- Volume 79, Issue 6, 1998
Volume 79, Issue 6, 1998
- Articles
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A proline-to-histidine substitution at position 225 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) sensitizes HIV-1 RT to BHAP U-90152
Two mutant virus strains in which the novel P225H mutation appeared in a V106A reverse transcriptase (RT)-mutated genetic background upon treatment of human immunodeficiency virus type 1 (HIV-1) with quinoxaline S-2720 were isolated. Surprisingly, the addition of the P225H mutation to the V106A RT mutant genetic background resensitized the V106A RT mutant virus to the nonnucleoside RT inhibitor (NNRTI) BHAP U-90152, but not to other NNRTIs. Construction of both recombinant viruses and recombinant RTs containing the V106A, P225H and V106A P225H mutations revealed that P225H was indeed responsible for the marked potentiation of the antiviral activity of BHAP against the P225H single-mutant virus and the V106A P225H double-mutant virus when compared to wild-type and V106A single-mutant viruses, respectively. An explanation for the markedly increased sensitivity of the P225H mutant HIV-1 RT to BHAP and not to the other NNRTIs was provided by the unique features of the X-ray structure of the RT-BHAP complex.
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Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kappaB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells
More LessTuberculosis has emerged as an epidemic, extended by the large number of individuals infected with human immunodeficiency virus type 1 (HIV-1). The major goal of this study was to determine whether the mycobacterial cell wall component mannose-capped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis (M. tuberculosis) could activate transcription of HIV-1 in T cells with the use of an in vitro cell culture system. These experiments are of prime importance considering that CD4-expressing T lymphocytes represent the major virus reservoir in the peripheral blood of infected individuals. Using the 1G5 cell line harbouring the luciferase reporter gene under the control of the HIV-1 LTR, it was first found that culture protein filtrates (CFP) from M. tuberculosis or purified ManLAM could activate HIV-1 LTR-dependent gene expression unlike similarly prepared CFP extracts devoid of ManLAM. The implication of protein tyrosine kinase(s), protein kinase A and/or protein kinase C was highlighted by the abrogation of the ManLAM-mediated activation of HIV-1 LTR-driven gene expression using herbimycin A and H7. It was also determined, using electrophoresis mobility shift assays, that M. tuberculosis ManLAM led to the nuclear translocation of the transcription factor NF-κB. M. tuberculosis ManLAM resulted in clear induction of the luciferase gene placed under the control of the wild-type, but not the κB-mutated, HIV-1 LTR region. Finally, the ManLAM-mediated activation of HIV-1 LTR transcription was found to be independent of the autocrine or paracrine action of endogenous TNF-α. The results suggest that M. tuberculosis can upregulate HIV-1 expression in T cells and could thus have the potential to influence the pathogenesis of HIV-1 infection.
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Dependence on host cell cycle for activation of human immunodeficiency virus type 1 gene expression from latency
Human immunodeficiency virus type 1 (HIV-1) establishes latent infection of a certain population of CD4 host cells which could be long-term reservoirs for HIV-1. The expression of viral genes in such long-term infected cells is strongly regulated by cellular status, such as the phase of the cell cycle or stage of cell differentiation. Here, viral gene expression in synchronized U1 cells, a monocytic cell clone latently infected with HIV-1, was characterized. The expression of HIV-1 antigens was detected exclusively at G2/M phase in U1 cells, irrespective of phorbol myristate acetate (PMA) treatment. The induction of HIV-1 gene expression in PMA-treated cells was due to the recruitment of NF-κB with DNA-binding activity at G2/M phase. Activated NF-κB was induced only by PMAtreatment during the late G1 to S, but not after entering G2 phase, indicating that the transcriptional factor(s) involved in viral gene expression is also largely regulated by the host cell cycle. In contrast, the enhancement of antigen expression by treatment with tumour necrosis factor-alpha (TNF-α) was cell cycle-independent. In fact, NF-κB was activated 2 h after TNF-α treatment at all stages of the cell cycle. Thus, the mechanisms of HIV-1 activation from latency in U1 cells by PMA and TNF-α treatment are different. The model system using U1 cells shown here may provide insight into the mechanisms responsible for HIV-1 gene expression from latency.
