- Volume 75, Issue 12, 1994
Volume 75, Issue 12, 1994
- Review Article
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- Animal
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Intracellular localization and DNA-binding activity of a class of viral early phosphoproteins in human fibroblasts infected with human cytomegalovirus (Towne strain)
More LessIndirect immunofluorescence (IF) with monoclonal antibody M23 prepared against the nuclei of human embryo lung (HEL) cells infected with human cytomegalovirus (HCMV) Towne strain showed that the M23 antigen reactive with the M23 antibody was localized within distinct foci throughout the nucleus of infected HEL cells shortly after infection, even at 2 h post-infection (p.i.). The foci increased in size by 24 h p.i. and then the IF patterns changed to show the nuclear inclusion body-like structures at 72 h p.i. Treatment with phosphono-acetic acid, a HCMV DNA replication inhibitor, resulted in a nuclear pattern similar to that observed shortly after infection. The double-labelled IF test revealed that the HCMV UL44 antigen essential for viral DNA replication colocalized with the M23 antigen in the same intranuclear structure shortly after infection whereas neither viral antigen appeared to colocalize in most cells later after infection. The M23 antibody immunoprecipitated four proteins, 34K, 43K, 50K and 84K, in infected cells. To examine whether these proteins correspond to four early phosphoproteins encoded by the HCMV strain AD169 genome, the Towne strain DNA sequence corresponding to that encoding both the 34K and 43K proteins of strain AD169 was determined and transiently expressed in COS-7, Vero and HEL cells. These proteins were detected by the M23 antibody within the foci of these nuclei as found in the nuclei of productively infected cells shortly after infection. In addition, the 34K, 43K and 50K proteins at least were shown to be DNA-binding proteins by double- and single-stranded DNA-cellulose column chromatography. The relationship of these proteins to the status of viral DNA replication is discussed.
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Down-regulation of integrin α1/β1 expression and association with cell rounding in human cytomegalovirus-infected fibroblasts
More LessHuman cytomegalovirus (HCMV) causes a c.p.e. characterized by rounding of the infected cell. Since interactions with the extracellular matrix may be involved in the cell rounding, we have analysed the expression of integrins, which are the main cell surface receptors involved in cell-substrate adhesion and spreading. By FACS analysis, a selective decrease in cell surface expression of α1/β1 integrin was observed in HCMV-infected fibroblasts. This decrease coincides with cell rounding. Immunoprecipitation studies and FACS analysis of permeabilized cells have further demonstrated that total levels of this integrin are decreased in infected cells, suggesting that the reduction in cell surface α1/β1integrin is not due to a defect in transport to the surface. Furthermore, we have ruled out the possibility that the observed decrease in α1/β1expression is caused by a cytokine released from the infected cells by showing that the reduction is abolished by inactivating the HCMV with u.v. irradiation, and that conditioned medium from HCMV-infected cells has no effect on expression of α1/β1integrin in uninfected cells. Concomitant with the reduction in α1/β1levels, the HCMV-infected fibroblasts show a reduced ability to adhere to laminin and collagen IV. Taken together the data indicate that de novo synthesis of HCMV protein(s) causes a decreased assembly/expression of α1/β1integrin, coincident with the well characterized morphological alterations of the infected cell.
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Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase
J. Conner, A. Cross, J. Murray and H. MarsdenThe large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0·4 μg/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 μg/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapep-tide YAGAWNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.
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Effects of herpes simplex virus type 1 infection on the plasma membrane and related functions of HeLa S3 cells
More LessIn this study we evaluated modifications of various structural and functional properties of the plasma membrane of HeLa S3 cells following infection by the lytic virus herpes simplex virus type 1 (HSV-1). Na+/K+-ATPase activity considerably decreased during the first few hours post-infection (p.i.), whereas Na+ and K+ concentrations were not significantly affected until a much later period. By 8 h p.i., a partial membrane depolarization in infected cells had occurred, as indicated by a small change in the transmembrane potential. HSV infection induced a time-dependent lipid peroxidation of HeLa cell plasma membranes temporally correlated with the progressive reduction in Na+/K+-ATPase activity. Moreover, a significant decrease of membrane fluidity appeared at a late phase of the viral replicative cycle probably representing cumulative membrane damage. These results demonstrate that HSV-1 infection induced the production of free radicals in nonphagocytic cells. Since lipid peroxidation begins at an early stage of the virus replicative cycle, it may be directly related to viral cytopathicity.
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A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus
More LessIn addition to the seven known homologous regions (hrs) of Autographa califomica multiple nucleocapsid polyhedrosis virus (AcMNPV) the HindIII-K fragment was also found to carry a putative ori, although this fragment does not contain an hr. Deletion analysis showed that this ori contains several segments essential for its activity and other ‘auxiliary’ sequences that enhance the ori activity. Sequence analysis identified several structures often found in other viral replication oris, such as palindromes and other repeated motifs. Although most of the auxiliary sequences of this ori were found to be deleted in the Bombyx mori nucleocapsid polyhedrosis virus genome, the essential part of this ori, containing the palindromes and the A/T-rich region, was retained. This and the fact that after prolonged serial passage of AcMNPV large replicating DNA molecules are found in which repeated sequences derived from the HindIII-K fragment accumulate are consistent with this region being a putative origin of AcMNPV DNA replication.
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Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organization and time point of replication
Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane. Nucleocapsids have a constant diameter of 33·7 ± 1·4 nm and a variable length of between 8 and 46 nm. Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp. When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp. By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 and 23 EcoRI clones were obtained. Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained. Part of a 16·8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development.
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Adenovirus protein-protein interactions: hexon and protein VI
More LessA variety of mastadenoviruses were denatured, their polypeptides separated by electrophoresis on SDS- polyacrylamide gels and transferred to nitrocellulose. The immobilized polypeptides were washed, incubated with buffers containing hexons from human adenoviruses (Ad) types 2, 5 and 12 and the location of bound hexons was detected with anti-hexon antibodies. It was found that hexons from any of the three human adenovirus types bound to protein VI from all the mastadenoviruses examined. Furthermore we found that hexon-VI binding was significantly greater than the interaction between hexon and the precursor to VI, pVI. This binding was susceptible to detergents and to changes in pH or salt concentration. A rabbit polyclonal antibody was raised against a recombinant protein derived from the middle third of pVI from Ad2 and was used to quantify the difference in binding and to demonstrate the presence of a single intermediate (designated iVI) in the processing of pVI to VI. The affinity between iVI and hexon was considerably greater in our assay than that of pVI but was less than that between hexon and VI. A complementary binding of recombinant iVI to immobilized hexons was also demonstrated. This latter interaction, however, was only observed when hexon preparations were not boiled prior to electrophoresis, substantiating the proposition that the recognition motif on the hexon was conformation- dependent. These results are discussed in the context of understanding further the molecular basis of protein- protein interactions between the structural proteins of adenoviruses and the factors involved in virion maturation.
