsets function in the induction of CTL responses, for the analysis of virus-induced immunosuppression and for the use of viruses or their products as therapeutic modalities.
-
-
-
Characterization and cloning of the African horsesickness virus genome
More LessThe dsRNA profiles of all nine African horsesickness virus (AHSV) serotypes were compared by agarose gel electrophoresis and PAGE. The agarose profiles were identical, but a unique profile was obtained for each of the nine serotypes by PAGE. Nine of the 10 dsRNA genome segments of AHSV-3 were cloned and the clones were used in dot-spot and Northern blot hybridization experiments to determine intra- and inter-serogroup nucleic acid similarities. Segments 1, 3, 4, 5, 7 and 8 were highly conserved in the AHSV serogroup and no genetic relationship with any of the other orbiviruses was observed. Of these segments 3, 5 and 8 showed the largest degree of cross-hybridization to the cognate genes of all the serotypes. These clones did not cross-hybridize to other orbiviruses such as epizootic haemorrhagic disease virus, bluetongue virus or equine encephalosis virus and are therefore recommended for use as group-specific probes for the identification of the AHSV serogroup. Genome segments 6 and 10 showed an intermediate degree of conservation, whereas segment 2 is serotype-specific and therefore probably codes for the outer capsid protein VP2.
-
-
-
Definition of adenovirus type 5 functions involved in the induction of chromosomal aberrations in human cells
More LessInfection of human embryonic kidney cells with adenovirus type 5 (Ad5) induces aberrations (gaps and breaks) in the cell chromosomes. We have conducted a study utilizing a large number of Ad5 mutants to identify the viral functions that are responsible for the occurrence of cytogenetic damage. The results of our investigation have indicated that expression of the gene products of the Ad5 early region 1A (E1 A) is necessary for the induction of chromosomal aberrations and that other early viral gene products do not appear to contribute to this phenotype. We have also shown that expression of both the major E1 A gene products, the 243 amino acid and the 289 amino acid proteins, is required for induction of damage at wild-type levels, although the 289 amino acid protein appears to retain detectable activity on its own. Lastly, we have observed that deletions in the amino-terminal region of the E1 A proteins and in the transactivating domain of the 289 amino acid protein prevent the occurrence of cytogenetic damage, whereas mutations elsewhere in the proteins do not affect this process.
-
-
-
Expression of the human papillomavirus type 16 genome in SK-v cells, a line derived from a vulvar intraepithelial neoplasia
More LessThe SK-v cells, established from a premalignant vulvar lesion, contain human papillomavirus type 16 (HPV- 16) sequences integrated at a single cellular site and derive from a cell clone present in vivo. Transcription of the HPV-16 genome in SK-v cells was analysed by cDNA heteroduplex mapping and sequencing, and by RNase mapping. Viral sequences were shown to be transcribed into virus-cell fusion messengers. The two major transcripts have a coding capacity for a truncated E6 protein, an E7 protein and an E1-E4 fusion protein, but differ in their 3′ virus-cell junction. Minor transcripts have a coding capacity for a full-length E6 protein and another truncated version of E6. The transcription pattern in the E6-E7 region was found to be the same both in SK-v cells and in CaSki cells, a line derived from an invasive cervical carcinoma. Immuno- precipitation experiments showed that the E6 protein (18K) and, predominantly, the E7 protein (20K) are expressed in SK-v cells as in CaSki cells. The E7 protein was found in a two- to threefold lower amount in SK-v cells, but showing the same half-life (about 1 h).
-
-
-
Purification and biochemical characterization of chicken anaemia agent
More LessChicken anaemia agent (CAA) was purified using differential centrifugation and successive cycles of equilibrium density gradient centrifugation using sucrose and CsCl. The purification method was dependent on the use of an antigen-detecting ELISA based on a CAA-specific monoclonal antibody. Virus particles banded at a density of 1·33 to 1·34 g/ml in CsCl and measured 23·5 ± 0· 8 nm in diameter. Purified preparations contained one major polypeptide (M r 50000) and a single-stranded, circular DNA (2.3 kb). CAA shares some of the biochemical characteristics possessed by porcine circovirus and the virus associated with psittacine beak and feather disease.