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Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity
Japanese studies have defined the discrete 22092248 amino acid region of the non-structural 5A protein (NS5A2209–2248) of hepatitis C virus genotype 1b (HCV 1b) isolates as the interferon sensitivity determining region (ISDR). European studies did not confirm these results since most of the ISDR sequences harboured an intermediate profile. Recently, a direct interaction between the NS5A protein, involving the ISDR, and the interferon-induced protein kinase (PKR) has been reported and presented as a possible explanation of HCV interferon resistance. In the present study, the entire NS5A amino acid sequence from 11 resistant and eight sensitive strains from European HCV 1b isolates was inferred from direct sequencing. The previously described important amino acid stretches and positions in NS5A were compared between the resistant and sensitive groups. Although some variations were observed, no clear differences could be directly correlated with the interferon sensitivity. However, sensitive strains were different, owing to more amino acid changes when compared to a consensus sequence from all strains. The carboxy- terminal region and especially the previously reported NS5A/V3 region showed most of the variations. Moreover, the conformational analysis of NS5A by secondary structure prediction allowed the differentiation of most sensitive strains from resistant ones. It was concluded that other regions different from ISDR were involved in resistance to interferon maybe via the interaction between NS5A and PKR.
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Infection of a chimpanzee with hepatitis C virus grown in cell culture
Culture supernatant harvested from Daudi cells, a lymphoplastoid cell line, after 58 days of infection with the H77 strain of hepatitis C virus (HCV), was inoculated into a chimpanzee. HCV RNA, as detected by RT-PCR, first appeared in the serum and liver 5 and 6 weeks, respectively, after inoculation. Peripheral blood mononuclear cells (PBMC) collected on week 7 were also positive for HCV RNA. The major sequences of hypervariable region 1 (HVR1) of the viral genome recovered from the inoculated chimpanzee were the ones which were the majority in the original H77 inoculum and not those which were in the majority in the culture supernatant. Only the sequence recovered from PBMC was the same as the major one found in the cell culture.
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Characterization of determinants involved in the feline infectious peritonitis virus receptor function of feline aminopeptidase N
More LessFeline aminopeptidase N (fAPN) is a major cell surface receptor for feline infectious peritonitis virus (FIPV), transmissible gastroenteritis virus (TGEV), human coronavirus 229E (HCV 229E) and canine coronavirus (CCV). By using chimeric molecules assembled from porcine, human and feline APN we have analysed the determinants involved in the coronavirus receptor function of fAPN. Our results show that amino acids 670–840 of fAPN are critically involved in its FIPV and TGEV receptor function whereas amino acids 135–297 are essential for the HCV 229E receptor function. We also demonstrate that a chimeric molecule assembled from human and porcine APN is able to act as a receptor for FIPV. This is surprising as neither human nor porcine APN by themselves mediate FIPV infection. These results suggest that different determinants in the APN protein are involved in mediating the coronavirus receptor function.
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The ectodomains but not the transmembrane domains of the fusion proteins of subtypes A and B avian pneumovirus are conserved to a similar extent as those of human respiratory syncytial virus
More LessThe fusion glycoprotein (FB) gene of five strains of the B subtype of avian pneumovirus (APV; turkey rhinotracheitis virus) has been sequenced. The length of the FB protein was 538 amino acids, identical to that of the F protein of subtype A virus, with which it had 74% and 83% overall nucleotide and deduced amino acid identities, respectively. The FB and FA ectodomains had 90% amino acid identity, very similar to the 91% identity between the ectodomains of the F proteins of subtype A and B human respiratory syncytial virus (HRSV). As with HRSV, the F2 polypeptide was less conserved (83% identity) than F1 (94%). In contrast to the ectodo- main, the transmembrane and cytoplasmic domains of the two APV subtypes were much less conserved (30% and 48% identity, respectively) than those of HRSV (92% and 87%, respectively). Comparisons within all the genera of the Paramyxoviridae (Pneumovirus, Morbillivirus, Paramyxovirus and Rubulla- virus) show that low amino acid identity between F protein transmembrane domains is a feature of different species of virus rather than of strain differences. This may indicate that the two subtypes of APV have evolved in different geographical regions and/or different avian species. This is the first report of an F gene sequence from a subtype B APV.
