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Volume 87,
Issue 9,
2006
Volume 87, Issue 9, 2006
- Animal
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- DNA viruses
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Evidence of ancient papillomavirus recombination
An open question amongst papillomavirus taxonomists is whether recombination has featured in the evolutionary history of these viruses. Since the onset of the global AIDS epidemic, the question is somewhat less academic, because immune-compromised human immunodeficiency virus patients are often co-infected with extraordinarily diverse mixtures of human papillomavirus (HPV) types. It is expected that these conditions may facilitate the emergence of HPV recombinants, some of which might have novel pathogenic properties. Here, a range of rigorous analyses is applied to full-genome sequences of papillomaviruses to provide convincing statistical and phylogenetic evidence that evolutionarily relevant papillomavirus recombination can occur.
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A granule cell neuron-associated JC virus variant has a unique deletion in the VP1 gene
More LessThe human polyomavirus JC (JCV) typically infects glial cells and is the aetiological agent of progressive multifocal leukoencephalopathy (PML), which occurs in immunosuppressed individuals. The full-length sequence of a granule cell neuron-tropic JCV variant, JCVGCN1, associated with lytic infection of granule cell neurons and cerebellar atrophy in a human immunodeficiency virus-infected patient with PML was determined and compared with the sequence of the JCV isolate from the classic PML lesions present in the hemispheric white matter of the same individual (JCVHWM). A unique deletion was found in the C terminus of the VP1 gene of JCVGCN1, which encodes the major capsid protein, resulting in a frame shift and a total change of the C-terminal amino acid sequence of this protein. This deletion was not present in JCVHWM, suggesting that this mutation may be instrumental in facilitating entry or replication of JCV into granule cell neurons.
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Cell death in bovine parvovirus-infected embryonic bovine tracheal cells is mediated by necrosis rather than apoptosis
The helper-independent bovine parvovirus (BPV) was studied to determine its effect on host embryonic bovine tracheal (EBTr) cells: whether the ultimate outcome of infection results in apoptotic cell death or cell death by necrosis. Infected cells were observed for changes marking apoptosis. Observations of alterations in nuclear morphology, membrane changes, apoptotic body formation, membrane phosphatidylserine inversions, caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. On the other hand, at the end of the virus replication cycle, infected cells released viral haemagglutinin and infectious virus particles, as would be expected from cell membrane failure. Moreover, the infected cells released lactate dehydrogenase (LDH), release of which is a marker of necrosis. LDH release into the cell medium correlated directly with viral m.o.i. and time post-infection. Furthermore, assessment of mitochondrial dehydrogenase activity was consistent with cell death by necrosis. Taken together, these findings indicate that cell death in BPV-infected EBTr cells is due to necrosis, as defined by infected-cell membrane failure and release of the cell contents into the extracellular environment.
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Gene organization and complete sequence of the Hyphantria cunea nucleopolyhedrovirus genome
More LessThe whole-genome sequence of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) was analysed. The entire nucleotide sequence of the HycuNPV genome was 132 959 bp long, with a G+C content of 45.1 mol%. A total of 148 open reading frames (ORFs) consisting of more than 50 aa were encoded by the genome. HycuNPV shares more than 122 ORFs with other lepidopteran group I NPVs, including Autographa californica MNPV, Bombyx mori NPV, Choristoneura fumiferana MNPV (CfMNPV), Choristoneura fumiferana defective NPV, Epiphyas postvittana MNPV and Orgyia pseudotsugata MNPV (OpMNPV). Six ORFs are identified as being unique to HycuNPV. Most of the HycuNPV ORFs showed higher similarity to CfMNPV and OpMNPV ORFs than to those of the other group I NPVs. HycuNPV encodes two conotoxin-like homologues (ctls), which are observed only in OpMNPV in group I NPVs. HycuNPV encodes three inhibitors of apoptosis (iaps), hycu-iap-1, hycu-iap-2 and hycu-iap-3, a feature that it shares only with CfMNPV. In addition, six homologous regions (hrs) are identified in the HycuNPV genome. These hrs are located in regions similar to those of the OpMNPV hrs, but different from most of the CfMNPV hrs. Based on the close phylogenetic relationship and conservation of group I NPV-specific genes, such as gp64, ie-2 and ptp-1, it is concluded that HycuNPV belongs to the group I NPVs and is most similar to CfMNPV or OpMNPV.