-
-
-
Further characterization of scrapie replication in PC12 cells
More LessThe rat pheochromocytoma cell line, PC 12, undergoes neuron-like morphological, biochemical and electrophysiological differentiation, in the presence of low concentrations of nerve growth factor (NGF). NGF- treated PC 12 cells have been shown previously to support 139A scrapie agent replication. In the present report we extended these findings and analysed the cellular conditions necessary for agent replication. Following the infection of differentiated PC 12 cells, scrapie replicated to relatively high titres as determined by an incubation period assay. The removal of NGF, which causes the gradual dedifferentiation of PC 12 cells, resulted in the inability of scrapie to replicate. The scrapie infectivity detected in PC 12 cultures is cell- associated and not released into the medium. Cells in infected cultures did not show any change in morphology when compared to cells in mock-infected cultures. Titration studies of scrapie infectivity in PC 12 cells have indicated that up to 4 LD50 units per cell can be obtained although a yield of 1 LD50 per cell was more common. Using an approximate m.o.i. of 1, only differentiated PC 12 cells supported 139A scrapie agent replication when compared to two other differentiated, neuronal cell types, indicating that PC 12 cells are more susceptible to agent replication. These studies support further the suitability of using differentiated PC 12 cells as an in vitro model to study scrapie agent replication.
-
-
-
Differential glycosylation of the protein (PrP) forming scrapie-associated fibrils
More LessPrP is a glycoprotein found in normal brain. In brain affected by scrapie it forms scrapie-associated fibrils (SAF). PrP from SAF shows considerable heterogeneity of size and charge on two-dimensional gels. It separates into six major regions, the three more acidic regions arising as a result of partial proteolytic degradation. The two more basic higher M r forms (M r 34000 and 29000) of PrP can be reduced in apparent M r to a lower M r form (M r 25000) with Peptide-N- glycosidase F. In addition, a series of lectins has been found to bind to PrP. Some bind preferentially to the higher M r forms whereas others bind more strongly to the lower M r form. Some of the heterogeneity of PrP is therefore due to differential N-glycosylation. We suggest that one or two N-linked carbohydrate chains are bound to the protein causing some of the differences in M r The major cause of heterogeneity of PrP is therefore proteolytic cleavage combined with differential glycosylation at the two potential N-glycosylation sites. The glycolipid moiety attached to PrP may be responsible for some lectin binding to all three bands. Using lectins as a probe to study potential differences in N-glycosylation we have looked at their binding to PrP isolated from SAF, from different strains of scrapie and from different regions of the same brain. No major differences in the N-glycan moieties were found.
-
-
-
Analysis of the nucleotide sequence of DNA from the region of the thymidine kinase gene of infectious laryngotracheitis virus; potential evolutionary relationships between the herpesvirus subfamilies
More LessWe have sequenced a 5·4 kb region of the genome from the avian herpesvirus infectious laryngotracheitis virus (ILTV) and identified genes homologous to the thymidine kinase (TK) gene, the capsid p40 gene of herpes simplex virus type 1 (HSV-1), a gene encoding a protein antigenic in Epstein-Barr virus infections plus one other highly conserved gene encoding a protein implicated in cell fusion in HSV-1. Computer analysis of the TK gene and the upstream overlapping gene sequence is used to produce trees showing potential evolutionary relationships between the herpesvirus subfamilies.