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Identification of regions of bovine respiratory syncytial virus N protein required for binding to P protein and self-assembly
More LessThe interaction of bovine respiratory syncytial virus (BRSV) nucleocapsid protein (N) with itself and phosphoprotein (P) was investigated using the yeast two-hybrid system. N-P interaction was abolished by any of a series of internal deletions or deletions at the C terminus. In contrast, removal of up to 32 amino acids from the N terminus had little effect. Interestingly, while removal of the C-terminal 32 amino acids ablated interaction, it was largely restored by a second deletion removing up to 32 amino acids from the N terminus. Many of these interactions of the BRSV N protein demonstrated a pattern that was similar to those occurring in the N protein of related viruses. N-N interaction was abolished by any of the internal deletions; however, removal of up to 32 amino acids from the N terminus or C terminus was tolerated and increased the strength of the interaction between the two N proteins.
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A host restriction-based selection system for influenza haemagglutinin transfectant viruses
During the 1996 influenza epidemic in Vienna we obtained influenza A virus specimens (Vienna/ 47/96, Vienna/81/96) which grow efficiently in African green monkey kidney (Vero) cells but not in embryonated chicken eggs. Amplification of the specimens in Vero cells resulted in progeny that agglutinated human but not chicken erythrocytes. Reassortment analysis suggested that the haem- agglutinin (HA) might be responsible for the host restriction. Vero cells were infected with the Vienna/47/96 virus and then transfected with reconstituted ribonucleoprotein complexes containing HA genes from egg-adapted strains. Subsequent selective passages in embryonated chicken eggs resulted in selection of transfectant viruses, growing in eggs and containing the transfected HAs. The results demonstrate that host restriction of the Vero-adapted Vienna/47/96 virus is due to its HA. Moreover, the experiments showed that the Vienna/47/96 strain can be used as helper virus for reverse genetics experiments.
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Identification and characterization of RNA-binding activities of avian reovirus non-structural protein sigmaNS
More LessCytoplasmic extracts prepared from avian reovirus (ARV) strain S1133-infected chicken embryo fibroblasts were examined for the presence of RNA- binding proteins in order to identify and characterize ARV RNA-binding proteins. Analysis of binding activity to poly(A)-Sepharose indicated that infected cells contained significant amounts of a protein that co-migrated with ARV protein σNS present in total virus-infected cell extracts. Determination of the N-terminal amino acid sequence of several peptide fragments generated by V8 protease digestion of the poly(A)-Sepharose- purified protein confirmed that this viral protein was σNS. Competition assays showed that singlestranded RNA from the unrelated avian pathogen infectious bursal disease virus was able to compete for binding of σNS to poly(A)-Sepharose. These data suggest that ARV σNS binds to single-stranded RNA in a nucleotide sequence non-specific manner and is functionally similar to its counterpart specified by mammalian reovirus.
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Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cell-cell fusion
During vaccinia virus (VV) morphogenesis intracellular mature virus (IMV) is wrapped by two additional membranes to form intracellular enveloped virus (IEV). IEV particles can nucleate the formation of actin tails which aid movement of IEVs to the cell surface where the outer IEV membrane fuses with the plasma membrane forming cell- associated enveloped virus (CEV) which remains attached to the cell, or extracellular enveloped virus (EEV) which is shed from the cell. In this report, we have used a collection of VV mutants lacking individual EEV-specific proteins to compare the roles of these proteins in the formation of IEV and IEV-associated actin tails and fusion of infected cells after a low pH shock. Data presented here show that p45–50 (A36R) is not required for IEV formation or for acid-induced cell-cell fusion, but is required for formation of IEV-associated actin tails. In contrast, gp86 (Δ 56R), the virus haemagglutinin, is not required for formation of either IEV or IEV- associated actin tails. Data presented also confirm that p37 (gene F13L), gp42 (B5R) and gp22–24 (A34R) are needed for formation of IEV-associated actin tails and for cell-cell fusion after low pH shock. The phenotypes of these mutants were not affected by the host cell type as similar results were obtained in a range of different cells. Lastly, comparisons of the phenotypes of VV strains Western Reserve, ΔA34R and ΔA36R demonstrate that actin tails are not required for low pH-induced cell-cell fusion.
