- Volume 82, Issue 2, 2001
Volume 82, Issue 2, 2001
- Animal: RNA Viruses
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The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus
More LessIn addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5–6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1–10 units/μg RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0·02 μg/μg RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)25, suggesting that the poly(A) sequence on the 3′ end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [3H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.
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The C-terminal residues of poliovirus proteinase 2Apro are critical for viral RNA replication but not for cis- or trans-proteolytic cleavage
More LessPoliovirus proteinase 2Apro is an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has been obtained that 2Apro is also involved in genome replication. All enteroviruses have a negatively charged cluster of amino acids at their C terminus (EE/D E/DAMEQ–NH2), a common motif suggesting function. When aligned with enterovirus sequences, the 2Apro proteinase of human rhinovirus type 2 (HRV2) has a shorter C terminus (EE…Q–NH2) and, indeed, the HRV2 2Apro cannot substitute for poliovirus 2Apro to yield a viable chimeric virus. Here evidence is provided that the C-terminal cluster of amino acids plays an unknown role in poliovirus genome replication. Deletion of the EEAME sequence from poliovirus 2Apro is lethal without significantly influencing proteinase function. On the other hand, addition of EAME to HRV2 2Apro, yielding a C terminus of this enzyme of EEEAMEQ, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. Poliovirus type 1 Mahoney 2Apro could be provided in trans to rescue the lethal deletion EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C terminus of poliovirus 2Apro is involved in particle formation. It is concluded that the C terminus of poliovirus 2Apro is an essential domain for viral RNA replication but is not essential for proteolytic processing.
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Molecular identification of new picornaviruses and characterization of a proposed enterovirus 73 serotype
More LessEnteroviruses (EV) have traditionally been identified by using serotype-specific antisera in a virus-neutralization test. Three EV strains isolated in California, USA, in 1955, 1964 and 1978, and a 1995 Oman isolate, were found to be antigenically related to one another; however, the strains were not neutralized by standard EV typing antisera, suggesting that they may represent a new EV serotype. The isolates were characterized genetically by RT–PCR coupled with amplicon sequencing and comparison to a database of enterovirus nucleotide sequences. The strains were 75·3 to 87·2% identical to one another in complete VP1 nucleotide sequence, but no more than 68% identical in sequence to the prototype strain of any EV serotype. Their complete capsid sequences were closely related to one another, but only distantly related to those of any EV prototype strain. The California and Oman isolates were most closely related to members of EV cluster B, suggesting that they are unclassified members (i.e. a new serotype) of cluster B. The complete genome sequence was determined for one isolate, CA55-1988, and the predicted polyprotein sequence was 86·5 to 89·2% identical to those of other cluster B EV and 56·7 to 61·9% identical to the polyprotein sequences of EV belonging to other clusters. Isolation of this new EV serotype from samples obtained on two continents and over a period of 40 years suggests continued circulation over a wide geographical area. In keeping with standard picornavirus nomenclature, we propose that this new serotype be named ‘enterovirus 73’ (EV73).
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Genetic reclassification of porcine enteroviruses
More LessThe genetic diversity of porcine teschoviruses (PTVs; previously named porcine enterovirus 1) and most serotypes of porcine enteroviruses (PEVs) was studied. Following the determination of the major portion of the genomic sequence of PTV reference strain Talfan, the nucleotide and derived amino acid sequences of the RNA-dependent RNA polymerase (RdRp) region, the capsid VP2 region and the 3′ non-translated region (3′-NTR) were compared among PTVs and PEVs and with other picornaviruses. The sequences were obtained by RT–PCR and 3′-RACE with primers based on the sequences of Talfan and available PEV strains. Phylogenetic analysis of RdRp/VP2 and analysis of the predicted RNA secondary structure of the 3′-NTR indicated that PEVs should be reclassified genetically into at least three groups, one that should be assigned to PTVs and two PEV subspecies represented by strain PEV-8 V13 and strain PEV-9 UKG410/73.
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Human immunodeficiency virus type 2 lentiviral vectors: packaging signal and splice donor in expression and encapsidation
More LessRetroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)–Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 105 transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1–5×107 TU/ml.