-
-
-
A mutant of herpes simplex virus type 1 immediate early polypeptide Vmw175 binds to the cap site of its own promoter in vitro but fails to autoregulate in vivo
More LessVmw175, the product of herpes simplex virus type 1 immediate early (IE) gene 3, is essential for viral replication. It is required for the activation of transcription from both early and late gene promoters and also for the repression of IE gene expression. Vmwl75 is able to bind specifically to certain DNA sequences, some of which (including that at the cap site of IE gene 3) contain the consensus sequence ATCGTC. The presence of this sequence at the cap site has been correlated with the ability of Vmw175 to autoregulate its own promoter. This report describes the characterization of five viruses with temperature-sensitive (ts) lesions in Vmwl75. Four of these mutants express Vmw175 which is ts in its ability to bind to DNA in vitro and to autoregulate IE-3 gene expression in the infected cell. Although Vmw175 produced by the remaining mutant, ts1225, fails to autoregulate IE-3 expression at the non-permissive temperature (NPT) its DNA-binding properties are indistinguishable from those of the wild-type protein. This suggests that the ability of Vmw175 to bind to the IE-3 cap site (as measured in vitro) is insufficient for autoregulation (in vivo). All five newly characterized ts mutants are partially permissive for early gene transcription at the NPT, although Vmw175 expressed by four of them is unable to bind to the IE-3 cap site sequence at elevated temperatures. This suggests that binding to one class of recognition sequences by Vmw175, as measured in vitro, is not absolutely required for the activation of early gene promoters during virus infection. The lesions in these five ts mutants lie in the carboxy- terminal third of the polypeptide; three of the mutations (those in tsl219, ts1221 and ts1225) were identified by DNA sequence analysis and were found to affect amino acid residues that are conserved in the homologous proteins from varicella-zoster virus and pseudorabies virus.
-
-
-
Immunological memory to herpes simplex virus type 1 glycoproteins B and D in mice
More LessMouse L cell lines constitutively expressing glycoproteins B or D of herpes simplex virus type 1 (LTKgB and LTKgD respectively) were used to study the longevity of the immune response to these viral glycoproteins in mice. Two immunizations with the cell lines were necessary to induce a persisting antibody response (present for over 200 days). Only LTKgD induced a neutralizing antibody response in mice and this also remained at high titres over 200 days after two inoculations. The presence in mice of precursor cytotoxic T lymphocytes specific for gB expressed in the L cells was also shown up to 270 days after immunization. Mice immunized with the cell lines showed an increased rate of virus clearance from the ear pinna, inoculation with LTKgD resulting in more clearance than LTKgB at 7 days post-immunization. This type of protection was reduced with time after inoculation, until by day 161 there was no significant difference in virus titres between immunized and control groups. However, LTKgD immunization protected against the establishment of latent infections in the ganglia of mice even up to 186 days postinoculation.
-
-
-
Structural and immunological characterization of human cytomegalovirus gp55-116 (gB) expressed in insect cells
More LessThe gene encoding the major envelope glycoprotein complex, gp55–116 (gB), of human cytomegalovirus (HCMV) was expressed at high levels in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the insect-derived gp55–116 was a protein of M r 150K which contained approximately 50K of N-linked oligosaccharides. The oligosaccharide linkages were almost exclusively endoglycosidase H- sensitive. The 150K protein was processed, presumably by proteolytic cleavage, to yield at least one of the previously defined cleavage products of the gp55–116. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells. Finally, the insect-derived gp55–116 was highly immunogenic in experimental animals and readily recognized by antibodies contained within HCMV- immune human serum, suggesting that this recombinant protein warrants further study as a potential HCMV subunit vaccine candidate.
-
-
-
Epitope analysis of glycoprotein I of pseudorabies virus
More LessA panel of 11 monoclonal antibodies (MAbs) raised against pseudorabies virus (PRV) was used to map epitopes on the virus glycoprotein I (gl). We employed three approaches to map epitopes on gl. By a competition binding assay, six groups of MAbs were defined as reacting with distinct antigenic domains on gl. To identify regions along the gl polypeptide chain encompassing the domains recognized by these MAbs, DNA fragments derived from the gI-coding region were cloned into pEX expression plasmids. The antigenic reactivity of the fusion proteins expressed in Escherichia coli was analysed by immunoblotting. Five antigenic domains were mapped within the first 238 amino acids of gl: domains A, B and D were mapped between amino acids 52 and 123 and domains C and E between amino acids 78 and 238. One MAb, representing domain F, did not react with the expressed fusion proteins. To assess the precise location and amino acid sequences of the epitopes, overlapping nonapeptides covering the amino acid sequence 52 to 238 were synthesized. The antibody-binding activity of these peptides was tested by an ELISA (Pepscan-method). Three antigenic domains were mapped: domain A was localized to amino acids 64 to 73 and 75 to 84, domain B to amino acids 52 to 67 and domain D to amino acids 68 to 82. Four MAbs representing antigenic domains C, E and F did not react in the Pepscan. Finally, sera from pigs infected experimentally with PRV reacted with the fusion protein of plasmid ps1 (amino acids 52 to 238).