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African swine fever: a disease characterized by apoptosis
More LessThe cell tropism, organ distribution and resultant pathology of African swine fever were compared in domestic pigs infected with lethal (Malawi) and sublethal (Malta) isolates of African swine fever virus (ASFV). After infections with both isolates, ASFV was predominantly localized in cells of the mononuclear phagocytic system and was not observed in endothelial cells in lymphoid tissue. More severe tissue destruction and cell depletion, associated with high levels of infected macrophages, were seen in lymphoid tissues from domestic pigs infected with the virulent Malawi isolate compared to the less virulent Malta isolate of ASFV. The abundant lymphocyte death was caused by apoptosis and not necrosis. In the spleen, as early as 3 days post-infection (p.i.), many lymphocytes in the B and T cell areas of the white and red pulp were apoptotic. Apoptosis in the T cells of the periarteriolar lymphoid sheaths in the spleen, however, occurred later, at 5–7 days p.i. In lymph nodes apoptosis was observed in T lymphocytes as early as 4 days p.i. and extended to B lymphocytes in the follicles later in infection. In pigs recovered from infection with the sublethal Malta isolate, virus was found to persist in lymph nodes and tonsils for up to 48 days p.i. and was located in cells, surrounded by apoptotic lymphocytes, in the paracortex of lymph nodes up to 32 days p.i. Taken together, these observations suggest that apoptosis of uninfected lymphocytes was induced by cytokines or apoptotic mediators released from ASFV infected macrophages.
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The pathogenesis of African swine fever in the resistant bushpig
More LessBushpigs and warthogs are natural reservoir hosts of African swine fever virus (ASFV) in the wild, showing no clinical signs of disease when infected with the same highly virulent isolates of ASFV that induce rapid, haemorrhagic death in domestic pigs. In contrast to domestic pigs, infection of bushpigs with Malawi isolate results in low levels of virus replication and lymphocyte apoptosis within the spleen, and a relatively low spread of virus to other lymphoid tissues. However, at 10 days post-infection, a high degree of apoptosis was seen in B lymphocytes of the B cell follicles in bushpig lymph nodes. Virus infected cells were present amongst the apoptotic B cells of these follicles, suggesting that indirect factors released from ASFV infected macrophages signal surrounding lymphocytes to enter apoptosis. The susceptibility/resistance of domestic pigs/bushpigs to ASFV may serve as a unique veterinary model for the recently emerging haemorrhagic disease of man.
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Restricted expression of Epstein-Barr virus (EBV)-encoded, growth transformation-associated antigens in an EBV- and human herpesvirus type 8-carrying body cavity lymphoma line
A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposi’s sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not colocalize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6,LMP1 and LMP2B. They were thus similar to type I Burkitt ’s lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used theQUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.
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Analysis of the hexon gene sequence of bovine adenovirus type 4 provides further support for a new adenovirus genus (Atadenovirus)
More LessThe putative hexon gene of bovine adenovirus type 4 (BAV-4), encoding 910 amino acid residues, has been identified and sequenced. A characteristic codon usage biased towards the use of AT-rich triplets was observed. Comparative analysis with other hexon sequences detected a high level of amino acid identity in the regions corresponding to the pedestals of the hexon. Substitutions, insertions and deletions were identified mainly in the variable regions forming the loops which are exposed on the outer surface of the virion. In these variable regions, BAV-4 shared similarity only with egg drop syndrome (EDS) virus and ovine adenovirus isolate 287 (OAV287). The close relationship of these viruses was also demonstrated by phylogenetic analysis of the hexon gene. In addition to the two groups of the Mastadenovirus and Aviadenovirus genera, a third cluster appeared comprising BAV-4, OAV287 and EDS virus.
