- Volume 85, Issue 4, 2004
Volume 85, Issue 4, 2004
- Animal
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- DNA viruses
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Autographa californica M nucleopolyhedrovirus early GP64 synthesis mitigates developmental resistance in orally infected noctuid hosts
More LessThe unusual early synthesis of the Autographa californica M nucleopolyhedrovirus (AcMNPV) budded virus (BV) structural protein GP64 is an important virulence factor during oral infection of Heliothis virescens larvae. Considering the breadth of the AcMNPV host range, the importance of early GP64 synthesis in orally infected permissive hosts (Trichoplusia ni and Spodoptera exigua) from subfamilies other than that of H. virescens was assessed. An AcMNPV recombinant, having wild-type early and late GP64 synthesis, was compared with one in which only late GP64 synthesis occurred. Early GP64 synthesis was found to have more of an effect on virulence in orally inoculated T. ni than S. exigua and that virulence was dependent on two factors: the ability of the host to slough occlusion-derived virus (ODV)-infected midgut cells and the rapidity with which BV was transmitted to the tracheal cells. In both host species, insects inoculated orally with the control virus transmitted BV to tracheal cells hours before those inoculated with the gp64 temporal mutant. Moreover, with early GP64 synthesis, the lag between the onset of viral gene expression in midgut and tracheal cells was only 3–4 h, supporting the conclusion that in these insects, the first systemic infections arose from ODV-derived nucleocapsids repackaged as BV. These results provide further empirical proof that early GP64 synthesis is a component of a unique and selectively advantageous baculovirus infection strategy for exploiting larval lepidopterans by counteracting developmental resistance.
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Identification of a virus trans-acting regulatory element on the latent DNA replication of Kaposi's sarcoma-associated herpesvirus
More LessLatency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays a pivotal role in the maintenance of the virus genome in latently infected cells. LANA1 links virus genomes to host chromosomes via a C-terminal DNA-binding domain which interacts with the sequences located in terminal repeats (TRs) of the virus genome and via an N-terminal chromosome-binding sequence which associates with the host chromosomes, respectively. Recent data suggest that LANA1 also actively participates in the replication of KSHV TR-containing plasmid in the transient DNA replication assay. In this report, it was found that C33A and COS-1, but not NIH/3T3, cell lines are permissive for the transient replication of KSHV TR-containing plasmid. Using several LANA1-deletion mutants, the minimum domain of LANA1 required for replication activity was also determined. In addition, the N terminus of LANA1 inhibited the transient replication systems of KSHV and Epstein–Barr virus (EBV) in transiently transfected 293 and 293T cells, but the C terminus of LANA1 specifically inhibited the transient replication system of KSHV in other cell lines. Consistent with previous reports, these data further emphasize the functional importance of the N terminus of LANA1 on replication from the KSHV latent origin of DNA replication.
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Novel herpesviruses of Suidae: indicators for a second genogroup of artiodactyl gammaherpesviruses
More LessFive novel herpesviruses were identified in suid species from Africa (common warthog, Phacochoerus africanus) and South-East Asia (bearded pig, Sus barbatus; babirusa, Babyrousa babyrussa) by detection and analysis of their DNA polymerase genes. Three of the novel species, P. africanus cytomegalovirus 1, P. africanus lymphotropic herpesvirus 1 (PafrLHV-1) and S. barbatus lymphotropic herpesvirus 1 (SbarLHV-1), were closely related to known beta- (porcine cytomegalovirus) and gammaherpesviruses [porcine lymphotropic herpesvirus (PLHV) 1 and 3] of domestic pigs. In contrast, two novel species, S. barbatus rhadinovirus 1 (SbarRHV-1) and Babyrousa babyrussa rhadinovirus 1 (BbabRHV-1), were more closely related to a ruminant gammaherpesvirus, bovine herpesvirus 4 (BoHV-4), than to the porcine gammaherpesviruses PLHV-1, -2, -3, PafrLHV-1 and SbarLHV-1. SbarRHV-1, BbabRHV-1 and BoHV-4 were therefore tentatively assigned to a novel genogroup of artiodactyl gammaherpesviruses. This latter genogroup may also contain an as yet undiscovered gammaherpesvirus of domestic pigs, thereby adding a concern to their use in xenotransplantation.