-
-
-
Activity of herpes simplex virus type 1-specified glycoprotein C antigenic site II epitopes reversibly modulated by peripheral fucose or galactose units of glycoprotein oligosaccharides
More LessWe have previously shown that the herpes simplex virus type 1 (HSV-l)-specified glycoprotein C (gC-1) produced in epitheloid cells contains epitopes of peptide nature, which are dependent on galactose of oligosaccharides for their expression. In the present communication we report that these epitopes are expressed in a mouse neuroblastoma cell line (Cl300) with low levels of galactosyl transferases. However, in place of galactose the glycoprotein from Cl300 cells was found to contain oligosaccharides with additional fucose units. Fucosidase treatment, but not galactosi-dase treatment, abolished the antigenic activity of the carbohydrate-dependent epitopes. Altogether the results indicated that the carbohydrate-dependent epitopes of gC-1 from Cl300 cells were stabilized by peripheral sugars of N-linked oligosaccharides rather than O-linked ones and that fucose could substitute for terminal galactose in promoting the activity of the carbohydrate-dependent epitopes. This is the first demonstration of the involvement of fucose in the establishment of a carbohydrate-dependent epitope of peptide nature. The results also demonstrated that reversible carbohydrate-peptide interactions were responsible for the activity of the carbohydrate-dependent epitopes.
-
-
-
Control of expression of the varicella-zoster virus major immediate early gene
More LessThe cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varieella-zoster virus (VZV) were investigated. The location of the IE mRNA 5′ terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions −131 and +57 were responsible for promoter activity, whereas sequences between −410 and −131 mediated the response to Vmw65. Two short elements in the −410 to −131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.
-
-
-
Macromolecular synthesis in varicella-zoster virus-infected epithelial and fibroblast cells
More LessReplication of varicella-zoster virus (VZV) was studied in a range of human cells (Chang conjunctival, HEp2 and Vero) and compared with that in HEL cells. Evidence for virus-specific protein and nucleic acid synthesis was obtained from each of these cell lines. In some cell lines, virus proteins were clearly visible by polyacrylamide gel electrophoresis both with and without immunoprecipitation. Multiple passage of infected cells enhanced both virus-specific protein synthesis and shutoff of host protein synthesis. No qualitative differences in the patterns of VZV-specific protein synthesis were detected. Approximately 2000 copies of the VZV genome were detected per infected HEL cell.
-
-
-
A herpes simplex virus type 1 variant, deleted in the promoter region of the latency-associated transcripts, does not produce any detectable minor RNA species during latency in the mouse trigeminal ganglion
In peripheral sensory ganglia latently infected with herpes simplex virus type 1 (HSV-1) transcription is restricted. A set of viral latency-associated transcripts, the LATs, have been characterized by Northern blotting and in situ hybridization. These transcripts have previously been mapped to a 3 kb region of the viral genome within the repeat long region. However, transcription from adjacent regions of the genome can be detected by in situ hybridization, which cannot be detected by Northern blotting. These RNAs are termed minor LATs or m-LAT. In this study we show that in ganglia latently infected with the HSV-1 variant 1704, which is deleted in one complete copy of the LAT gene and in the promoter and 5′ portion of the other copy, m-LATs are not detected by in situ hybridization. Furthermore, the levels of DNA in nervous system tissue latently infected with the parental and the 1704 variant virus are similar. Thus we propose that the sequence elements necessary for initiating transcription or stabilizing m-LATs are within the region deleted in variant 1704 that codes for the promoter and the 5′ end of the LATs.