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Complementation of a fibre mutant adenovirus by packaging cell lines stably expressing the adenovirus type 5 fibre protein
More LessAdenovirus-based gene therapy vectors now in use cannot be targeted to specific cell types in vivo and are immunogenic, properties which limit their clinical utility. Improved vectors lacking the genes for viral structural proteins may overcome these limitations. We have developed cell lines which stably express the adenovirus type 5 (Ad5) fibre protein in its native trimeric form. These cells can complement an Ad5 mutant with a defect in the fibre gene, and are capable of incorporating the Ad5 fibre into particles of a different Ad serotype. As the fibre protein is responsible for the initial binding of virus to cells, packaging cell lines expressing different or modified fibre proteins will be useful in studying the mechanism by which adenovirus infects different cell types.
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MHC class I molecules are enriched in caveolae but do not enter with simian virus 40
More LessSimian virus 40 (SV40) binds to MHC class I molecules anywhere on the cell surface and then enters through caveolae. The fate of class I molecules after SV40 binding is not known. Sensitivity of 125I-surface-labelled class I molecules to papain cleavage was used to distinguish internalized class I molecules from class I molecules remaining at the cell surface. Whereas the caveolae-enriched membrane microdomain was found to also be enriched for class I molecules, no internalized papain-resistant 125I-surface-labelled class I molecules could be detected at any time in either control cells or in cells preadsorbed with saturating amounts of SV40. Instead, 125I-surface-labelled class I molecules, as well as preadsorbed 125I-labelled anti-class I antibodies, accumulated in the medium, coincident with the turnover of class I molecules at the cell surface. The class I heavy chains that accumulated in the medium were truncated and their release was specifically prevented by the metalloprotease inhibitor 1,10-phenanthroline. Thus, whereas class I molecules mediate SV40 binding, they do not appear to mediate SV40 entry.
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Taxonomic characteristics of fijiviruses based on nucleotide sequences of the oat sterile dwarf virus genome
More LessSequence determination of full-length cDNA clones of genomic segments 7–10 (S7-S10) of oat sterile dwarf fijivirus (OSDV) revealed that the 5′ and 3′ ends of the plus strands of these segments had the same conserved terminal sequences, 5′ AACGAAAAA and UUUUUUUAGUC 3′. These sequences are similar, but not identical, to the conserved terminal nucleotide sequences of the genomic segments of rice black streaked dwarf fijivirus (RBSDV) and maize rough dwarf fijivirus (MRDV). The coding strands of S7 and S10 each contained two large nonoverlapping open reading frames (ORFs), as do RBSDV S7 and S9, MRDV S6 and S8 and Nilaparvata lugens reovirus (NLRV; a putative member of Fijivirus) S9. These results strongly suggest that the dicistronic nature of certain genomic segments is characteristic of fijiviruses. Computer analyses revealed sequence homology between RBSDV S7 ORF2, MRDV S6 ORF2 and OSDV S7 ORF2, suggesting that this protein is conserved among plant fijiviruses. No counterparts were found in the genome of NLRV, which is a nonphytopathogenic insect reovirus. Furthermore, phylogenetic trees derived from multiple sequence alignments of each of the homologous proteins from OSDV, RBSDV, MRDV and NLRV suggest that NLRV did not evolve from either Fijivirus group 2 (RBSDVand MRDV) or group 3 (OSDV).