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Analysis of type-restricted and cross-reactive epitopes on virus-like particles of human papillomavirus type 33 and in infected tissues using monoclonal antibodies to the major capsid protein
A panel of six monoclonal antibodies recognizing at least three different antigenic regions has been raised against the LI major capsid protein of human papillomavirus type 33 (HPV-33), which is associated with cervical carcinoma. The antigenic sites defined by these antibodies have been mapped and classified as type- restricted or broadly cross-reactive using bacterially expressed LI fusion proteins of a variety of HPV types. Conformational and linear epitopes have been distinguished using native and denatured virus-like particles. HPV infection of genital lesions has been analysed using both monoclonal antibodies and DNA amplification by PCR. The antibodies obtained should be useful to probe the structure of HPV capsids and to develop a general assay for the detection and classification of productive HPV infections.
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Mutations in the carboxy terminus of adeno-associated virus 2 capsid proteins affect viral infectivity: lack of an RGD integrin-binding motif
More LessUsing site-directed mutagenesis, we tested whether a potential integrin-binding site, (composed of the amino acids RGD) which is predicted in the adeno-associated virus 2 (AAV-2) capsid open reading frame (ORF), plays a role in the infectivity of AAV-2. Nucleotide sequencing of wild-type and mutant capsid protein-coding sequences, however, revealed discrepancies with the published sequence data at several positions, including a frameshift in the carboxy terminus which cancels the RGD motif and extends the capsid ORF by 27 amino acis. This sequence was confirmed by protein sequencing of proteolytic fragments of VP3. Thus, the virus mutant (pTAV-p), in which the intention was to exchange D of the putative RGD motif for E, resulted in replacing I480 by S in the newly established ORF. A second virus mutant (pTAV-d), in which the intention was to delete the RGD peptide, in fact gave a shift into the ORF of the originally published sequence. The pTAV-p mutant showed a strongly reduced infectivity compared to wild- type AAV-2, whereas pTAV-d was not infectious at all. Neither mutant accumulated viral ssDNA as detected by Hirt extraction. Analysis of virus particle formation and subcellular localization of the capsid proteins revealed a defect of the mutant capsid proteins in capsid assembly. This shows that the newly established C-terminal sequence of the AAV capsid proteins plays an important role in viral assembly.
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Interleukin-10 inhibits initial reverse transcription of human immunodeficiency virus type 1 and mediates a virostatic latent state in primary blood-derived human macrophages in vitro
More LessInterleukin-10 (IL-10), a product of T lymphocytes, B cells and macrophages, participates in Th-2 immune responses and modulates macrophage functions including possible interactions with pathogens. We have found that Chinese hamster ovary cell-derived human recombinant (hr) IL-10 inhibits human immunodeficiency virus type 1 strains Ada and Ba-L (HIV-1ADA and HIV-lBa-L) replication in primary tissue culture- derived macrophages in a dose-dependent manner. Inhibition by IL-10 treatment (> 5 U/ml) was effective 72 h before or 24 h after infection and cytokine activity blocked by anti-hrIL-10 antibody (19F1), or lost after heat inactivation of IL-10. Viral production was measured by determining p24 and reverse transcriptase levels while reverse transcription kinetics for the long terminal repeat (LTR) and gag were assessed at timed intervals after infection and quantified by 32P endlabelling. IL-10 inhibited early steps of infection without modulating cell surface CD4+ levels. The onset of LTR reverse transcription was delayed by 4 to 8 h and the number of LTR transcripts was decreased by 77% at 24 h and by 87 % 48 h after infection. IL-10 effects were reversible; after cytokine washout, cells treated before infection showed lower levels of virus compared with those treated after infection. IL-10 biological activity was confirmed in three virus-independent assays. These results demonstrate IL-10 decreases HIV-1 reverse transcription upon macrophage infection and subsequently mediates viral latency in vitro. Therefore, IL-10 may be involved in the effective control of HIV-1-infected macrophages in vivo.
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Comparison of the expression and phosphorylation of the non-structural protein NS2 of three different orbiviruses: evidence for the involvement of an ubiquitous cellular kinase
More LessThe non-structural protein NS2 of epizootic haemorrhagic disease (EHD), bluetongue (BT) and African horsesickness (AHS) viruses has each been expressed to high levels using a baculovirus vector gene expression system. It was found that the recombinant baculovirus- expressed EHDV NS2 protein was resolved as a doublet following PAGE. Peptide mapping of these protein bands indicated that they were identical. The difference in the sizes of the NS2 protein bands could not be attributed to the phosphorylation of NS2 or other posttranslational modification such as N-glycosylation and remains obscure. The EHDV, BTV and AHSV baculo- virus-expressed NS2 proteins were all phosphorylated in vitro without the addition of an exogenous kinase. An unphosphorylated form of EHDV NS2, obtained by expressing the NS2 gene as a fusion protein in Escherichia coli cells, could be phosphorylated in vitro by a protein kinase associated with the cytoplasm of insect cells. The phosphorylated version of this protein was found to be significantly less efficient in binding ssRNA, compared to the unphosphorylated version.
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Molecular biology of rotaviruses. IX. Conservation and divergence in genome segment 5
L. Xu, Y. Tian, O. Tarlow, D. Harbour and M. A. McCraeNucleotide sequencing of RNA segment 5 from seven strains of group A rotavirus has been carried out to investigate the extent of diversity and conservation, as well as possible selective pressures involved in driving the fixation of sequence changes in this gene. Analyses of the derived sequences revealed that sequence conservation could not be correlated either with rotavirus serotype or the species of origin of the virus strain. These sequences together with other published and unpublished sequences of this gene have raised the total number available for comparison to 17. Alignment of all the available sequences revealed that only 88 amino acid positions (17·6%) in the protein encoded by gene 5 (VP5) are absolutely conserved but that the metalbinding motif reported by others is conserved in all sequences. Despite the high degree of sequence divergence, alignment of secondary structure predictions for VP5 showed a high level of conservation, suggesting that constraints on sequence divergence may operate at the level of overall higher-order structure of the encoded protein.The new rotavirus gene 5 sequences appearing in this paper have been deposited with the EMBL sequence database and given the following accession numbers: Z12105 (bovine strain B223), Z12106 (human strain Hochi), Z12107 (porcine strain OSU), Z12108 (bovine strain UKtc), Z32534 (human strain St 3), Z32535 (simian strain RRV), Z32552 (human strain 69M).