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The rapamycin sensitivity of human T-cell leukaemia virus type I-induced T-cell proliferation is mediated independently of the polypyrimidine motifs in the 5′ long terminal repeat
More LessThe immunosuppressant rapamycin can regulate the translation of a subset of messenger RNAs, a phenotype which has been linked to the presence of a polypyrimidine motif [C(N)4–14] downstream of the mRNA cap structure. T-cell clones naturally infected with transcriptionally active human T-cell leukaemia virus, type I (HTLV-I) undergo autologous proliferation; this phenotype is inhibited by rapamycin but not FK506, which reverses the rapamycin effect. Within the R region of the HTLV-I 5′ long terminal repeat (LTR) there are seven polypyrimidine motifs. We sought to determine if these were involved in the sensitivity of proliferation to the presence of rapamycin. Here we illustrate the generation of an in vitro model of this rapamycin-sensitivity and the analysis of LTR mutants which were created to determine the importance of the polypyrimidine motifs. Reporter gene assays suggest the effect is independent of the polypyrimidine motifs in the virus leader sequence.
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Measles virus and canine distemper virus target proteins into a TAP-independent MHC class I-restricted antigen-processing pathway
After infection of CEM174.T2 cells [deficient for the transporter of antigen presentation (TAP)] with measles virus (MV) the nucleocapsid protein is recognized by Ld-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated by colocalization with the lysosomal-associated membrane protein 1 (LAMP-1). Similarly, the nucleocapsid protein of canine distemper virus (CDV) is recognized in a TAP-independent fashion. In addition, a recombinant MV expressing bacterial β-galactosidase protein is able to introduce the recombinant antigen into the TAP-independent pathway whereas a vaccinia virus expressing β-galactosidase is not. These data and a report about TAP-independent recognition of parainfluenza virus type 1 suggest that members of the Paramyxoviridae family regularly introduce viral proteins into the TAP-independent antigen-processing pathway.
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- Animal: DNA Viruses
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The UL14 protein of herpes simplex virus type 2 translocates the minor capsid protein VP26 and the DNA cleavage and packaging UL33 protein into the nucleus of coexpressing cells
The herpes simplex virus type 2 (HSV-2) gene UL14 encodes a 32 kDa protein which is a minor component of the virion tegument and is expressed late in infection. The UL14 protein shows varied localization patterns in HSV-2-infected and singly expressing cells, suggesting the possibility that it is multifunctional. We have investigated the influence of the UL14 protein on the intracellular localization of capsid proteins and DNA cleavage and packaging proteins in coexpressing cells. VP26 is the minor capsid protein; it binds to hexons of the outer capsid shell and is predominantly cytoplasmic upon sole expression. We have found that VP26 coexpressed with the UL14 protein showed mutual and predominant relocation into the nucleus. At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32 and UL33) that are required for DNA cleavage and packaging. We have found that the UL33 protein, which was also cytoplasmic by sole expression, was relocated to the nucleus upon expression with the UL14 protein, which again seemed to be a result of mutual influence. Coexpression experiments also suggested the possibility of a mutual influence between the UL14 and UL17 proteins, and the UL17 protein and VP26. Our results suggest that the UL14 protein can influence the intracellular localization patterns of a number of proteins belonging to the capsid or the DNA encapsidation machinery.
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Varicella-zoster virus gH:gL contains a structure reactive with the anti-human gamma chain of IgG near the glycosylation site
Varicella-zoster virus (VZV) glycoproteins were purified from infected cells using monoclonal antibodies and gH:gL was found to react with antibodies to the γ chain of human IgG (h-IgG), whereas gE:gI and gB did not. When gH:gL was captured by concanavalin A, it lost reactivity with the anti-h-IgG γ chain (anti-h-γ-IgG). gH:gL reacted with anti-h-γ-IgG in an ELISA assay and gave a K d value of 2·16×10−7 M in a BIAcore assay. The K d value of the human monoclonal antibody to gH (TI-57) used for the purification of gH:gL was 4·45×10−10 M. Virus pretreated with anti-h-IgG was five times more resistant to neutralization with TI-57. Although the nature of the binding was not clear, gH:gL bound to anti-h-γ-IgG. If this interaction results from immunological similarity between gH:gL and h-IgG, it may cause immune evasion in the pathogenesis of VZV infection.
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The tegument protein ppUL25 of human cytomegalovirus (CMV) is a major target antigen for the anti-CMV antibody response
More LessA viral protein of approximately 82 kDa is the only structural protein of human cytomegalovirus (CMV) that is strongly immunogenic during natural infection and the corresponding gene of which is still unknown. In this work, strong evidence is presented that this protein is the product of UL25.
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Herpesvirus saimiri protein StpB associates with cellular Src
More LessSubgroup B isolates of Herpesvirus saimiri are less efficient in T lymphocyte transformation when compared with subgroups A or C. Here it is shown that subgroup B strain SMHI encodes a protein, StpB, at a position equivalent to those of the ORFs for the saimiri transforming proteins (Stp) of subgroups A and C. StpB shares little similarity with StpA or StpC, but interacts with the SH2 domain of cellular Src, as does StpA. Thus, factors other than c-Src binding determine the efficiency of primary T cell transformation by Herpesvirus saimiri.