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Establishment of cell lines from the wasp Hyposoter didymator (Hym., Ichneumonidae) containing the symbiotic polydnavirus H. didymator ichnovirus
Cell lines derived from polydnavirus-associated wasps should constitute a valuable tool for investigations of polydnavirus replication, but none is yet available. In this work, we describe the first cell lines, named Hd-AA, -AD, -BBA and -K, to have been established from the ichneumonid wasp Hyposoter didymator, associated with the polydnavirus H. didymator ichnovirus (HdIV). Southern blot analysis indicated that the viral DNA was present in all four cell lines and co-localized with high molecular mass DNA, probably the wasp chromosomes. Northern blot analysis of mRNAs extracted from the AA cell line showed transcription of some HdIV-encoded genes, although at low level. The effects of ecdysone treatment, HdIV re-infection and 42 °C heat-shock were analysed in the AA cell line. No effect was detected at the DNA (virus replication) or RNA (gene expression) levels, which may be due to the limitation of the present available tools.
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Productive human herpesvirus 6 infection causes aberrant accumulation of p53 and prevents apoptosis
More Lessp53 plays an important role in tumour suppression in cells exposed to some genotoxic stresses. We found that the p53 protein level was increased in a variety of cell lines infected with human herpesvirus 6 (HHV-6). Because the elevation in p53 began very soon after infection (4 h) and did not occur with UV-inactivated virus infection, it appeared to require the expression of one or more viral immediate-early (IE) genes. To elucidate the mechanism of p53 induction, we investigated its regulation at the protein level. Pulse–chase analysis showed that the stability of p53 increased in HHV-6-infected cells. In addition, the ubiquitination of p53 decreased after infection, indicating that the stability of p53 was increased through deubiquitination. We showed by confocal microscopy that the additional p53 mainly localized to the cytoplasm and that p53 was retained in the cytoplasm even after UV irradiation, but that it translocated into the nucleus in mock-infected cells. Furthermore, DNA fragmentation analysis, a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and annexin V staining showed that infected cells were resistant to UV-induced apoptosis. These results lead us to propose that HHV-6 has a mechanism for retaining p53 within the cytoplasm and protects the infected cells from apoptosis.
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Proliferation and differentiation in isogenic populations of peripheral B cells activated by Epstein–Barr virus or T cell-derived mitogens
More LessHuman B cells isolated from peripheral blood were activated and induced to proliferate by either Epstein–Barr virus (EBV) or the T cell-derived mitogens CD40 ligand (CD40L) plus interleukin (IL)-4. Although both populations initially proliferated as B-blasts, significant differences were revealed over a longer period. EBV infection resulted in continuously proliferating lymphoblastoid cell lines (LCLs), whereas most of the CD40L/IL-4-stimulated B cells had a finite proliferative lifespan of 3–4 weeks. Cell cycle analysis, trypan blue staining and Western blot analysis for cleavage of poly(ADP-ribose) polymerase (PARP) all demonstrated that the decrease in proliferation in CD40L/IL-4-stimulated B cells is not due to cell death. Instead, these cells arrest, accumulate in G0/G1 and undergo alterations in cell surface marker expression, cellular morphology and immunoglobulin production, all consistent with plasmacytoid differentiation. In contrast, B cells infected with EBV continued to proliferate and retained a blast-like phenotype. Differences in both cytokine production and the expression of cell cycle regulators were identified between the two B-cell populations, which might contribute to the differentiation of the CD40L/IL-4-stimulated B cells and suggest potential mechanisms by which EBV may overcome this. The study has also identified a window of opportunity during which a comparison of isogenic populations of EBV- and mitogen-driven B blasts can be made.