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Identification of the nucleic acid binding domain of the rotavirus VP2 protein
More LessThe bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for doublestranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified singleshelled particles.
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Characterization of a virus variant produced by L cells persistently infected with lymphocytic choriomeningitis virus
More LessContinuous cultivation of murine L cells infected with lymphocytic choriomeningitis virus strain Armstrong leads to production of L(Arm) cells, which produce a predominantly cell-associated attenuated variant, the L(Arm) virus. The relatively few infectious particles that are released have lost the ability to form plaques on L cells and to cause illness in mice even if inoculated intracerebrally. Based on equal protein Mr s, antigenicity and protein kinase activity, essentially identical results were obtained for the purified Armstrong and L(Arm) viruses. There was also no difference in production and release of particles with the potential to cause homologous interference. Such particles consisted of two types, one of which was highly susceptible to u.v.- irradiation, the other was highly resistant. In the case of the L(Arm) virus interfering particles, it appears that the u.v.-irradiation-susceptible forms represented infectious virus. Purified L(Arm) virus particles contained considerable quantities of subgenomic forms of (small) S- and (large) L-RNA and their complementary counterparts, which all appeared to be replicated autonomously in an unenriched manner.
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Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm
More LessThe Bunyamwera bunyavirus (BUN) M RNA genome segment encodes three proteins, two glycoproteins termed G1 and G2 and a non-structural protein called NSm, in the form of a polyprotein precursor that is co-translationally cleaved to give the mature proteins. Indirect immunofluorescence experiments have shown that these proteins localize to the Golgi complex in BUN-infected cells. We have used a recombinant vaccinia virus (vTF7-3), which expresses bacteriophage T7 RNA polymerase, to drive the expression of plasmids containing either the entire BUN M segment cDNA or fragments that encode the G1, G2 and NSm proteins separately under control of the T7 promoter. After transfection of these plasmids into vTF7-3-infected cells, correctly sized and processed proteins were detected by immunoprecipitation with BUN-specific antibodies. Immunofluorescence experiments showed that G1, G2 and NSm localized to the Golgi when transiently expressed from the full-length cDNA. When G2 or NSm were expressed separately they also localized to the Golgi, but when G1 was expressed alone a staining pattern typical for the endoplasmic reticulum was obtained. However coexpression of G2 and G1 from independent plasmids resulted in G1 localizing to the Golgi. In contrast translocation of G1 to the Golgi was not observed when G1 was coexpressed with NSm, although NSm itself was still detected in the Golgi. Similar results were obtained when the proteins were expressed from transfected plasmids containing the G2-, NSm- or G1-coding sequences under control of the cytomegalovirus immediate-early promoter. The localization of G1 to the Golgi when coexpressed with G2 was confirmed by the loss of endoglycosidase H (endo H) sensitivity of G1 after approximately 60 min in a pulse-chase experiment; G1 remained sensitive to endo H when expressed either alone or in combination with NSm. These results suggest that G2 contains the Golgi targeting and/or retention signals and that G1 has to interact with this protein to localize to this cellular compartment.
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The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell
More LessFlaviviruses elicit a humoral immune response to two virus-encoded, membrane-associated glycoproteins. One is the major virion surface envelope protein (E), which is recognized by antibody, whereas the other is a secreted, heavily glycosylated non-structural protein (NS1). Inoculation with either protein can give rise to a protective immune response, as can the passive transfer of E and NS1 monospecific monoclonal antibodies. Experiments reported here demonstrate that the secreted form of NS1, whether from cells infected with tick-borne encephalitis virus (TBEV) or from cells infected with a defective recombinant adenovirus containing the NS1 gene, occurs chiefly as a pentamer or hexamer and occasionally as a decamer or dodecamer. Intracellular forms of this protein however occur only as dimers. The higher M r forms secreted from the cell are exquisitely sensitive to detergent, suggesting they are held together by hydrophobic bonds. Both intracellular and extracellular forms of the dimer can be dissociated by heat, but at different temperatures. Unlike similar proteins from mosquito-bome viruses, NS1 from TBEV-infected cells cannot be dissociated at ambient temperatures by extremes of pH. Studies on the antigenic structure of this protein show it to have several highly conserved epitopes, confirming similar earlier conclusions from amino acid sequence analyses.
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Classical swine fever: genetic detection and analysis of differences between virus isolates
More LessTwo pairs of oligonucleotide primers were designed that specifically amplified regions of the classical swine fever virus genome. These products, corresponding to a 671 bp portion of the genes encoding the El and E2 (gp33 and gp55) proteins and a 1090 bp portion of the putative polymerase gene, were amplified from eight virus isolates which had been responsible for a series of classical swine fever outbreaks in Italy involving both domestic pigs and wild boar. For each virus the fragments were partially sequenced to give 475 bp of the E1/E2 glycoprotein and 212 bp of the putative polymerase gene sequences. The data from each set of fragments were compared with one another and with reference strains. This allowed us confidently to assign most of the viruses to one of three subgroups. An analysis of the same viruses with a panel of monoclonal antibodies was much less informative. The subgrouping of the isolates suggested that, in this region of Italy, there had been at least two separate introductions of classical swine fever over a 7 year period and that virus had been transmitted between domestic pigs and wild boar. A consensus nucleotide sequence derived from the glycoprotein fragments of all the viruses examined revealed conservation at the wobble position of some codons.
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Analysis of NS3-mediated processing of the hepatitis C virus non-structural region in vitro
More LessThe protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3−5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.
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Functional reconstitution in lipid vesicles of influenza virus M2 protein expressed by baculovirus: evidence for proton transfer activity
More LessThe influenza virus M2 protein was expressed from a recombinant baculovirus in Spodoptera frugiperda Sf9 cells, purified and reconstituted into artificial membrane vesicles. The specific inhibitor amantadine overcame the toxic activity of the protein and boosted the rate of M2 synthesis by a factor of 10, allowing yields of about 1 mg of purified M2 protein per g of Sf9 cells. M2 protein expressed in this system was phosphorylated and palmitoylated and displayed properties similar to the authentic virus protein. Purified wild-type M2 protein and an amantadine-resistant mutant M2 (M2δ) with a deletion in the trans-membrane domain (amino acids 28 to 31) were incorporated into lipid vesicles, which were loaded with the fluorescent pH indicator pyranine. On imposition of an ionic gradient, M2 caused a decrease in intravesicular pH, which was susceptible to inhibition by 0·1 to 1 μm-rimantadine or N-ethyl-rimantadine. M2δ behaved similarly but exhibited the expected drug resistance. These experiments indicate that isolated M2 functions as an ion channel and demonstrates in vitro M2-mediated proton translocation.