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Open reading frame 132 of Heliocoverpa armigera nucleopolyhedrovirus encodes a functional per os infectivity factor (PIF-2)
Open reading frame 132 (Ha132) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is a homologue of per os infectivity factor 2 (pif-2) of Spodoptera exigua multiple nucleopolyhedrovirus. Sequence analysis indicated that Ha132 encoded a protein of 383 aa with a predicted molecular mass of 44.5 kDa. Alignment of HA132 and its baculovirus homologues revealed that HA132 was highly conserved among baculoviruses, with 14 absolutely conserved cysteine residues. RT-PCR indicated that Ha132 was first transcribed at 24 h post-infection. Western blot analysis showed that a 43 kDa band was detectable in HearNPV-infected HzAM1 cells from 36 h post-infection. Western blots also indicated that HA132 was a component of the occlusion-derived virus, but not of budded virus. Deletion of Ha132 from HearNPV abolished per os infectivity, but had no effect on the infectivity of the budded virus phenotype.
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- Plant Viruses
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An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo
More LessIt was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)CP,148 CR upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem–loop (SL) region of CP subgenomic promoters (TMV U1-CP–GFP/SL-mut and crTMV-CP–GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP–GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP–GFP/SL-mut, whereas a small amount of CP–GFP synthesis was obtained in crTMV-CP–GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.
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Domains of tobacco mosaic virus movement protein essential for its membrane association
More LessA series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains.
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Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions
The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3–20 of TYLCV CP (CP-NLS) or aa 84–106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA–NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin α.
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Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type
The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5′ regions of the two genomic RNAs contain a ‘cap’ structure and poly(A) tails were identified in the 3′-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.
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Potato virus X RNA-mediated assembly of single-tailed ternary ‘coat protein–RNA–movement protein’ complexes
Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5′-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP–RNA interactions resulting in head formation prevailed over TGBp1–RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5′ end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP–RNA STPs are produced, due to encapsidation of the 5′-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.
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Random mutagenesis of wheat streak mosaic virus HC-Pro: non-infectious interfering mutations in a gene dispensable for systemic infection of plants
More LessMutations within the HC-Pro coding region of Wheat streak mosaic virus (WSMV) were introduced by misincorporation during PCR and evaluated for phenotype within the context of an infectious clone. Nine synonymous substitutions and 15 of 25 non-synonymous substitutions had no phenotypic effect. Four non-synonymous substitutions, including one that reverted consistently to wild type, resulted in attenuated systemic infection. Six non-synonymous substitutions and one nonsense substitution abolished systemic infectivity. Mutants bearing the GUS reporter gene were evaluated for the ability to establish primary infection foci. All attenuated mutants and two systemic infection-deficient mutants produced localized regions of GUS expression on inoculated leaves 3 days post-inoculation. In vitro assays revealed that mutants able to establish infection foci retained HC-Pro proteinase activity. Among mutants unable to establish infection foci, HC-Pro proteinase activity was retained, reduced or absent. As a complete HC-Pro deletion mutant can infect plants systemically, certain substitutions in this dispensable gene probably prevented infection of WSMV via interference.
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In vitro-reassembled plant virus-like particles for loading of polyacids
More LessThe coat protein (CP) of certain plant viruses may reassemble into empty virus-like particles (VLPs) and these protein cages may serve as potential drug delivery platforms. In this paper, the production of novel VLPs from the Hibiscus chlorotic ringspot virus (HCRSV) is reported and the capacity to load foreign materials was characterized. VLPs were readily produced by destabilizing the HCRSV in 8 M urea or Tris buffer pH 8, in the absence of calcium ions, followed by removal of viral RNA by ultrahigh-speed centrifugation and the reassembly of the CP in sodium acetate buffer pH 5. The loading of foreign materials into the VLPs was dependent on electrostatic interactions. Anionic polyacids, such as polystyrenesulfonic acid and polyacrylic acid, were successfully loaded but neutrally charged dextran molecules were not. The molecular-mass threshold for the polyacid cargo was about 13 kDa, due to the poor retention of smaller molecules, which readily diffused through the holes between the S domains present on the surface of the VLPs. These holes precluded the entry of large molecules, but allowed smaller molecules to enter or exit. The polyacid-loaded VLPs had comparable size, morphology and surface-charge density to the native HCRSV, and the amount of polyacids loaded was comparable to the weight of the native genomic materials. The conditions applied to disassembly–reassembly of the virions did not change the structural conformation of the CP. HCRSV-derived VLPs may provide a promising nano-sized protein cage for delivery of anionic drug molecules.
