Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies.
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The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy. (+info)
Cyclophilin A incorporation is not required for human immunodeficiency virus type 1 particle maturation and does not destabilize the mature capsid.
(34/5443)
The cellular protein cyclophilin A (CypA) is packaged into human immunodeficiency virus type 1 (HIV-1) virions through a specific interaction with the capsid (CA) domain of the Gag polyprotein. CypA is important for infectivity, but its role in viral replication is currently unknown. Previous reports suggested that CypA promotes uncoating or enhances maturation. We analyzed the morphology and capsid stability of HIV-1 variants defective in CypA binding and of virus grown in the presence of cyclosporin. Both cyclosporin treatment and alteration of Gly89 or Pro90 in the CypA-binding site of CA caused a 5- to 20-fold decrease in CypA incorporation. Virus produced from cyclosporin-treated cells and variants G89V and G89A were 10- to 100-fold less infectious but exhibited normal virion morphologies with regular cone-shaped capsids. Irregular capsid morphologies and lower infectivities were observed for some other variants in the CypA-binding region. Decreased CypA incorporation did not reduce the kinetics of intracellular polyprotein processing or of virus release. No increase in immature particles was observed. These results suggest that CypA does not promote virion maturation. Furthermore, detergent stripping of virus particles with various CypA contents revealed no difference in capsid stability. Based on these results and those reported in the accompanying paper, it appears likely that CypA also is not an uncoating factor. Alternative models for CypA function are discussed. (+info)
Intratype sequence variation among clinical isolates of the human papillomavirus type 6 L1 ORF: clustering of mutations and identification of a frequent amino acid sequence variant.
(35/5443)
Human papillomavirus type 6 (HPV-6) is the causative agent of condyloma acuminata, a common sexually transmitted disease. Virus-like particles (VLPs) assembled from the L1 major capsid protein represent promising candidates for prophylactic vaccines. However, any intratype sequence variation among HPV-6 L1 ORFs will influence which sequence is used for a vaccine according to its prevalence in the population and its propensity for VLP production. Therefore, we have analysed the entire L1 nucleotide sequence of 17 clinical isolates of HPV-6 from the London area. We found 28 positions where changes from the prototype HPV-6b L1 occurred, showing that HPV-6 L1 intratype variation is greater than previously reported. The most frequently observed substitutions are clustered into three discrete regions: R1 (nt 5920-6075), R2 (nt 6590-6670) and R3 (nt 7070-7230). Indeed, most of the nucleotide substitutions within the HPV-6 L1 reported worldwide also map to these regions. The R3 region contains predominantly non-silent substitutions, the most common of which is a G-to-C substitution at position 7079. This results in a Glu-to-Gln change at aa 431, although this change had no effect on VLP yield or stability. This substitution defines a new HPV-6 L1 amino acid sequence that is more abundant in the isolates examined than any other reported sequence. (+info)
Several genes in Chlorella virus strain CVG-1 encode putative virion components.
(36/5443)
We have started to characterize the capsid components of European Chlorella virus isolate CVG-1, a member of the Pbi subgroup of the Phycodnaviridae. The major coat protein, Vp49, was biochemically characterized and the amino acid sequence of the N terminus was determined. Subsequently, the corresponding gene was isolated from CVG-1 genomic DNA. Sequence data were compared to those available from PBCV-1 and other Chlorella virus isolates representing the NC64A subgroup of the Phycodnaviridae. The major coat proteins of all strains are homologous and similar in size, but apparently differ in their degree of glycosylation. Like PBCV-1, the major coat protein of CVG-1 is part of a gene family, as two open reading frames with high similarity to Vp49 were also isolated and characterized in this study. The predicted amino acid sequences of the CVG-1 and PBCV-1 virus genes examined show, with one exception, a divergence of about 25%. Taking into account that corresponding genes of NC64A viruses are almost identical, this divergence supports the original placement of the NC64A and Pbi viruses into separate subgroups of the Phycodnaviridae. (+info)
The tobacco mosaic virus particle: structure and assembly.
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A short account is given of the physical and chemical studies that have led to an understanding of the structure of the tobacco mosaic virus particle and how it is assembled from its constituent coat protein and RNA. The assembly is a much more complex process than might have been expected from the simplicity of the helical design of the particle. The protein forms an obligatory intermediate (a cylindrical disk composed of two layers of protein units), which recognizes a specific RNA hairpin sequence. This extraordinary mechanism simultaneously fulfils the physical requirement for nucleating the growth of the helical particle and the biological requirement for specific recognition of the viral DNA. (+info)
Tobacco mosaic virus particle structure and the initiation of disassembly.
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The structure of an intact tobacco mosaic virus (TMV) particle was determined at 2.9 A resolution using fibre diffraction methods. All residues of the coat protein and the three nucleotides of RNA that are bound to each protein subunit were visible in the electron density map. Examination of the structures of TMV, cucumber green mottle mosaic virus and ribgrass mosaic virus, and site-directed mutagenesis experiments in which carboxylate groups were changed to the corresponding amides, showed that initial stages of disassembly are driven by complex electrostatic interactions involving at least seven carboxylate side-chains and a phosphate group. The locations of these interactions can drift during evolution, allowing the viruses to evade plant defensive responses that depend on recognition of the viral coat protein surface. (+info)
Tobacco mosaic virus and the study of early events in virus infections.
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In order to establish infections, viruses must be delivered to the cells of potential hosts and must then engage in activities that enable their genomes to be expressed and replicated. With most viruses, the events that precede the onset of production of progeny virus particles are referred to as the early events and, in the case of positive-strand RNA viruses, they include the initial interaction with and entry of host cells and the release (uncoating) of the genome from the virus particles. Though the early events remain one of the more poorly understood areas of plant virology, the virus with which most of the relevant research has been performed is tobacco mosaic virus (TMV). In spite of this effort, there remains much uncertainty about the form or constituent of the virus that actually enters the initially invaded cell in a plant and about the mechanism(s) that trigger the subsequent uncoating (virion disassembly) reactions. A variety of approaches have been used in attempts to determine the fate of TMV particles that are involved in the establishment of an infection and these are briefly described in this review. In some recent work, it has been proposed that the uncoating process involves the bidirectional release of coat protein subunits from the viral RNA and that these activities may be mediated by cotranslational and coreplicational disassembly mechanisms. (+info)
Vaccinia virus WR gene A5L is required for morphogenesis of mature virions.
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The vaccinia virus WR A5L open reading frame (corresponding to open reading frame A4L in vaccinia virus Copenhagen) encodes an immunodominant late protein found in the core of the vaccinia virion. To investigate the role of this protein in vaccinia virus replication, we have constructed a recombinant virus, vA5Li, in which the endogenous gene has been deleted and an inducible copy of the A5 gene dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG) for expression has been inserted into the genome. In the absence of inducer, the yield of infectious virus was dramatically reduced. However, DNA synthesis and processing, viral protein expression (except for A5), and early stages in virion formation were indistinguishable from the analogous steps in a normal infection. Electron microscopy revealed that the major vaccinia virus structural form present in cells infected with vA5Li in the absence of inducer was immature virions. Viral particles were purified from vA5Li-infected cells in the presence and absence of inducer. Both particles contained viral DNA and the full complement of viral proteins, except for A5, which was missing from particles prepared in the absence of inducer. The particles prepared in the presence of IPTG were more infectious than those prepared in its absence. The A5 protein appears to be required for the immature virion to form the brick-shaped intracellular mature virion. (+info)