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Defective interfering type A equine influenza virus (H3N8) protects mice from morbidity and mortality caused by homologous and heterologous subtypes of influenza A virus
More LessIntranasal administration of defective interfering A/equine/Newmarket/7339/79 (H3N8) influenza virus (DI EQV) protected mice from otherwise lethal intranasal infection with homologous virus (EQV) or with the heterologous subtypes A/WSN (H1N1) or A/PR/8/34 (H1N1). Such protected mice showed little or no sign of clinical disease. Disease with only low mortality resulting from a ‘low’ dose of WSN was completely prevented with a 100-fold lower dose of DI EQV (4 haemag-glutinating units/ml or 12 ng virus/mouse), indicating that there was a roughly proportional relationship between the protective dose of DI virus and the infecting inoculum. DI EQV-protected mice continued to gain weight at the normal rate, whereas those treated with inactivated DI EQV ceased putting on weight for about 7 days and were still underweight nearly 3 weeks later. Unlike DI WSN, DI EQV inhibited multiplication of infectious WSN in the lungs by 20 to 60-fold. Intranasal DI EQV on its own gave little protection to mice challenged 24 days later with EQV suggesting that it was only weakly immunogenic. DI EQV afforded significant protection when given up to 5 days before live virus challenge indicating that the DI genome remained active in the respiratory tract for this period of time.
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Neutralization escape mutants of type A influenza virus are readily selected by antisera from mice immunized with whole virus: a possible mechanism for antigenic drift
More LessIt is not fully understood how antigenic drift of the haemagglutinin of type A influenza virus in man occurs in the presence of the expected polyclonal antibody response to the five antigenic sites, A to E. Here we show that 12 % (11/92) of sera from mice which had mounted a secondary immune response to inactivated influenza virus were able to select escape mutants. No escape mutant was selected with serum from nonimmunized mice (0/65). Selection required only a single passage, and escape mutants were identified by their reaction with monoclonal antibodies (MAbs); all but one had altered reactivity at site A. Most of the site A escape mutants (7/10) were conventional in character and did not react in haemagglutination-inhibition (HI) or neutralization assays with the identifying MAb. The HA genes of three of these were part sequenced and had a predicted single amino acid substitution (Gly-144 → Glu) in site A. The other escape mutants (3/10) had a small (2-fold) reduction in HI and neutralization to the site A MAb, but no amino acid substitution in site A. The final mutant was a conventional site B escape mutant. To model antisera which selected escape mutants, we constructed ‘pseudo-immune sera’ using mixtures of two neutralizing MAbs in which the first MAb was held at a constant high concentration (1000 HIU/ml). Escape mutants could be selected to the first MAb when the titre of the second MAb was reduced to a low but still inhibiting concentration (1 to 3 HIU/ml). Mixtures of three MAbs also selected escape mutants with similar facility provided that the second and third MAbs were reduced to a similar low concentration. Thus it is possible that the ability of an antiserum to select escape mutants is due to the neutralizing antibody response being biased to an epitope/cross-reacting epitopes within a single antigenic site. However, when escape mutants were reacted in HI assay with their selecting antiserum, the maximum difference from the titre with wt virus was 75 %. The findings of this study may be relevant to the understanding of antigenic drift in type A human influenza virus, and to immune-driven antigenic variation in other virus infections.
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Identification of a second protein encoded by influenza C virus RNA segment 6
More LessInfluenza C virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 6. Unspliced mRNA from this RNA segment, which has not been previously identified, can potentially encode a polypeptide that contains an additional 132 amino acids on the carboxy terminus of the M1 protein. Here the nucleotide sequences of RNA segment 6 of four influenza C strains, isolated in Japan between 1964 and 1988, were compared with the previously determined sequence of C/Ann Arbor/1/50. The results indicated that the deduced amino acid sequence of the carboxy-terminal 132 amino acid domain is conserved fairly well although it is more divergent than the M1 protein sequence. Examination of RNA segment 6-specific mRNAs also showed that unspliced mRNA is present, although in small quantities (~ 13 % of spliced mRNA), in influenza C virus-infected cells. To search for a polypeptide encoded by the unspliced mRNA, the extra carboxy-terminal domain was expressed in Escherichia coli as the glutathione 5-transferase fusion protein, and rabbit immune serum was raised against the purified fusion protein. Immunoprecipitation experiments with this antiserum revealed that a previously unrecognized protein of apparent M r ~ 18000, designated CM2, is synthesized in influenza C virus-infected cells.
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Increased binding activity of measles virus to monkey red blood cells after long-term passage in Vero cell cultures
More LessRecent field isolates of measles virus (MV) obtained by using B95-8 cells have been reported not to agglutinate African green monkey red blood cells (AGM-RBC). Vero cell-adapted, plaque-forming strains derived from three field isolates at the third passage in Vero cell cultures (T8Ve-3, T11Ve-3 and N13Ve-3) also exhibited markedly decreased binding activity, as determined by infectivity-absorption and haemadsorption tests. On the other hand, binding activity of the respective strains at the twentieth passage (T8Ve-20, T11Ve-20 and N13Ve-20) increased to practically the same level as that of the Edmonston strain, a standard strain of MV passaged long-term. A membrane immunofluorescence test revealed that the decreased binding activity to AGM-RBC of T8Ve-3, T11Ve-3 and N13Ve-3 was not due to decreased expression of the haemagglutinin (H) protein on the cell surface. The deduced amino acid sequence of the H protein synthesized in T11Ve-3-infected cells was identical to that in T11Ve-20-infected cells, although a single amino acid alteration was observed when T8Ve-3 was compared with T8Ve-20. Similarly, approximately half of the N13Ve-20-infected cells synthesized an H protein identical to that produced in N13Ve-3-infected cells, and nevertheless, exhibited markedly increased haemadsorption. The present results suggest that a viral protein(s) other than the H protein contributed to the binding activity of MV to AGM-RBC.
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Fusion properties of cells constitutively expressing human parainfluenza virus type 4A haemagglutinin-neuraminidase and fusion glycoproteins
We established HeLa cell lines that constitutively expressed the fusion (F) and/or haemagglutinin-neuraminidase (HN) glycoproteins of human parainfluenza virus type 4A (PIV-4A) and used them to analyse the roles of these glycoproteins in virus-induced cell fusion. No syncytium formation occurred, even in HeLa cells expressing both the F and HN proteins (HeLa-4aF+HN cells). Also no syncytium was found in a mixed culture of cells expressing the F protein (HeLa-4aF) and the HN protein (HeLa-4aHN). Syncytia were observed in HeLa-4aF cells transfected with the HN gene, but no syncytium formation was found in HeLa-4aHN cells transfected with the F gene. Co-cultivation of HeLa-4aF + HN cells with HeLa-4aF cells generated large polykaryocytes, whereas co-cultivation with HeLa-4aHN cells induced no cell fusion. Infection of HeLa-4aF cells with PIV-4A generated large syncytia and degenerated nuclei, whereas little or no polykaryocytes were found in HeLa-4aHN cells infected with PIV-4A. From the above findings, the following conclusions were drawn: (i) the expression of both the F and HN proteins in the same cell is necessary for cell fusion; (ii) the expression of the F protein alone enhances susceptibility to cell fusion; (iii) the constitutive expression of the HN protein promotes resistance to paramyxovirus-induced cell fusion.