-
-
-
Trans-complementation of the C gene of human and the P gene of woodchuck hepadnaviruses
More LessA 5 bp insertion was introduced into the BstEII site at nucleotide 2815 in DNA of hepatitis B virus (HBV) and a mutant HBV genome was produced, which coded for envelope and core proteins, but not for DNA polymerase, due to a frameshift. Cultured hepatoma cells (HepG2) were simultaneously transfected with a plasmid harbouring a tandem dimer of the mutant HBV DNA and another plasmid harbouring a tandem dimer of DNA of woodchuck hepatitis virus or duck hepatitis B virus. The replication of mutant HBV DNA, incapable of encoding DNA polymerase, was accomplished by cotransfecting woodchuck hepatitis virus DNA, but not by duck hepatitis B virus DNA. These results indicated a trans-complementation of the C and P genes in mammalian hepadnaviruses beyond a species barrier.
-
-
-
Mutations of the human papillomavirus type 16 E7 gene that affect transformation, transactivation and phosphorylation by the E7 protein
More LessThe human papillomavirus type 16 (HPV-16) E7 gene cooperates with an activated ras oncogene to transform primary rodent cells and is important in the immortalization of cervical keratinocytes. We have generated a series of point mutations within the E7 gene and show that mutation of residues serine 31 and serine 71 affect the phosphorylation of the E7 protein, but do not alter its ability to cooperate with ras. Further mutations which alter cysteine residues in a -Cys-X-X-Cys- motif decrease transformation markedly, although they do not abolish it entirely. All the mutations generated displayed a decreased ability to transactivate the adenovirus E2 promoter. These results show that neither phosphorylation of E7 nor its ability to transactivate are required for transformation by E7.
-
-
-
A baculovirus expression vector derived from the basic protein promoter of Autographa californica nuclear polyhedrosis virus
More LessThe basic protein of Autographa californica nuclear polyhedrosis virus (AcMNPV) is associated with virus DNA in virion nucleocapsids and is produced in infected cells during the late phase of gene expression. A transfer vector was constructed containing the β-galactosidase gene, under the control of a copy of the putative bask protein promoter, in place of the polyhedrin gene within the AcMNPV EcoRI fragment I. After cotransfection of Spodoptera frugiperda cells with the transfer vector and infectious AcMNPV DNA, polyhedrin-negative recombinant viruses were selected which expressed high levels of β-galactosidase. Radiolabelling of infected cell proteins showed that β-galactosidase was expressed at the same time as the viral basic protein, between 8 to 24 h post-infection, with a peak synthesis at 12 to 15 h. These results demonstrated that the temporal regulation of the basic protein promoter was not affected by its position within the virus genome. Furthermore, a new baculovirus vector system is now available for high level expression of foreign genes at earlier times in infected cells.
-
-
-
Demonstration of enzyme activities required for cap structure formation in infectious bursal disease virus, a member of the birnavirus group
More LessThe putative viral transcriptase p90 in infectious bursal disease virus (IBDV) was shown to form an enzyme- guanylate intermediate which is indicative of guanylyl- transferase activity. The p90-nucleotide bond is most likely to be a phosphodiester linkage, as it resisted treatment with HC1 and NH2OH but was sensitive to NaOH. This is in contrast to phosphoamide bonds formed by reovirus cores. Methyltransferase activity was also demonstrated in IBDV, and is closely associated with transcription, suggesting that p90 may be a multifunctional enzyme.
-
-
-
The genome type of human parvovirus B19 strains isolated in Japan during 1981 differs from types detected in 1986 to 1987: a correlation between genome type and prevalence
More LessThe genome DNAs of 12 strains of human parvovirus B19, isolated in Japan at two different times, 1981 and 1986 to 1987, were molecularly cloned in the plasmid pUC18. The cloned B19 DNAs were analysed by cleaving with restriction endonucleases, and were classified into several groups by genome type. The restriction endonuclease cutting patterns of B19 strains isolated during 1981 were similar to that of the group IV genome type, and the patterns of those isolated later were similar to that of group II, suggesting a correlation between the genome type and the prevalence. We conclude that the prevalences of B19 infection in Japan during 1981 and in 1986 to 1987 were caused by viruses differing in genome type, and that B19 viruses with similar genome types disseminated widely in Japan during each prevalence.