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- Jgv Direct
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Role of human cytomegalovirus UL131A in cell type-specific virus entry and release
The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169 with wild-type UL131A in cis in an ectopic position restored endothelial cell tropism. The UL131A protein was found in virions in a complex with gH. Coinfection of fibroblasts with UL131A-negative and -positive viruses restored the endothelial cell tropism of UL131A-negative virions by complementing the virions with UL131A protein. Virus entry into endothelial cells, but not into fibroblasts, was blocked by an antipeptide antiserum to pUL131A. AD169, cis-complemented with wild-type UL131A, showed an impaired release of infectious particles from fibroblasts. A comparable defect in virus release was observed when UL131A was expressed ectopically in a virus background already expressing an intact copy of UL131A. In contrast, virus release from infected endothelial cells was not affected by UL131A. These data suggest a dual role for pUL131A in virus entry and virus exit from infected cells.
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Coronaviruses in bent-winged bats (Miniopterus spp.)
More LessA novel group 1 coronavirus was previously identified in bent-winged bats (Miniopterus spp.). Here, results are described from our ongoing surveillance of these bats for coronaviruses. These findings show that group 1 coronaviruses are endemic in these bat populations in Hong Kong. Genetic analysis of these viruses indicates that there are at least four different, but closely related, group 1 coronaviruses (bat-CoV 1A, 1B, HKU7 and HKU8) circulating in bent-winged bats. Phylogenetic analysis revealed that these group 1 bat coronaviruses have descended from a common ancestor and that these viruses have been established in these bats for a long period of time. These data provide a better understanding of the emergence and evolution of coronaviruses. Bat-CoV 1A and 1B were detected in apparently healthy Miniopterus magnater and Miniopterus pusillus, respectively, on repeated sampling occasions at a single habitat, suggesting that these viruses have established a persistent infection in these populations.
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A humanized murine monoclonal antibody protects mice either before or after challenge with virulent Venezuelan equine encephalomyelitis virus
More LessA humanized monoclonal antibody (mAb) has been developed and its potential to protect from or cure a Venezuelan equine encephalomyelitis virus (VEEV) infection was evaluated. The VEEV-neutralizing, protective murine mAb 3B4C-4 was humanized using combinatorial antibody libraries and phage-display technology. Humanized VEEV-binding Fabs were evaluated for virus-neutralizing capacity, then selected Fabs were converted to whole immunoglobulin (Ig) G1, and stable cell lines were generated. The humanized mAb Hy4-26C, designated Hy4 IgG, had virus-neutralizing capacity similar to that of 3B4C-4. Passive antibody protection studies with purified Hy4 IgG were performed in adult Swiss Webster mice. As little as 100 ng Hy4 IgG protected 90 % of mice challenged with 100 intraperitoneal (i.p.) mean morbidity (MD50) doses of virulent VEEV (Trinidad donkey) 24 h after antibody transfer; also, 500 μg Hy4 IgG protected 80 % of mice inoculated with 100 intranasal MD50 doses of VEEV. Moreover, 10 μg passive Hy4 IgG protected 70 % of mice from a VEEV challenge dose as great as 107 i.p. MD50. Hy4 IgG also protected mice from challenge with another epizootic VEEV variety, 1C (P676). Importantly, therapeutic administration of the humanized mAb to mice already infected with VEEV cured 90 % of mice treated with Hy4 IgG within 1 h of VEEV inoculation and 75 % of mice treated 24 h after virus infection.
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Volumes and issues
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