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Proteasome inhibitor induces nucleolar translocation of Epstein–Barr virus-encoded EBNA-5
More LessWe have previously shown that Epstein–Barr virus (EBV)-encoded EBNA-5 is localized to PML bodies (PODs) in EBV-immortalized lymphoblastoid cell lines (LCLs). Here we have extended our study of the subnuclear localization of EBNA-5 and found a strict co-localization with PML in LCLs and in BL lines with an immunoblastic, LCL-like phenotype. Moreover, GFP–EBNA-5 accumulated in PML bodies upon transfection into LCLs. In contrast, transfection of cell lines of non-immunoblastic origin with an EBNA-5 expression construct showed preferential localization of the protein to the nucleoplasm. Since PML is involved in proteasome-dependent protein degradation, we investigated the total levels and sub-cellular localization of EBNA-5 upon inhibition of proteasome activity. We found that a proteasome inhibitor, MG132, induced the translocation of both endogenous and transfected EBNA-5 to the nucleoli in every cell line tested. The total EBNA-5 protein levels were not affected by the proteasomal block. EBNA-5 forms complexes with heat shock protein Hsp70. The proteasome inhibitor induced a rise in total levels of Hsp70 and dramatically changed its homogeneous nuclear and cytoplasmic distribution into nucleolar and cytoplasmic. This effect was EBNA-5-independent. The nucleolar localization of Hsp70 was enhanced by the presence of EBNA-5, however. EBNA-5 also enhanced the nucleolar translocation of a mutant p53 in a colon cancer line, SW480, treated with MG132. The coordinated changes in EBNA-5 and Hsp70 localization and the effect of EBNA-5 on mutant p53 distribution upon MG132 treatment might reflect the involvement of EBNA-5 in the regulation of intracellular protein trafficking associated with the proteasome-mediated degradation.
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Characterization of hepatitis B virus genotypes among Yucpa Indians in Venezuela
The complete genome sequences of hepatitis B virus (HBV) from 12 HBV-infected Yucpa Indians of Venezuela, a group with highly endemic HBV, were amplified and sequenced. The 12 isolates were closely related to each other, with 98·6–100% nucleotide identity. A phylogenetic tree based on the complete genome indicated clearly that they were genotype F. Three individuals had evidence of infection with two different HBV deletion mutants. In two individuals, a three amino acid deletion was identified just prior to the ‘a’ determinant loop of the S region. A third individual was infected with virus that contained a complete core reading frame and a population that contained a deletion in the middle of the core region. These results indicate that genotype F HBV is present in the Venezuelan Yucpa Amerindians and the complete genome sequence allowed the identification of two unique deletion mutants in a limited set of samples.
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Vaccination of chronic hepatitis B virus carriers with preS2/S envelope protein is not associated with the emergence of envelope escape mutants
More LessPreS2/S vaccination of chronic hepatitis B virus (HBV) carriers led to a reduction in HBV replication or clearance of virus in 30% of treated patients. This study assessed whether vaccinotherapy of chronic HBV carriers induced the selection of escape mutants in the envelope ‘a’ determinant and whether envelope genetic variability might affect the response to vaccination. No amino acid differences were observed in the ‘a’ determinant between sequences obtained before and after treatment (five responders and seven non-responders). However, alignment with HBV prototype sequences revealed seven amino acid changes. Two mutations (T140S and P127L) diverged from subtype variations. In the complete envelope sequence (five non-responders and five responders), ten amino acid modifications were detected between sequences obtained before and after treatment. The absence of any common mutations did not enable the definition of a hot spot of mutations implicated in the response to vaccination. Moreover, vaccinotherapy does not induce the selection of escape mutants in the ‘a’ determinant.
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Sequence comparison of an Australian duck hepatitis B virus strain with other avian hepadnaviruses
The genome of an Australian strain of duck hepatitis B virus (AusDHBV) was cloned from a pool of congenitally DHBV-infected-duck serum, fully sequenced and found by phylogenetic analyses to belong to the ‘Chinese’ DHBV branch of the avian hepadnaviruses. Sequencing of the Pre-S/S gene of four additional AusDHBV clones demonstrated that the original clone (pBL4.8) was representative of the virus present in the pool, and a head-to-tail dimer of the clone was infectious when inoculated into newly hatched ducks. When the published sequences of 20 avian hepadnaviruses were compared, substitutions or deletions in the polymerase (POL) gene were most frequent in the 500 nt segment encoding the ‘spacer’ domain that overlaps with the Pre-S domain of the Pre-S/S gene in a different reading frame. In contrast, substitutions and deletions were rare within the adjacent segment that encodes the reverse transcriptase domain of the POL protein and the S domain of the envelope protein, presumably because they are more often deleterious.