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Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7, S8, S9 and S10
More LessThe proteins encoded by rice black streaked dwarf fijivirus (RBSDV) genomic segments 7–10 (S7-S10) were characterized. Open reading frames (ORFs) from these segments were expressed as fusion proteins in Escherichia coli. Antibodies raised against the expressed products were used as probes to determine whether the viral ORFs encode structural proteins. In Western blots, antibodies to the expressed S8 and S10 products reacted with a core capsid (65 kDa) and a major outer capsid (56 kDa) protein, respectively, while none of the antibodies to S7 and S9 products reacted with structural proteins. Antisera to RBSDV S7 ORF1 and S9 ORF1 each detected a single protein of the predicted size in total protein extracts from infected rice plants and viruliferous Laodelphax striatellus. Immuno- electron microscopy revealed that antibodies to RBSDV S7 ORF1 and RBSDV S9 ORF1 reacted with tubular structures and viroplasm, respectively, in sections of both infected maize plants and viruliferous L. striatellus. Antisera to ORF2 of S7 and S9 failed to detect any proteins in the infected tissue using either Western blotting or immuno-electron microscopic techniques.
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Characterization of cis-acting elements affecting strength and phloem specificity of the coconut foliar decay virus promoter
More LessDuring replication in its host plant, coconut foliar decay virus (CFDV) remains restricted to the phloem tissue. Previous /n v/vo studies on subgenomic CFDV DNA had provided evidence for the phloem specificity of the CFDV promoter. Here, new promoter constructs are described which are distinguished by the presence or absence of various cis - acting signals and which gave rise to a 16-fold higher reporter gene (β -glucuronidase) activity (reaching 30% of the cauliflower mosaic virus 35S promoter) in tobacco protoplasts, while the phloem specificity in transgenic tobacco plants was conserved. Surprisingly, the CFDV stem-loop structure dramatically influenced transcriptional efficiency. From these studies and sequence comparisons with other phloem-specific promoters, ci s-signals involved in CFDV promoter strength and tissue specificity were identified.
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Defective forms of cotton leaf curl virus DNA-A that have different combinations of sequence deletion, duplication, inversion and rearrangement
More LessTobacco and tomato plants inoculated at least 9 months previously with a Pakistani isolate of cotton leaf curl virus (CLCuV-PK), a whitefly-transmitted geminivirus, contained substantial amounts of circular dsDNA molecules that were mostly about half the size of CLCuV-PK dsDNA-A. They appeared to be derived from CLCuV-PK DNA-A by various combinations of sequence deletion, duplication, inversion and rearrangement and, in a few instances, insertion of sequences of unknown origin. Each of ten tobacco plants contained a different predominant form of such a defective molecule; however, all the forms contained the intergenic region and part of the AC1 (Rep) gene. Some of the forms contained novel open reading frames and might have a role in the evolution of variant geminiviruses. The defective components were not detected at 3 months after the original culture of CLCuV-PK was transmitted by whiteflies (Bemisia tabaci) from cotton to tomato but were present after a further 6 months. They were transmitted, along with full-length DNA-A, between tobacco and tomato plants by grafting and by B. tabaci.
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Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants
Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Hetero- encapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy. Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission. However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs. Transgenic Nicotiana bent- hamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT. Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.
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Role of the helper component in vector-specific transmission of potyviruses
More LessFour aphid species were tested for their ability to transmit tobacco etch (TEV) and turnip mosaic (TuMV) potyviruses. Myzus persicae and Aphis gossypii transmitted both viruses efficiently from infected plants, whereas Lipaphis erysimi transmitted only TuMV and Myzus ascalonicus was a poor or non-transmitter of either virus. Similar electrically monitored probing patterns were produced by M. persicae, L. erysimi and M. ascalonicus, ruling out behavioural differences as the cause of differential transmission. Transmission results similar to those from infected plants were obtained when these aphids acquired homologous virus/helper component (HC) mixtures through membranes. With heterologous virus/HC mixtures, M. persicae remained a highly efficient vector and M. ascalonicus a non-vector, but L. erysimi became an efficient vector of TEV if acquired in the presence of TuMV HC and A. gossypii transmitted both viruses less efficiently when acquired with TuMV HC. Transmission was highly correlated with the retention of virus in the stylets, as determined by autoradiography of 125I-labelled virions. The results show that constituent(s) of or in the food canal of different aphid species differ in their ability to interact with specific HCs, leading to qualitative or quantitative differences in ability to retain and subsequently transmit specific potyviruses.