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Evidence that the paramyxovirus simian virus 5 can establish quiescent infections by remaining inactive in cytoplasmic inclusion bodies
More LessFollowing infection of BALB/c fibroblastic (BF) cells with simian virus 5 (SV5) only low levels of infectious virus were produced and the majority of cells survived the infection. However at 1 day post-infection (p i ), near normal levels of all the virus proteins were synthesized and the virus genome was replicated. RNA analysis of the infected cells revealed that the levels of viral genomic RNA remained high over 5 days of infection, but that viral mRNA levels were significantly reduced by 3 days p.i. There was no evidence for the accumulation of defective genomes over this period. The reduction in mRNA levels was reflected by a concomitant decrease in the rate of ongoing viral protein synthesis. Despite the apparent decrease in viral transcription, comparative measurements of the relative levels of the different virus proteins at various times p.i. revealed that the levels of the P and NP proteins were similar at 1 and 5 days p.i. but the levels of Y, M and F declined. Immunofluorescence analysis supported this data showing that at later times p.i., although there were some cells which were positive for all the viral proteins, a high proportion of cells were strongly positive for NP and P but negative for M, F and HN proteins. In these cells, NP and P were often located in discrete cytoplasmic foci. A series of cell lines were established from BF cells that had been infected at high multiplicity. Immunofluorescence studies showed that only a minority of cells in these cell lines were infected. This suggests that upon cell division, in a proportion of cells, virus replication was not taking place; otherwise it would be expected that all the daughter cells would remain infected. However, upon co-cultivation of these cells with Vero cells (cells that are fully permissive for SV5 replication), non-defective virus could be recovered. Virus cytoplasmic inclusion bodies could still be detected in a small proportion of BF cells that had been infected at high m.o.i. and passaged 10 times over a 12 week period, and again low levels of infectious virus could be recovered from these cells. It is proposed that in these persistently infected cells, the majority of virus genomes reside in an inactive form in cytoplasmic inclusion bodies but from which virus may occasionally be reactivated. Studies on cell clones obtained from a Vero cell line persistently infected with SY5 for over 100 passages (in which there are large numbers of defective genomes) also showed that not all daughter cells remained infected upon cell division. At later times p.i. in BF cells, when the majority of cells were positive for only NP and P, the cells became more resistant to cell-mediated immune lysis. The significance of these results in terms of the biology and immunology of paramyxovirus infections is discussed.
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Identification of an amino acid change that affects N protein function in vesicular stomatitis virus
More LessTsW16B is a temperature-sensitive mutant of vesicular stomatitis virus. Others have shown that it is temperature-sensitive for replication in vivo and for transcription in vitro and that these phenotypes are probably due to mutation of the N (nucleocapsid) gene. Five independent revertants were isolated from tsW16B based on their ability to grow at 39 °C. The thermosensitivity of in vitro transcription by these revertants was similar to that of the wild-type virus [Wt (HR) ] from which tsW16B was derived. Fractionation-reconstitution studies of two revertants indicated that the reversion was in the N or P (phosphoprotein) gene. The N and P genes of ts(HR), tsW16B, and these two revertants were sequenced. There were no differences between the P genes. Comparison of the predicted N protein sequences of ts (HR), tsW16B and the two revertants indicated that the growth and in vitro transcription phenotypes of ttW16B were due to a change of amino acid residue 238 from threonine to isoleucine. The amino acid at position 238 in the other three revertants also showed an exact reversion to threonine. Amino acid residue 238 lies in a domain of the N protein which is highly conserved among vesiculoviruses.
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Molecular cloning and sequence analysis of the phosphoprotein, nucleocapsid protein, matrix protein and 22K (M2) protein of the ovine respiratory syncytial virus
More LessRespiratory syncytial viruses (RSVs) cause serious respiratory tract disease in infants and children worldwide and similar disease in calves. Strains have been isolated from other ruminant animals such as sheep and goats, but these viruses have not been characterized at the molecular level. In this study, we report the cloning and sequencing of four structural genes coding for the phosphoprotein, nucleocapsid (N) protein, matrix (M) protein and 22K protein of an ovine RSV strain. Comparisons of these sequences with those of bovine and human RSV show that the M and N proteins are the most conserved between ruminant RSV strains and the N protein is the most conserved protein between human and ruminant RSV strains. The attachment G glycoprotein and the small hydrophobic protein are the most divergent proteins among human and ruminant RSV subgroups.
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Characterization of a secreted form of measles virus haemagglutinin expressed from a vaccinia virus recombinant
More LessThe measles virus (MV) haemagglutinin (HA) is a class 2 glycoprotein by means of which the virus particle attaches to the host cell receptor. We have previously expressed this glycoprotein as a vaccinia recombinant virus and have shown that the HA glycoprotein synthesized is indistinguishable from that coded by MV. In the present study, we report that in RK13 cells a soluble form (sHA) of the HA is secreted into the medium. We show by SDS-PAGE and sucrose density gradient centrifugation that the sHA is a dimer and is smaller than the cell-associated form. Using a variety of inhibitors the production of sHA was shown to be a late event, probably occurring at the membrane; only fully glycosylated molecules were found in sHA. Finally, we demonstrate that sHA retains its antigenicity with conformation-dependent MAbs and its receptor recognition function. We conclude that sHA is a valuable tool for use in studies of the structure and function of the MV HA glycoprotein.
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Nucleotide sequence comparisons of the fusion protein gene from virulent and attenuated strains of rinderpest virus
More LessWe have cloned and sequenced the entire fusion (F) protein gene of the RBOK vaccine strain of rinderpest virus and the coding regions for the F genes of two mild field isolates of the virus from Africa. Analysis of the nucleotide and the predicted amino acid sequences showed that the vaccine virus was more than 99% identical in the protein coding region to the virulent Kabete O strain from which it was derived, whereas the field isolates differed by 10 to 12% from each other and from the vaccine strain. No changes were found in the F protein which could explain attenuation of the vaccine; however, each of the mild field isolates had amino acid changes in important functional areas which may be related to their attenuated phenotype.