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Genetic analysis of full-length genomes and subgenomic sequences of TT virus-like mini virus human isolates
The phylogenetic relationship between the complete genomic sequences of ten Japanese and one French isolate of TT virus-like mini virus (TLMV) was investigated. Analysis of the variability of the nucleotide sequences and the detection of signature patterns for overlapping genes suggested that ORFs 1 and 2 are probably functional. However, this was not the case for a putative third ORF, ORF3. Throughout the viral genome, several nucleotide or amino acid motifs that are conserved in circoviruses such as TT virus (TTV) and chicken anaemia virus were identified. Phylogenetic analysis distinguished three main groups of TLMV and allowed the identification of putative recombination breakpoints in the untranslated region. TLMV genomes were detected by PCR in the plasma of 38/50 French blood donors tested and were also identified in peripheral blood mononuclear cells, faeces and saliva. A phylogenetic study of 37 TLMV strains originating from France, Japan and Brazil showed that groupings were not related to geographical origin.
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- Insect
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Epizootiology and transmission of a newly discovered baculovirus from the mosquitoes Culex nigripalpus and C. quinquefasciatus
Reports of mosquito baculoviruses are extremely uncommon and epizootics in field populations are rarely observed. We describe a baculovirus that was responsible for repeated and extended epizootics in field populations of Culex nigripalpus and C. quinquefasciatus over a 2 year period. These mosquito species are important vectors of St Louis and Eastern equine encephalitis in the United States. Our initial attempts to transmit this baculovirus to mosquitoes in the laboratory were unsuccessful. A salt mixture similar to that found in water supporting infection in the field was used in laboratory bioassays and indicated that certain salts were crucial to transmission of the virus. Further investigations revealed conclusively that transmission is mediated by divalent cations: magnesium is essential, whereas calcium inhibits virus transmission. These findings represent a major advancement in our understanding of the transmission of baculoviruses in mosquitoes and will allow characterization of the virus in the laboratory. In addition, they can explain, in great part, conditions that support epizootics in natural populations of mosquitoes that vector life-threatening diseases of man and animals.
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Morphological and molecular evidence that Culex nigripalpus baculovirus is an unusual member of the family Baculoviridae
We present evidence that a newly discovered mosquito virus from Culex nigripalpus is an unusual member of the family Baculoviridae. Development of this virus was restricted to nuclei of midgut epithelial cells in the gastric caeca and posterior stomach. The globular occlusion bodies were not enveloped, measured around 400 nm in diameter, occurred exclusively in nuclei of infected cells and typically contained four, sometimes up to eight, virions. The developmental sequence involved two virion phenotypes: an occluded form (ODV) that initiated infection in the midgut epithelial cells, and a budded form that spread the infection in the midgut. Each ODV contained one rod-shaped enveloped nucleocapsid (40×200 nm). The double-stranded DNA genome was approximately 105–110 kbp with an estimated GC content of 52%. We have sequenced approximately one-third of the genome and detected 96 putative ORFs of 50 amino acids or more including several genes considered to be unique to baculoviruses. Phylogenetic analysis of the amino acid sequences of DNApol and p74 placed this virus in a separate clade from the genera Nucleopolyhedrovirus and Granulovirus. We provisionally assign this virus in the genus Nucleopolyhedrovirus, henceforth abbreviated as CuniNPV (for Culex nigripalpus nucleopolyhedrovirus), and suggest that, awaiting additional data to clarify its taxonomic status, it may be a member of a new genus within the family Baculoviridae.
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P34.8 (GP37) is not essential for baculovirus replication
More LessPrevious reports have indicated that p34.8 (gp37) may be essential for the replication of Autographa californica nucleopolyhedrovirus (AcMNPV) because no virus with inactivated p34.8 was isolated. We have ascertained the requirement for this gene by attempting to inactivate it with a large insertion [the gene encoding GFP (green fluorescent protein)] or by deleting all the conserved domains from the open reading frame (ORF). The gene encoding GFP was inserted into the NotI site of the p34.8 ORF and a viral plaque containing the insertion was propagated in SF-21 cells. Similarly, 531 bp (NotI–XbaI) containing all conserved domains were deleted from the ORF. All mutants were authenticated by PCR amplification, restriction endonuclease analysis, DNA sequencing, and Southern and Northern blot analysis. It was found that inactivation of p34.8 of AcUW1-LacZ (AcMNPV containing a lacZ gene in the p10 locus) had no effect on the biological property of virus, such as virulence and kinetics. These two independent methods showed that p34.8 is not essential for replication and that this locus could provide another site for the engineering of baculoviruses.