-
-
-
Differentiation of vaccine and wild mumps viruses using the polymerase chain reaction and dideoxynucleotide sequencing
More LessParts of the F gene from 16 mumps viruses derived from vaccines and clinical isolates were amplified using the polymerase chain reaction and their nucleotide sequences were determined. Over a region of 111 nucleotides, eight regions of variability were detected with a maximum of six (5·4%) changes occurring between any two virus strains. The Jeryl Lynn and Urabe vaccine strains were clearly different from each other and from wild virus isolated from cases of nonvaccine-associated mumps. In contrast, viruses isolated from the cerebrospinal fluid and throat in cases of meningitis and parotitis following vaccination with the Urabe strain were identical to this strain. We conclude that the vaccine was the source of these infections.
-
-
-
Genetic evidence for multiple functions of the matrix protein of vesicular stomatitis virus
More LessTsO82, a spontaneous temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV) isolated in chick embryo fibroblasts (CEFs), complements the five prototype ts mutants of the virus. The data presented here indicate that the defect in tsO82 is localized in the M gene. The mutation changed a methionine to an arginine at position 51 of the M protein. Only true revertants could be isolated, and their frequency was low, perhaps due to the type of substitution required to return to the wild-type phenotype. TsO82 does not exhibit hypertranscription, in contrast to the data reported for all of the other ts mutants affected in the M protein. Moreover, tsO82 is conditionally ts, since it grows normally in BHK-21 cells at all temperatures. It exhibits no c.p.e. at the non-permissive temperature in CEFs. Our data argue for multiple functions of the M protein of VSV, the domain affected by the tsO82 mutation possibly being implicated both in the shut-off of cellular RNA synthesis, and for the recognition of a cellular factor required for efficient viral RNA synthesis.
-
-
-
Localization of P protein binding sites on the Sendai virus nucleocapsid
More LessPrevious studies have shown that the molecules of P protein associated with transcriptionally active Sendai virus nucleocapsids are arranged in discrete clusters. Our study investigates whether or not this localized distribution is due to the existence of only a few P protein binding sites on the nucleocapsid core. We used immunoelectron microscopy to examine whether additional P proteins could bind at locations between the groups of endogenous P proteins. To differentiate between endogenous and added proteins, we constructed a recombinant gene which instructs the in vitro synthesis of a chimeric protein containing the carboxyl-terminal nucleocapsid-binding region of P protein, fused to chloramphenicol acetyltransferase (CAT). Immunogold labelling, using an antibody to the CAT moiety, revealed at the electron microscope level, that the chimeric product bound to nucleocapsids at many sites located over the entire length of the nucleocapsid. This indicated that the localized distribution of P protein molecules is not due to a limited number of P protein binding sites on the nucleocapsid core.
-
- Plant
-
-
-
In vitro synthesis of biologically active transcripts of tomato black ring virus satellite RNA
More LessSynthetic transcripts of tomato black ring virus satellite RNA (TBRV satRNA), isolate L, were prepared from cDNA cloned in the Bluescribe transcription vector. Transcripts with 49 (T49L) or two (T2GL) extra nucleotides at their 5′ ends and 42 extra nucleotides at their 3′ ends were able to induce, but to different extents, the synthesis in vitro of the satRNA- encoded 48K protein. However, when inoculated into Chenopodium quinoa together with TBRV L genomic RNAs, only T2GL was biologically active, in the presence or absence of a 5′ cap analogue in the transcription reactions. Analysis of the 5′ and 3′ termini of the satRNA isolated from plants showed that nonviral extensions were not maintained in the transcript progeny.
-
-
-
-
Homologies between the genomes of a carlavirus (lily symptomless virus) and a potexvirus (lily virus X) from lily plants
cDNA clones complementary to the 3′-terminal regions of the genomic RNAs of the carlavirus lily symptomless virus and the potexvirus lily virus X (LVX) have been sequenced. The carlavirus RNA sequence contains five open reading frames (ORFs) coding for proteins of Mr 25374, 11631, 6960, 32041 (coat protein) and 16121, which are all very similar in size, amino acid sequence and relative position in the genome to proteins encoded by two different potato carlaviruses. The first four of these proteins also show considerable amino acid sequence similarity to proteins encoded by RNA of potexviruses, and the relative position of the ORFs on the carlavirus genome strongly resembles that in the potexvirus genomes. The LVX cDNA clone contains three ORFs encoding proteins of M r 23574, 11767 and 21569 (coat protein). A small ORF immediately 5′ of the coat protein ORF, which has been found in other potexvirus genomes, is not present in the LVX genome. Thus, the data confirm the close taxonomic relationship between carlaviruses and potexviruses and reveal some differences in genome organization among the potexviruses.
