Transmission of polyhedral viruses between different insect species.
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Animals↗
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In vitro and in vivo identification of structural and sequence elements in the 5' untranslated region of Ectropis obliqua picorna-like virus required for internal initiation.
Ectropis obliqua picorna-like virus (EoPV) is a newly described insect virus that is classified as a putative member of the genus Iflavirus. The virus possesses a large, positive-sense RNA genome encoding a single polyprotein that shares physicochemical properties with those of members of the family Picornaviridae. The 5' untranslated region (5' UTR) plays an important role in picornavirus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation. To investigate translation in EoPV, an extensive range of mutations were engineered within the 5' UTR and the effects of these changes were examined in vitro and in vivo by using a bicistronic construct. Results showed that deletions within the first 63 nt had little impact on IRES activity, whilst core IRES function was contained within stem-loops C and D, as their removal abrogated IRES activity significantly. In contrast to these findings, removal of stem-loop G containing two cryptic AUGs caused a remarkable increase in IRES activity, which was further investigated by site-directed mutagenesis at these two positions. It was also confirmed that initiation of protein synthesis occurs at AUG6 (position 391-394) and not at the AUG immediately downstream of the polypyrimidine tract. Mutation of the polypyrimidine tract (CCTTTC) had a slight effect on EoPV IRES activity. Furthermore, mutations of the RAAA motif led to a decrease in IRES activity of approximately 40 % in vitro, but these results were not supported by in vivo experiments. In conclusion, this study reveals that the EoPV IRES element is unique, although it has features in common with the type II IRESs.
5' Untranslated Regions↗
Baculovirus vectors: novel mammalian cell gene-delivery vehicles and their applications.
Various methods have been developed to transfer and express genes in mammalian cells. Each method, whether virally, non-virally, or physically-based, has unique favorable features, but also drawbacks with respect to meeting desired and specific needs. Baculoviruses have been used since 1983 to express recombinant genes controlled by strong insect-virus promoters in their natural host (insect) cells. Today this is a well-established and easy to handle system for producing large quantities of recombinant proteins for numerous purposes. In 1995 it was first published that recombinant baculoviruses are able to deliver genes into mammalian cells. These genes are expressed provided that they are controlled by a promoter which is active in mammalian cells. Since then, various vector variants have been developed and numerous potential and meaningful applications have been described. It is not surprising that the use of baculovirus vectors as mammalian cell gene delivery vectors is constantly increasing and that the system is undergoing permanent improvements. Based on the convenience of the system to transfer genes into mammalian cells, baculoviruses can be applied in cell-based assays for drug screening to overcome the long periods of time required to generate stable cell lines. Baculovirus vectors are able to deliver very large DNA sequences into mammalian cells and vectors for toxic gene products can also be generated. In addition, baculoviruses are valuable tools for launching viral infection in cases where there is no appropriate cell culture system. Moreover, recent research has shown that the vectors can be applied in vivo. Depending on the design of the study, baculovirus vectors allow for sustained gene expression or are able to induce an immune response directed against the delivered and/or displayed gene product. The latter offers the opportunity to generate monoclonal antibodies against certain proteins that have failed by other means. In addition, it points to the potential usefulness of baculovirus vectors as new kinds of vaccines. Baculovirus vectors are therefore considered an enabling technology for various product opportunities.
Animals↗
Virus stability and protein-nucleic acid interaction as studied by high-pressure effects on nodaviruses.
In this work, we evaluate the stability, dynamics and protein-nucleic acid interaction in Flock House virus (FHV). FHV is an RNA insect virus, non-enveloped, member of the family Nodaviridae. It is composed of a bipartite single-stranded RNA genome packaged in an icosahedral capsid of 180 copies of an identical protein (alpha protein). A fundamental property of many animal viruses is the post-assembly maturation required for infectivity. FHV is constructed as a provirion, which matures to an infectious virion by cleavage of alpha protein into beta and gamma subunits. We used high pressure, temperature and chemical denaturing agents to promote perturbation of the viral capsid. These effects were monitored by spectroscopy measurements (fluorescence, light scattering and CD) and size-exclusion chromatography. The data showed that FHV was stable to pressures up to 310 MPa at room temperature. The fluorescence emission and light scattering values showed small changes that were reversible after decompression. When we combined pressure and sub-denaturing urea concentrations (1 M), the changes were more drastic, suggesting dissociation of the capsid. However, these changes were reversible after pressure release. The complete dissociation of FHV could be observed only under high urea concentrations (10 M). There were no significant changes in emission spectra up to 5 M urea. FHV also was stable when we used temperature treatments (high and low). We also compared the effects of urea and pressure on FHV wild type and cleavage-defective mutant VLPs (virus-like particles). The VLPs and authentic particles are distinguishable by protein-RNA interactions, since VLPs pack cellular RNA and native particles contain viral RNA. Our results demonstrated that native particles are more stable than VLPs to physical and chemical treatments. Our data point to the specificity of the interaction between the capsid protein and the viral RNA. This specificity is crucial to the stability of the particle, which makes this interaction an excellent target for drug development.