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Evidence that assembly of a potyvirus begins near the 5′ terminus of the viral RNA
More LessA search for the first region in the genomic RNA of a potyvirus to be encapsidated during the assembly of virus particles in vivo has been undertaken. Protoplasts were collected at various times after inoculation and fragments of viral RNA that were protected from nuclease degradation were isolated from extracts and identified by RT-PCR procedures. Nuclease-resistant fragments of viral RNA were not detected in protoplasts that had been infected for 30 min. However, such fragments were present in protoplasts collected 40 min after inoculation and these contained a region at or near the 5′ terminus of the viral RNA. Protoplasts that had been infected for 45 min or longer contained full-length viral RNA in a nuclease-resistant form. These results suggest that assembly of virus particles begins with the interaction of coat protein subunits with the 5′ terminal region of progeny viral RNA molecules.
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Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR
More LessCitrus psorosis virus (CPV) is a multicomponent ssRNA virus with a coat protein of approximately 48 kDa. The viral genome is encapsidated in short and long particles that are readily separated by sucrose density-gradient centrifugation. CPV particles are spiral filaments that are referred to as spiroviruses (SV). A cDNA library of purified short particles from isolate CPV-4 was prepared in a Lambda vector and screened for expression of the coat protein gene (CPG) with a monoclonal antibody to the coat protein. Sequencing of immunopositive clones indicated a single ORF encoding a 49 kDa protein. This ORF, when expressed in E. coli, gave a protein identical in size and immunoreactivitytothe CPV coat protein. A full-length clone of the CPG was transcribed and used in Northern hybridization assays to establish that short particle RNA of CPV is negative sense and contains the CPG. Moreover, the CPG was not found on RNA extracted from long particles or on the sedimentable dsRNA from CPV infected tissue. RT-PCR assays were developed for the amplification of a 600 bp fragment of CPG and for the complete CPG (1317 bp). The 600 bp fragment from a biologically and serologically different isolate, CPV-6, was cloned, sequenced and found to share 86% (nucleotide) and 96% (amino acid) identity with CPV-4. BLAST analysis of sequences from CPV-4 and CPV-6 detected no significant nucleic acid or protein similarity with any known viral sequences.
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The nucleotide sequence of satellite tobacco necrosis virus strain C and helper-assisted replication of wild-type and mutant clones of the virus.
More LessThe complete nucleotide sequence of satellite tobacco necrosis virus strain C (STNV-C) was determined. The genome has a similar overall organization to two STNV isolates studied previously but differs significantly from them in the secondary structure of the translated and untranslated regions (UTRs). STNV-C RNA is naturally uncapped and contains 1221 nt: 101 nt in the 5′ UTR, 606 nt in the capsid protein (CP) coding region and 514 nt in the 3′ UTR. Using the known sequences of STNV-C and tobacco necrosis virus strain D (TNV-D) RNAs, full-length cDNA clones of both RNAs were constructed. Synthetic transcripts derived from STNV-C cDNA clones only replicated in plants and protoplasts when co-inoculated with TNV-D transcripts. A number of mutant clones in both the 3′ and the 5′ STNV-C RNA UTRs were constructed which disrupted putative cis-acting elements recognized by helper virus polymerase. Deletion analysis revealed an essential requirement of all 3′ and 5′ proximal sequences in the STNV-C UTRs for replication. However, an internal region in the 3′ UTR could be deleted without loss of infectivity. Likewise, the entire STNV-C CP-encoding region could be deleted and replaced with a marker gene of a similar size without loss of transcript accumulation in plants.
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The ecdysteroid UDP-glucosyltransferase gene of Autographa californica nucleopolyhedrovirus alters the moulting and metamorphosis of a non-target insect, the silkworm, Bombyx mori (Lepidoptera, Bombycidae).