-
-
-
The nucleotide sequence of the genomic RNA of kennedya yellow mosaic tymovirus-Jervis Bay isolate: relationships with potex- and carlaviruses
More LessThe nucleotide sequence of the genomic RNA of kennedya yellow mosaic tymovirus-Jervis Bay isolate (KYMV-JB) has been determined. The genome of KYMV-JB is 6362 nucleotide residues long and encodes three major open reading frames. The genomic organization and the encoded proteins of KYMV-JB are very similar to those of three other tymoviruses that have recently been reported. Sequence comparisons revealed that the possible replicase proteins of tymoviruses are closely related to those of potexviruses and carlaviruses, suggesting a close evolutionary relationship among these viruses, despite differences in their genome organization and particle morphology.
-
-
-
A tomato spotted wilt-like virus with a serologically distinct N protein
M. D. Law and J. W. MoyerA serologically distinct member of the tomato spotted wilt virus (TSWV) group was isolated from the hybrid flower crop New Guinea impatiens (Impatiens sp.) and termed TSWV-I. TSVW-I type isolates have frequently been detected in a wide variety of flower crops throughout the United States. TSWV-shares many characteristics with TSWV, such as symptomatology and possession of three ssRNA species (L, M and S of 8·3 kb, 5·2 kb and 3·4 kb, respectively) and three structural proteins (G1, G2 and N of 78K, 52K and 28K respectively). The TSWV-I G1 and G2 glycoproteins were serologically related to the respective proteins of TSWV, but the TSWV-I nucleocapsid or N protein was serologically unrelated to that of TSWV. Hybridization analysis under high stringency conditions revealed no hybridization between clones of TSWV-1 S and M and the S and M RNAs of TSWV, respectively and in addition, a TSWV S clone hybridized only with TSWV S RNA. The cytopathology of TSWV-1 also differed from that of TSWV. TSWV-1- infected tissue primarily contained filamentous structures arranged in paracrystalline arrays, which were also observed by immunosorbent electron microscopy of tissue extracts. The filamentous structures were only trapped by TSWV-1 antibodies. The conserved serological relatedness between TSWV types for G1 and G2, but not N, is consistent with serological analyses of the nairovirus and phlebovirus genera of the Bunyaviridae, the virus family that TSWV most closely resembles.
-
-
-
Cucumovirus transmission by the aphid Myzus persicae is determined solely by the viral coat protein
More LessVirus particles were reassembled in vitro from the proteins of two cucumoviruses, the aphid-transmissible V strain of tomato aspermy virus (VTAV) or the non aphid-transmissible M strain of cucumber mosaic virus (MCMV) with the RNAs of either virus or that of tobacco mosaic virus. Particles reassembled from VTAV protein and RNAs from any of the three viruses were transmitted by the aphid, Myzus persicae, following virus acquisition through membranes. However particles reassembled from MCMV protein and RNAs from any of the three viruses were not transmitted by the aphids. These results indicate that the transmission of cucumoviruses by aphids is determined solely by properties of their protein coats.