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Genetic reassortment of influenza C viruses in man
We reported previously that the antigenicity of the haemagglutinin-esterase (HE) glycoprotein of the human influenza C virus strain C/Nara/1/85 was indistinguishable from that of strain C/Nara/82. However, the ribonuclease T1-oligonucleotide map of total virion RNA of C/Nara/1/85 differed remarkably from the map of C/Nara/82, resembling instead the map of C/Nara/ 2/85, which has an HE antigenicity dissimilar to C/Nara/82 and C/Nara/1/85. This observation raised the possibility that C/Nara/1/85 might have arisen by reassortment from two viruses closely related to C/Nara/ 82 and C/Nara/2/85, respectively. Here, we compared the total nucleotide sequence of the HE gene and partial sequences of the other genes of C/Nara/1/85 with those of C/Nara/82 and C/Nara/2/85. The results suggest that C/Nara/1/85 has inherited HE and NP genes from a C/Nara/82-related virus and the PB2, PB1, PA, M and NS genes from a C/Nara/2/85-related virus.
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Nucleotide sequence of hepatitis C virus (type 3b) isolated from a Japanese patient with chronic hepatitis C
The genomic sequences of many hepatitis C virus (HCV) isolates have been reported and a variety of virus genotypes have been classified based on homology in the conserved regions. We have previously identified five distinct genotypes (1a, 1b, 2a, 2b and 3b) in Japanese patients with chronic HCV infection by comparing the sequences of the NS5 region. The complete nucleotide sequence for five genotypes (1a, 1b, 1c, 2a and 2b) have already been reported and we report here the complete nucleotide sequence of genotype 3b. The isolate (HCV Tr) was 9439 nucleotides long, excluding the poly(U) tract at its 3′ end, and encodes a single long open reading frame of 3023 amino acids. Total nucleotide sequence homologies were 68·4 to 68·7%, 68·3 to 69·0%, 67·2%, 65·8 % and 65·6 % compared with type 1a, 1b, 1c, 2a and 2b genomes, respectively. The amino acid sequences of these five genotypes were highly homologous in the core, NS3 and NS5B regions, but the E2/NS1 region, which contains hypervariable regions 1 and 2, and the NS5A region were poorly conserved. Although it was possible to detect antibody against the relatively homologous core and NS3 regions by ELISA, the presence of divergent protein structures must be taken into account in the development of a vaccine.
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Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3
More LessThe gene encoding the outer capsid protein, VP2, of African horsesickness virus serotype 3 (AHSV-3) has been sequenced in its entirety from cDNA clones of the segment 2 RNA, and compared with the previously published VP2 gene sequence of AHSV-4. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50·5 % identity to the amino acid sequence of AHSV-4 VP2. Two areas of high variability (approximately 35 % identity) were identified. The N-proximal variable region (amino acids 128 to 309) exhibited significant hydro-philicity, suggesting a possible role in the determination of the serotype-specific immune response. VP2 of AHSV-3 has furthermore been expressed in a baculo- virus expression system. The expressed protein was shown to react specifically with an anti-AHSV-3 serum in Western blots. Antibodies raised in rabbits and guinea-pigs against the recombinant VP2 neutralized the virus in a plaque reduction assay, confirming the identity of VP2 as the major neutralization-specific antigen of AHSV.
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Genetic analysis of NSP1 genes of human rotaviruses isolated from neonates with asymptomatic infection
More LessThe nucleotide sequences of the genomic RNA segments 5 (gene 5) encoding the non-structural protein NSP1 of rotavirus strains M37 and ST3, isolated from neonates with asymptomatic infection, were determined. The sequences were similar overall (95 % identity) as were the deduced amino acid sequences of NSP1 (93 %). However, the M37 and ST3 NSP1 proteins shared only 82% and 81% identity, respectively, with the corresponding protein from another strain isolated from a neonate with asymptomatic infection (1321). Differences (of between 15% and 31 %) were found in comparison with NSP1 sequences of rotaviruses isolated from older children with symptomatic infection (Wa, DS1 and IGV-80–3). Using an M37 gene 5-derived probe, Northern hybridization analysis of total genomic RNA extracted from viruses isolated from older children (Wa, RV4, RV5 and P) and neonates (M37, ST3, RV3 and 1076), representatives of the most common human G and P types, further indicated that while the gene 5 alleles of strains M37, ST3 and RV3 had a high degree of identity, no significant identity between 1076 and M37 was observed. In addition, cross-hybridization between the M37 probe and RNA of strains from older children (Wa, RV4 and P) was evident. Thus, neonatal human rotavirus strains do not carry a common NSP1 gene.
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Virus neutralization reveals antigenic variation among feline immunodeficiency virus isolates
More LessBy using a focus reduction assay in CrFK feline fibroblast cells, virus neutralizing antibodies (VNA) to feline immunodeficiency virus (FIV) were demonstrated in cats that had been naturally or experimentally infected with FIV. The antigenic relatedness of four strains of FIV, divergent in nucleotide sequence within the env gene, was investigated by neutralization following adaptation of each virus for growth in CrFK cells. Two of the viruses were from The Netherlands (FIV/AM-4 and AM-6), one was from the U.K. (FIV/GL-8) and one was from the U.S.A. (FIV/PET). Reaction of the viruses in the neutralization assay with cat antibodies to homologous or heterologous strains indicated that while there was a degree of cross-reactivity between all four, there were consistent differences suggesting the existence of FIV neutralization subtypes. In particular, FIV/PET and FIV/AM-6 were closely related but FIV/PET and FIV/GL-8 were clearly distinct. VNA from naturally infected cats in the field showed a pattern of reactivity against FIV/PET and FIV/GL-8 that confirmed the antigenic diversity of FIV.
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Feline immunodeficiency virus can productively infect cultured endothelial cells from cat brain microvessels
Feline immunodeficiency virus (FIV) provokes a disease in cats characterized by histopathological lesions similar to those observed in AIDS patients. In order to determine whether endothelial cells from brain microvessels are involved in the central nervous system disease to the same extent as macrophages and microglia, cells were isolated from healthy cat brains, cultured and infected in vitro with the FIV Villefranche IFFA 1/88 strain. The isolated cells displayed typical endothelial cell ultrastructural features and were characterized further by von Willebrand factor-labelling and the binding of specific lectins such as Ulex europaeus lectin on their membrane. They were also able to take up acetylated low density lipoproteins. Two weeks after infection, significant amounts of FIV p24 antigen were detected by indirect immunofluorescence in syncytia and single cells. Concomitantly, the same antigen could be detected in the culture medium of the infected cells by an ELISA technique. Numerous viral particles as well as different steps in the process of viral budding were observed under transmission electron microscopy. The synthesis of FIV p24 antigens still occurred in cells in which replication was blocked in the G2 phase with taxol. Our results suggest the possibility of a productive infection of brain microvascular endothelial cells by FIV in vivo, which could lead to important perturbations in the functions of the blood-brain barrier.