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Peroral infectivity of non-occluded viruses of Bombyx mori nucleopolyhedrovirus and polyhedrin-negative recombinant baculoviruses to silkworm larvae is drastically enhanced when administered with Anomala cuprea entomopoxvirus spindles
More LessNon-occluded viruses (NOVs) of Bombyx mori nucleopolyhedrovirus (BmNPV) are poorly infectious to silkworm larvae when administered by peroral inoculation, although they are highly infectious when injected into the insect haemocoel. In the present study, it is demonstrated that NOVs of BmNPV became highly infectious even through peroral inoculation when administered with spindles (proteinaceous structures) of Anomala cuprea entomopoxvirus (AcEPV). Marked enhancement of peroral infectivity of NOVs by AcEPV spindles (nearly 1000-fold higher in the strongest case) was observed in all growth stages of silkworm larvae tested (2nd to 5th instar). Similarly, peroral infectivity of polyhedrin-negative recombinants of BmNPV, which do not produce polyhedra, was also enhanced remarkably by AcEPV spindles. In contrast, spheroids (proteinaceous structures containing AcEPV virions) did not enhance the peroral infectivity of either NOVs or the recombinant BmNPV in silkworm larvae.
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Negative regulatory regions of the PAT1 promoter of Hz-1 virus contain GATA elements which associate with cellular factors and regulate promoter activity
More LessThe persistence-associated transcript 1 (PAT1) is actively expressed during persistent infection with Hz-1 virus, while transcription of the rest of the viral genes is shut down. Previously, results of a series deletion of the PAT1 promoter suggested that the regions from nucleotides −312 to −212 and nucleotides −158 to −90 negatively regulate the promoter activity. Here, the negative regulatory effect of the −312/−90 fragment was confirmed using a heterologous IE0 promoter of Autographa californica multiple nucleopolyhedrovirus. Further, the negative regulation of the −312 to −212 region was orientation-independent. The results of electrophoresis mobility shift assays showed that cellular protein(s) bind specifically to DNA fragments −312/−212 and −158/−90. In each of these fragments, a GATA element was identified by computer-assisted analysis. Mutating both GATA elements in the −312/−90 fragment completely eliminated its negative effect on IE0 promoter activity, while mutating only one of these elements had little or no effect. Together, these results suggest that the GATA element has a negative regulatory role on the IE0 and PAT1 promoters.
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- Plant
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Cell-to-cell movement of potato virus X involves distinct functions of the coat protein
Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.ΔCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.ΔCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.ΔCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.
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Banana bunchy top nanovirus DNA-1 encodes the ‘master’ replication initiation protein
More LessBanana bunchy top nanovirus has a multicomponent, circular single-stranded DNA genome comprising at least six integral components, BBTV DNA-1 to -6, which have been consistently associated with bunchy top disease worldwide. At least three other components, BBTV S1, S2 and Y, which have been isolated from Taiwanese BBTV isolates, do not appear to be integral components. We show here that both BBTV DNA-1 and S1, which encode replication initiation (Rep) proteins, were capable of self-replication when bombarded into banana embryogenic cell suspensions. However, only BBTV DNA-1 was capable of directing the replication of two other BBTV genomic components, namely BBTV DNA-3 which encodes the coat protein, and DNA-5 which encodes a retinoblastoma binding-like protein. These results indicate that (i) BBTV DNA-1 is the minimal replicative unit of BBTV and encodes the ‘master’ viral Rep and (ii) BBTV S1 is possibly a satellite DNA which is unable to replicate integral BBTV components.
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- Other Agents
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Heat stability of prion rods and recombinant prion protein in water, lipid and lipid–water mixtures
More LessPrion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27–30, and the corresponding recombinant protein, rPrP(90–231), were autoclaved in water, bovine lipid or lipid–water mixtures for 20 min at temperatures from 100 to 170 °C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or lipid–water as well as for rPrP(90–231) in lipid–water. The presence of lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in pure lipid gave different results – not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in pure lipid exceeded that in lipid–water mixtures. Degradation factors larger than 104 were reached at 170 °C in the presence of lipids and at 150 °C in the absence of lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 °C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.
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Volumes and issues
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