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Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling
The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130–132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N130) to aspartic acid resulted in a mutant (N130D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N130 is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N130A) was severely impaired in Gq/11-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in Gi/0-mediated signalling. Another protein that contained a modified NRY motif, R131A, did not show constitutive activity, whereas mutants Y132F and Y132A displayed similar activities to the WT receptor. This indicated that residue R131 is critical for pR33 function in vitro, whereas Y132 is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.
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- Plant Viruses
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Compatibility of the movement protein and the coat protein of cucumoviruses is required for cell-to-cell movement
More LessFor the cell-to-cell movement of cucumoviruses both the movement protein (MP) and the coat protein (CP) are required. These are not reversibly exchangeable between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV). The MP of CMV is able to function with the TAV CP (chimera RT), but TAV MP is unable to promote the cell-to-cell movement in the presence of CMV CP (chimera TR). To gain further insight into the non-infectious nature of the TR recombinant, RNA 3 chimeras were constructed with recombinant MPs and CPs. The chimeric MP and one of the CP recombinants were infectious. The other recombinant CP enabled virus movement only after the introduction of two point mutations (Glu→Lys and Lys→Arg at aa 62 and 65, respectively). The mutations served to correct the CP surface electrostatic potential that was altered by the recombination. The infectivity of the TR virus on different test plants was restored by replacing the sequence encoding the C-terminal 29 aa of the MP with the corresponding sequence of the CMV MP gene or by exchanging the sequence encoding the C-terminal 15 aa of the CP with the same region of TAV. The analysis of the recombinant clones suggests a requirement for compatibility between the C-terminal 29 aa of the MP and the C-terminal two-thirds of the CP for cell-to-cell movement of cucumoviruses.
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Heterologous expression of the modified coat protein of Cowpea chlorotic mottle bromovirus results in the assembly of protein cages with altered architectures and function
More LessWe have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification. Expression of the structural protein of Cowpea chlorotic mottle bromovirus (CCMV) using the yeast-based Pichia pastoris heterologous expression system resulted in the assembly of particles that were visibly indistinguishable from virus particles produced in the natural host. We have shown that a collection of non-infectious CCMV coat protein mutants expressed in the P. pastoris system assemble into viral protein cages with altered architectures and function. This provides an alternative to other heterologous expression systems for production of viral structural proteins in which expression has resulted in unassembled cages. Heterologous expression in P. pastoris further enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.
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Interaction of VPg-Pro of Turnip mosaic virus with the translation initiation factor 4E and the poly(A)-binding protein in planta
The viral protein linked to the genome (VPg) of Turnip mosaic virus (TuMV) interacts in vitro with the translation eukaryotic initiation factor (eIF) 4E. In the present study, we investigated the consequence of TuMV infection on eIF4E expression. Two isomers are present in plants, namely eIF4E and eIF(iso)4E. Expression of the latter was detected in both TuMV-infected and mock-inoculated Brassica perviridis plants, but expression of eIF4E was found only in infected plants. Membranes from TuMV-infected or mock-inoculated tissues were separated by sucrose gradient centrifugation and fractions were collected. Immunoblot analyses showed that 6K2-VPg-Pro/VPg-Pro polyproteins were associated with endoplasmic reticulum membranes and were the viral forms likely to interact with eIF(iso)4E and eIF4E. In planta interaction between 6K2-VPg-Pro/VPg-Pro and eIF(iso)4E/eIF4E was confirmed by co-purification by metal chelation chromatography. The poly(A)-binding protein (PABP) was also found to co-purify with VPg-Pro. Direct interaction between VPg-Pro and PABP was shown by an ELISA-based binding assay. These experiments suggest that a multi-protein complex may form around VPg-Pro of TuMV.
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Volumes and issues
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Volume 105 (2024)
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