Capsid↗
Analysis of genetic information of an insect picorna-like virus, infectious flacherie virus of silkworm: evidence for evolutionary relationships among insect, mammalian and plant picorna(-like) viruses.
We synthesized the cDNAs of an insect picornavirus, infectious flacherie virus of silkworm (IFV), genomic RNA and inserted it into a bacterial plasmid (pUC119). The 9,650 nucleotides (nts) sequence except for the poly(A) tail was obtained from the cloned cDNAs, and the sequence integrity was confirmed by primer extension and direct RNA sequencing. The sequence has a large open reading frame (ORF) of 9,255 nts (3,085 codons) flanked by the short 5' non-coding region (156 nts) and by the rather long 3' non-coding (239 nts). The structural proteins VP3, 4, 1 and 2 were located at the N-terminus of the polyprotein in this order and were preceded by a tentative small peptide. Computer analysis identified the sequences similar to the consensus sequences of 2C (helicase?), 3C (protease), and 3D (RNA polymerase) conserved among mammalian and plant picorna(-like) viruses. In addition, the predicted genome organization of IFV was quite similar to those of picornaviruses. Further analyses of the characteristics of the genome structure and a tentative phylogenetic tree constructed on the basis of the amino acid sequence similarity emphasized the evolutionary relationships among the insect and plant viruses.
Adenoviridae↗
The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation.
In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol. wt. protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol. wt. protein (p10) whose function is unknown. The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs. We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells. Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined. The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells. The same promoter was active, however, in AcNPV-infected S. frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus. This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells. The nature of these products was unknown. The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene. Since Drosophila DNA or S. frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter. Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetyltransferases↗
Nucleotide sequence analysis shows that Rhopalosiphum padi virus is a member of a novel group of insect-infecting RNA viruses.
Rhopalosiphum padi virus (RhPV) is an aphid virus that has been considered a member of the Picornaviridae based on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, and to a partial sequence from the 3' end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV, Drosophila C virus, Plautia stali intestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.
Amino Acid Sequence↗
Midgut and viral associated proteases of Heliothis armigera.
The role played by the gut juice of insects in the infective process of insect viruses was examined. Analysis of larval gut extract of Heliothis armigera by SDS-PAGE revealed protease activity associated with components of molecular weights 48,000 and 94,000. Proteases were found to be associated with occlusion bodies and virions of both nuclear polyhedrosis virus (NPV) and cytoplasmic polyhedrosis virus (CPV) infecting H. armigera. CPV occlusion bodies were dissolved by gut juice extract at pH 8.0, trypsin and chymotrypsin at pH 8.0, and carbonate-chloride solution at pH 10.5. Trypsin treatment was selective for occlusion bodies of CPV at pH 8.0, whereas solutions more alkaline than pH 10.0 without added enzymes were adequate to digest NPV occlusion bodies. This property was used to identify and separate the two types of viruses from a mixed infection. Gut extract proteases have characteristics similar to those of trypsin.
Animals↗
Enzymatic characterization of a trypsin-like serine protease encoded by the genome of cell fusing agent virus.
Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosquito cell line. Based on viral morphology, phenotypic and phylogenetic studies, CFAV had been tentatively assigned to the genus Flavivirus (family Flaviviridae). The determination of the CFAV polyprotein complete sequence showed a putative serine protease domain analogue to the flaviviral NS2B/NS3 complex. This complex had been extensively studied, because it represented one of the main targets for antiflavivirus therapy development. We report herein the biochemical characterization of CFAV DeltaNS2B-NS3pro protease complex. CFAV polyprotein sequence was computationally analysed to identify the amino-acid regions involved in protease activity. We designed, expressed and purified a catalytically active protease whose enzymatic properties were determined using fluorogenic substrates. Our results showed that, despite the low level of conservation of its amino-acid sequence, CFAV protease exhibited physico-chemical properties of other flaviviruses (high pH value requirement for optimal activity, inhibition by salt and preference for substrates featuring a basic residue at P(1) position).
Amino Acid Sequence↗
Parvovirus replication.
The members of the family Parvoviridae are among the smallest of the DNA viruses, with a linear single-stranded genome of about 5 kilobases. Currently the family is divided into three genera, two of which contain viruses of vertebrates and a third containing insect viruses. This review concentrates on the vertebrate viruses, with emphasis on recent advances in our insights into the molecular biology of viral replication. Traditionally the vertebrate viruses have been distinguished by the presence or absence of a requirement for a coinfection with a helper virus before productive infection can occur, hence the notion that the dependoviruses (adeno-associated viruses [AAV]) are defective. Recent data would suggest that not only is there a great deal of structural and genetic organizational similarity between the two types of vertebrate viruses, but also there is significant similarity in the molecular biology of productive replication. What differs is the physiological condition of the host cell that renders it permissive. Healthy dividing cells are permissive for productive replication by autonomous parvoviruses; such cells result in latent infection by dependoviruses. For a cell to become permissive for productive AAV replication, it must have been exposed to toxic conditions which activate a latent AAV genome. Such conditions can be caused by helper-virus infection or exposure to physical (UV light) or chemical (some carcinogens) agents. In this paper the molecular biology of replication is reviewed, with special emphasis on the role of the host and the consequences of viral infection for the host.