-
-
-
Double-stranded RNAs isolated from plant tissue infected with red clover necrotic mosaic virus correspond to genomic and subgenomic single-stranded RNAs
More LessRed clover necrotic mosaic dianthovirus (RCNMV) has a genome of two ssRNA species, RNA 1 (4.0 kb) and RNA 2 (1.4 kb). Double-stranded RNA was isolated from Phaseolus vulgaris plants infected separately with five isolates of RCNMV from Czechoslovakia, Sweden, Scotland and England. In each case three species of dsRNA, designated A, B and C in order of increasing mobility, were resolved by gel electrophoresis in non-denaturing conditions. The mobilities of dsRNA species A and B were similar for all the isolates, but the mobilities of the different species of dsRNA C showed some variation. Southern blotting followed by hybridization with cloned probes showed that dsRNA species A and B are related to RNA 1, whereas dsRNA C is related to RNA 2. In gel electrophoresis in denaturing conditions, RNA from dsRNA A had the same mobility as ssRNA 1, whereas RNA from dsRNA species B and C had the same mobility as ssRNA 2. It is concluded that dsRNAs A and C are the doublestranded forms of the genomic RNA 1 and RNA 2 respectively and that dsRNA B is the double-stranded form of a subgenomic RNA which is derived from RNA 1 and has the same size as RNA 2.
-
Volumes and issues
-
Volume 105 (2024)
-
Volume 104 (2023)
-
Volume 103 (2022)
-
Volume 102 (2021)
-
Volume 101 (2020)
-
Volume 100 (2019)
-
Volume 99 (2018)
-
Volume 98 (2017)
-
Volume 97 (2016)
-
Volume 96 (2015)
-
Volume 95 (2014)
-
Volume 94 (2013)
-
Volume 93 (2012)
-
Volume 92 (2011)
-
Volume 91 (2010)
-
Volume 90 (2009)
-
Volume 89 (2008)
-
Volume 88 (2007)
-
Volume 87 (2006)
-
Volume 86 (2005)
-
Volume 85 (2004)
-
Volume 84 (2003)
-
Volume 83 (2002)
-
Volume 82 (2001)
-
Volume 81 (2000)
-
Volume 80 (1999)
-
Volume 79 (1998)
-
Volume 78 (1997)
-
Volume 77 (1996)
-
Volume 76 (1995)
-
Volume 75 (1994)
-
Volume 74 (1993)
-
Volume 73 (1992)
-
Volume 72 (1991)
-
Volume 71 (1990)
-
Volume 70 (1989)
-
Volume 69 (1988)
-
Volume 68 (1987)
-
Volume 67 (1986)
-
Volume 66 (1985)
-
Volume 65 (1984)
-
Volume 64 (1983)
-
Volume 63 (1982)
-
Volume 62 (1982)
-
Volume 61 (1982)
-
Volume 60 (1982)
-
Volume 59 (1982)
-
Volume 58 (1982)
-
Volume 57 (1981)
-
Volume 56 (1981)
-
Volume 55 (1981)
-
Volume 54 (1981)
-
Volume 53 (1981)
-
Volume 52 (1981)
-
Volume 51 (1980)
-
Volume 50 (1980)
-
Volume 49 (1980)
-
Volume 48 (1980)
-
Volume 47 (1980)
-
Volume 46 (1980)
-
Volume 45 (1979)
-
Volume 44 (1979)
-
Volume 43 (1979)
-
Volume 42 (1979)
-
Volume 41 (1978)
-
Volume 40 (1978)
-
Volume 39 (1978)
-
Volume 38 (1978)
-
Volume 37 (1977)
-
Volume 36 (1977)
-
Volume 35 (1977)
-
Volume 34 (1977)
-
Volume 33 (1976)
-
Volume 32 (1976)
-
Volume 31 (1976)
-
Volume 30 (1976)
-
Volume 29 (1975)
-
Volume 28 (1975)
-
Volume 27 (1975)
-
Volume 26 (1975)
-
Volume 25 (1974)
-
Volume 24 (1974)
-
Volume 23 (1974)
-
Volume 22 (1974)
-
Volume 21 (1973)
-
Volume 20 (1973)
-
Volume 19 (1973)
-
Volume 18 (1973)
-
Volume 17 (1972)
-
Volume 16 (1972)
-
Volume 15 (1972)
-
Volume 14 (1972)
-
Volume 13 (1971)
-
Volume 12 (1971)
-
Volume 11 (1971)
-
Volume 10 (1971)
-
Volume 9 (1970)
-
Volume 8 (1970)
-
Volume 7 (1970)
-
Volume 6 (1970)
-
Volume 5 (1969)
-
Volume 4 (1969)
-
Volume 3 (1968)
-
Volume 2 (1968)
-
Volume 1 (1967)