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Phylogenetic classification of human T cell leukaemia/lymphoma virus type I genotypes in five major molecular and geographical subtypes
Proviral DNA was obtained from ex vivo peripheral blood mononuclear cells of 75 human T cell leukaemia/ lymphoma virus type I (HTLV-I)-infected individuals who were either asymptomatic or had adult T cell leukaemia or tropical spastic paraparesis/HTLV-I-asso- ciated myelopathy. Amplified long terminal repeats (LTRs) were analysed for restriction fragment length polymorphisms (RFLPs). The results, together with previously published LTR data (a total of 180 specimens analysed), showed the presence of 12 different RFLP profiles with four major molecular subtypes. Furthermore, a fragment of 413 bp (nucleotides 22 to 434) of the U3/R region was sequenced for 12 new HTLV-I specimens originating from Central and West Africa (8 cases), Iran (1 case), Caribbean (2 cases) and Reunion Island (1 case). Phylogenetic analysis using three different techniques (maximum parsimony, neighbourjoining and UPGMA) comparing these 12 strains (including four new African HTLV-I variants) with the 30 published partial HTLV-I LTR sequences (nt 120 to 434) showed the existence of clusters of molecular variants in discrete geographical areas. The topology of the phylogenetic trees is thought to reflect HTLV-I evolution and the migrations of virally infected populations in the recent or distant past. Furthermore, there was a nearly perfect concordance between the clustering based on the LTR sequence homologies and the LTR RFLP subtypes suggesting that this rapid and simple technique is well suited to the investigation of HTLV-I molecular epidemiology. These results allow a new phylogenetic classification of HTLV-I genotypes into five major molecular subtypes: Cosmopolitan (C) subtype widespread all over the world, Japanese (J) subtype, West African (WA) subtype, Central African (CA) subtype and Melanesian (M) subtype.
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The granulin gene region of Cryptophlebia leucotreta granulosis virus: sequence analysis and phylogenetic considerations
More LessThe nucleotide sequence of a 2·2 kb genome region of the baculovirus Cryptophlebia leucotreta granulosis virus (C1GV) encompassing the granulin gene and a second open reading frame, designated ORF909, was determined. The putative granulin ORF comprises 744 nucleotides encoding a polypeptide with a predicted M r of 29·3K. The 5′ leader of the granulin gene contains a canonical late baculovirus promoter (ATAAG) and differs only in two nucleotide positions from the sequence of Cydia pomonella granulosis virus (CpGV). Sequence comparison with the granulin genes of different granulosis viruses indicated a very close relationship between C1GV and CpGV of about 96% amino acid identity. ORF909 is 909 nucleotides in length and encodes a potential protein of M r 36·2K. It contains two zinc finger-like motifs, similar to ME53, which was previously described for Autographa cali- fomica multiple nucleocapsid nuclear polyhedrosis virus. ORF909 protein is significantly shorter than ME53 protein, lacking more than 105 amino acids of the amino terminus of ME53. Both genes contain an early and a late promoter motif suggesting a similar temporal regulation. Homologous sequences to ORF909 are also present upstream of the granulin genes of other granulosis viruses (GVs) indicating that this ORF is common in GVs.
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A modified hepatitis B virus surface antigen with the receptor-binding site for hepatocytes at its C terminus: expression, antigenicity and immunogenicity
More LessA modified hepatitis B virus (HBV) surface antigen, the SA-28 protein, was constructed and expressed by recombinant vaccinia virus in mammalian cells. This protein was composed of a PreSl region-derived peptide (amino acids 21 to 47) that contained the hepatocyte receptor-binding site, joined to the C terminus of the major S protein at amino acid position 223. This modified surface antigen could be efficiently assembled into particles with a density of 1·23 g/ml and could be secreted from several mammalian cell lines. The results of immunoprecipitation revealed that the SA-28 protein was recognized by both the anti-S protein antibody and the anti-PreSl antibody. A strong antibody response, against both the S protein and PreSl epitopes, was induced in BALB/c mice immunized by the SA-28 particles, indicating good immunogenicity. These results suggested that the HBV surface antigen consisting of the SA-28 protein could be a promising candidate as a new HBV vaccine with higher efficacy.
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ICP34.5 influences herpes simplex virus type 1 maturation and egress from infected cells in vitro
More LessWe have previously demonstrated that efficient replication of mutant herpes simplex virus which fails to synthesize the polypeptide ICP34.5 is cell type and cell state dependent. ICP34.5 negative viruses do not grow in stationary state mouse embryo fibroblast 3T6 cells whereas the growth kinetics in BHK cells are indistinguishable from those of wild-type. We now demonstrate that this defect is not due to an inability of mutant virus to adsorb to 3T6 cells but rather to an inability to spread from the initially infected cells. Electron microscopic studies with wild-type HSV in both BHK and 3T6 cells revealed virus particles equally distributed between nucleus and cytoplasm, and additionally in the extracellular matrix. In BHK cells infected with the ICP34.5 negative mutant 1716, virus is likewise distributed between nucleus and cytoplasm but in 50 % of the infected cells there is marked delamination and swelling of the nuclear membrane. In addition there is evidence of a significant number of particles trapped between the nuclear lamellae. When 1716 is used to infect 3T6 cells, over 90% of the virus particles are confined to the nuclei and the number of infected cells remains constant between 24 and 48 h with no increase in the proportion of extracellular virus. Failure to express ICP34.5 appears therefore to result in a defect in virus maturation and egress from the nuclei of infected cells. Egress of HSV from the nuclei to the extracellular space is thought to occur via two pathways. We postulate that lack of expression of ICP34.5 results in one of these pathways being blocked. In BHK cells this leads to overloading of the alternative pathway with a buildup of particles in the nuclear lamellae and associated endoplasmic reticulum. In stationary state 3T6 cells, it appears that there is no functional alternative pathway. We conclude that ICP34.5 exerts an effect on HSV maturation by controlling the passage of virus through infected cells.