The Autographa californica nucleopolyhedrovirus (AcMNPV) does not infect the silkworm and molecular studies on silkworm insusceptibility have not been performed. In cultured cells of the silkworm, the expression of viral genes has been reported. The expression of AcMNPV genes and their effect in vivo and in vitro was studied. In this study, the early gene, the ecdysteroid UDP-glucosyltransferase (egt) gene of AcMNPV, which inactivates the insect moulting hormone by sugar conjugation, was examined to determine whether it would alter the growth of the silkworm. Using wild-type (wt) AcMNPV, the egt gene deletion virus (vEGTDEL), and the virus carrying the egt promoter-lacZ cassette in vEGTDEL (vEGTZ), the egt promoter-driven expression in cultured cells and in nonproductive infection of the silkworm was characterized. Infection of cultured cells with vEGTZ at three different doses occurred in a single cell manner. When budded wt AcMNPV was injected into the fourth and fifth instar larvae, an increase in the amount of virus occurred and caused abnormal larval growth, which resulted in the prolongation or skipping of the larval instar, premature pupation, or death during the pupal stage. For infection of the fourth instar larvae, precocious metamorphosis was observed. When the same amount of vEGTDEL was injected, the alteration of growth did not occur. These results suggest that the egt gene was expressed in the primary infected cells of the silkworm, and that the EGT was secreted into the haemocoel, which significantly altered larval growth.
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The single-nucleocapsid nucleopolyhedrovirus of Buzura suppressaria encodes a P10 protein.
The p10 gene of Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was identified by virtue of its localization downstream from the Autographa californica (Ac) MNPV p26 homologue. The BusuNPV p10 gene encodes a protein of 94 amino acids. The amino acid sequence contains domains characteristic of baculovirus P10 proteins, e.g. a coiled-coil domain, a proline-rich motif and a positively charged C terminus. The highest amino acid homologies were found with the Spodoptera littoralis (Spli) NPV and Spodoptera exigua (Se) MNPV P10 proteins. An AcMNPV recombinant expressing the BusuNPV P10 formed fibrillar structures in the cytoplasm of Spodoptera frugiperda cells. BusuNPV P10 could not fully replace AcMNPV P10 in its nuclear disintegration function, since polyhedra were not efficiently liberated from infected cells late in infection. The BusuNPVp26 gene encodes a protein of 263 amino acid residues with 70% amino acid similarity with SeMNPV P26. Downstream of the BusuNPV p10 gene, the gene for the occlusion-derived virus protein ODVP-6e is located. This is unlike the situation in many other NPVs, including SeMNPV, where the p10 gene neighbours the p74 gene. The data presented here suggest that although the p10 gene is not conserved in sequence, evolutionary pressure preserves the structure of P10 and hence its function. These data also indicate that all NPVs, MNPVs as well as SNPVs, contain this gene.
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Specificity of multiple homologous genomic regions in Spodoptera exigua nucleopolyhedrovirus DNA replication.
More LessThe region upstream of the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) ubi-quitin gene contains four near-identical 68-bp-long palindromic repeats. This region, named Sehr6 and located at map unit (m.u.) 88 of the SeMNPV genome on pSeEcoRI-2.2, showed structural homology to previously identified homologous regions (hrs) in a number of other baculoviruses. Hrs function as enhancers of transcription and as putative origins (oris) of baculovirus DNA replication. Five additional hrs (Sehr1-Sehr5) were identified on the SeMNPV genome by Southern blot hybridization with an 18-bp-long oligonucleotide complementary to a sequence conserved within the arms of the four palindromic repeats of Sehr6. Sehr1-Sehr6 were dispersed on the SeMNPV genome at m.u. 8·0, 30·0, 38·5, 51·0, 77·0 and 88·0, respectively. Sequence analysis of these hrs confirmed the presence of palindromic repeats, highly similar to those found in pSeEcoRI-2.2. The number of palindromes varied from one (Sehr4) to nine (Sehr1) per hr. The Sehrs are all present in non-coding regions of the SeMNPV genome and also contain multiple putative transcription recognition sequences. Plasmids containing either of the Sehrs replicated in an SeMNPV-dependent DNA replication assay. The Sehrs were unable to replicate in an AcMNPV-dependent DNA replication assay. This was in contrast to the previously observed SeMNPV non-hr type ori, which replicated in the presence of both AcMNPV and SeMNPV. These data suggest that the replication of SeMNPV and the role of hrs in this process is highly specific.
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