Animals↗
Mechanisms of plant virus evolution.
Plant viruses utilize several mechanisms to generate the large amount of genetic diversity found both within and between species. Plant RNA viruses and pararetroviruses probably have highly error prone replication mechanisms, that result in numerous mutations and a quasispecies nature. The plant DNA viruses also exhibit diversity, but the source of this is less clear. Plant viruses frequently use recombination and reassortment as driving forces in evolution, and, occasionally, other mechanisms such as gene duplication and overprinting. The amount of variation found in different species of plant viruses is remarkably different, even though there is no evidence that the mutation rate varies. The origin of plant viruses is uncertain, but several possible theories are proposed. The relationships between some plant and animal viruses suggests a common origin, possibly an insect virus. The propensity for rapid adaptation makes tracing the evolutionary history of viruses difficult, and long term control of virus disease nearly impossible, but it provides an excellent model system for studying general mechanisms of molecular evolution.
Journal Article↗
Electron microscope observations of the membrane surrounding polyhedral inclusion bodies of insects.
Paracrystalline inclusion bodies (polyhedra) which occlude insect viruses are bounded by two membrane units which are closely appressed. The membranes appear to be similar to those of cisternae found in the nucleus. These cisternae were observed associated with polyhedra in various stages of condensation and appression.
Animals↗
The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences.
The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12. This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements.
Amino Acid Sequence↗
Assessment of the application of baculoviruses for control of Lepidoptera.
Baculoviruses, among other insect viruses, are regarded as safe and selective bioinsecticides, restricted to invertebrates. They have been used worldwide against many insect pests, mainly Lepidoptera. Their application as microbial pesticides, however, has not met their potential to control pests in crops, forests, and pastures, with the exception of the nuclear polyhedrosis virus of the soybean caterpillar (Anticarsia gemmatalis), which is used on approximately 1 million ha annually in Brazil. Problems that have limited expansion of baculovirus use include narrow host range, slow killing speed, technical and economical difficulties for in vitro commercial production, timing of application based on frequent host population monitoring, variability of field efficacy due to climatic conditions, and farmers' attitudes toward pest control, which have been based on application of fast-killing chemical insecticides. Farmer education regarding use of biological insecticides and their characteristics is considered one of the major actions necessary for increased use of baculoviruses. Strategies to counteract some of the limitations of baculoviruses, especially their slow killing activity, have been investigated and are promising. These include the use of chemical or biological substances added to virus formulations and genetic engineering of the viruses themselves to express insect toxins or hormones. Such strategies can enhance viral activity and increase speed of kill as well as reduce larval feeding activity. The use of baculoviruses against Lepidoptera is reviewed, with the utilization of the nuclear polyhedrosis virus of A. gemmatalis in Brazil serving as a case-study.
Journal Article↗
Identification and sequencing of the spheroidin gene of Choristoneura biennis entomopoxvirus.
Entomopoxviruses are a class of insect viruses whose virions are embedded in cytoplasmic occlusion bodies. The major component of these protective complexes is a protein called spheroidin. An open reading frame encoding the spheroidin gene of Choristoneura biennis entomopoxvirus has been identified and sequenced in our laboratory. This protein coding region is 1023 nucleotides long and specifies a polypeptide of 38,500 Da. Spheroidin was purified by SDS polyacrylamide gel electrophoresis, electroeluted, and its amino terminus sequence was determined on a gas phase sequencer. We observed that the first 20 N-terminal amino acids were absent in the mature processed form of the spheroidin molecule. Examination of these 20 residues revealed their hydrophobic nature and close resemblance to the consensus signal peptide sequence which is commonly found on membrane proteins. The DNA sequence of the spheroidin gene predicted a processed polypeptide with a molecular weight of 36 kDa. However, spheroidin was observed to aggregate in complexes composed of 50-kDa monomers. Intermolecular disulfide bonds were shown to play major roles in the formation and structure of these viral occlusion bodies. The difference in molecular weight between the predicted protein and its counterpart in infected cells is likely due to post-translational modifications. Indeed, two potential asparagine-linked glycosylation sites are present on the spheroidin molecule. The 5' flanking regions of the spheroidin gene and the vaccinia major core protein precursor gene P4b were shown to share substantial homology.
Amino Acid Sequence↗
Replication of the gonad-specific virus Hz-2V in Ld652Y cells mimics replication in vivo.
A newly discovered, nonoccluded insect virus, known as gonad-specific virus or Hz-2V, was found to replicate differently in two insect cell lines derived from ovarian tissues (Tn-368 cells from Trichoplusia ni and Ld652Y from Lymantria dispar). Differences between these two cell lines were observed in virus plaque forming ability, rate of viral DNA replication, time course of infectious virus production, and the mechanism of virus release from infected cells. Replication of Hz-2V in Ld652Y cells was more productive and more closely resembled in vivo virus replication.
Animals↗
Replication of a nuclear polyhedrosis virus in an established insect cell line.
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Animals↗
A study of the long virus rods associated with insect granuloses.
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In Vitro Techniques↗