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Evidence for two groups of banana bunchy top virus isolates
More LessBanana bunchy top virus (BBTV) DNA component 1 from isolates from 10 different countries was cloned and sequenced and the sequences were aligned and compared. This analysis indicated two groups: the South Pacific group (isolates from Australia, Burundi, Egypt, Fiji, India, Tonga and Western Samoa) and the Asian group (isolates from the Philippines, Taiwan and Vietnam). The mean sequence difference within each group was 1·9 to 3·0% and between isolates from the two groups was approximately 10%, but some parts of the sequences differed more than others. However, the protein encoded by the major open reading frame, which is probably a replicase, differed by approximately 5%. The region from the beginning of the stem-loop sequence to the potential TATA box was identical in all isolates except for a two nucleotide change in the Western Samoan isolate and a single change in that of the NSW isolate. These results, together with other evidence, suggest that BBTV has spread to bananas after the initial movement of bananas from the Asian Pacific regions to Africa and the Americas.
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Papaya ringspot potyvirus: isolate variability and the origin of PRSV type P (Australia)
More LessThe GenBank accession numbers for the sequence data reported in this paper are U14736 to U14744.
We have sequenced the coat protein gene of nine isolates of papaya ringspot virus (PRSV) including six Australian and three Asian isolates and compared these with four previously reported sequences of PRSV. There was up to 12 % sequence variation between isolates at the nucleotide level. However, there was no significant difference between the sequences obtained from Australian isolates irrespective of whether they were PRSV type P (cucurbit or papaya infecting) or PRSV type W (cucurbit infecting) and these isolates were more closely related to one another than to any other isolate. These results imply that PRSV-P, first recorded in Australia in 1991, arose locally from PRSV-W (first recorded in Australia in 1978) rather than being introduced. Further, there was no consistent sequence difference between PRSV-P and PRSV-W isolates that would obviously account for their host range difference.
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Capsid protein properties of cowpea aphid-borne mosaic virus and blackeye cowpea mosaic virus confirm the existence of two major subgroups of aphid-transmitted, legume-infecting potyviruses
More LessA study of the capsid proteins of different legume-infecting potyviruses using specific monoclonal antibodies on immunoblots of crude extracts from infected plants revealed that cowpea aphid-borne mosaic virus (CAMV) and blackeye cowpea mosaic virus (B1CMV) have coat protein M r values of 32K and 35K, respectively. Immunoblot comparisons of BICMV, peanut stripe mosaic virus (PStV), bean common mosaic virus (BCMV) and azuki bean mosaic virus (AzMV) revealed equal reactivity of their 35K coat proteins. Similar comparisons between CAMV and the necrotic strain of BCMV (isolate NL3) showed a serological relationship between their 32K coat proteins, results providing the first evidence of a possible similarity between CAMV and BCMV NL3. Peptides from trypsin digests of the coat proteins of several of these legume-infecting potyviruses were analysed by HPLC. Comparison of the peptide profiles confirmed the serological results in distinguishing the two subgroups. Peptide profiles of coat protein from BICMV, PStV, AzMV and BCMV were almost identical, results suggesting that they could be considered as strains of one virus. In contrast, peptide profiles of various CAMV serotypes and BCMV NL3 were distinct from the first group and exhibited limited similarities to each other.
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The nucleotide sequence of potato mop-top virus RNA 2: a novel type of genome organization for a furovirus
More LessParticles of isolate T of potato mop-top furovirus (PMTV) contain three RNA species (6·5, 3·0 and 2·5 kb). Hybridization tests with cloned cDNA probes showed that none of these species was derived from another. RNA 2 (2962 nt), which was sequenced, has non-coding regions of 368 nt and 285 nt at the 5′ end and 3′ end, respectively. Near the 5′ terminus, nucleotides 46 to 110 are able to form a stem-loop structure, the stem of which has 23 bp with only one mismatch and one unpaired nucleotide. From the 5′ end, the four open reading frames encode proteins of 5IK, 13K, 21K and 8K. The first three of these have sequence similarity to the triplegene-block proteins of other viruses, particularly barley stripe mosaic hordeivirus. The 51K protein contains a putative NTP-binding motif and the 13K and 21K proteins each contain two hydrophobic regions separated by a hydrophilic region. The 8K protein is rich in cysteine. PMTV differs from other furoviruses in having a tripartite genome. Its RNA 2 differs in gene content from the RNA 2 of soil-borne wheat mosaic virus, which lacks a triple gene block, and from that of beet necrotic yellow vein virus, which has a coat protein gene and read-through domain to the 5′ side of its triple gene block. The gene arrangement in PMTV is therefore novel for a furovirus.
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Nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus, the prototype of the tenuiviruses
More LessThe complete nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus (RSV), was determined using two sets of overlapping cDNA clones. RNA segment 1 comprises 8970 nucleotides and on the viral complementary sequence has a single long open reading frame coding for a protein of 2919 amino acids with an estimated M r of 336860. Amino acid sequence comparisons of the putative protein indicated strong homology (30% amino acid identity over about 1500 residues) with the L protein of the genus Phlebovirus of the Bunyaviridae, but no detectable similarity with other members of the Bunyaviridae. However, weak similarity was detected with the L protein of Tacaribe arenavirus. The highly homologous sequence domain includes the conserved motifs of the putative RNA-dependent RNA polymerase. The data presented here, along with previous work clearly show significant similarities in genome organization, structure and expression between RSV and members of the genus Phlebovirus of the Bunyaviridae. Taken together, we propose that tenui-viruses should be included in the Bunyaviridae under the genus Tenuivirus.
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Nucleotide sequence and structural features of the Group III citrus viroids
More LessThe nucleotide sequence and secondary structure of two representative variants from the Group III citrus viroids, CVd-IIIa (297 bases) and CVd-IIIb (294 bases) were determined. The variants are related to the apple scar skin viroid (ASSVd) family. Although smaller in size than any of the ASSVd-related viroids, the central conserved region as well as most of the terminal conserved region of ASSVd is retained. The rod-like structural configuration (characteristic of ASSVd) of the variants as predicted by minimum free energy analysis is presented.
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The closely related citrus ringspot and citrus psorosis viruses have particles of novel filamentous morphology
More LessSome properties of the particles of citrus ringspot virus (CtRSV) and the related citrus psorosis-associated virus (CPsAV) are described. The particles of CtRSV have been reported to be sinuous linear structures about 10 nm in diameter and of two lengths (300 to 500 nm and 1500 to 2500 nm) representing ‘top’ and ‘bottom’ sedimentation components. We show that these particles are collapsed double-stranded forms of nucleocapsid-like, highly flexuous open circles formed of filaments 3 to 4 nm in diameter. Top-component filaments had contour lengths of 600 to 1000 nm, i.e. twice that reported for the corresponding collapsed form. Bottom-component filaments had contour lengths about four times longer than those of top-component filaments. The structures suggest that CtRSV represents a new genus (possibly family) related to the tenuiviruses. However, we failed to demonstrate any serological relationship between CtRSV and several tenuiviruses; moreover, the capsid protein sizes and host ranges are quite different. We offer the name Ophiovirus for the proposed new genus.
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