The structure of tomato aspermy virus by X-ray crystallography

The three-dimensional structure of tomato aspermy virus (TAV) has been solved by X-ray crystallography and refined to an R factor of 0.218 for 3.4-40 A data (effective resolution of 4A). Molecular replacement, using cucumber mosaic virus (Smith et al., 2000), provided phases for the initial maps used for model building. The coat protein of the 280 A diameter virion has the canonical "Swiss roll" beta-barrel topology with a distinctive amino-terminal alpha-helix directed into the interior of the virus where it interacts with encapsidated RNA. The N-terminal helices are joined to the beta-barrels of protein subunits by extended polypeptides of six amino acids, which serve as flexible hinges allowing movement of the helices in response to local RNA distribution. Segments of three nucleotides of partially disordered RNA interact with the capsid, primarily through arginine residues, at interfaces between A and B subunits. Side chains of cys64 and cys106 form the first disulfide observed in a cucumovirus, including a unique cysteine, 106, in a region otherwise conserved. A positive ion, putatively modeled as a Mg(+)ion, lies on the quasi-threefold axis surrounded by three quasi-symmetric glutamate 175 side chains.     

Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture

There has been major progress in our understanding of hepatitis C virus (HCV) molecular virology in recent years. An essential prerequisite for this progress was the availability of functional molecular HCV clones, that serve as a starting point in order to establish cell culture systems. The first of these was the HCV replicon system, which used self-replicating subgenomic viral RNAs. However, these replicons only recapitulated the intracellular life cycle, and did not support production of infectious virus: this became possible with the identification of an HCV isolate that, for unknown reasons, replicates to very high levels in a human hepatoma cell line. Cells containing this genome release virus particles that are infectious in cell culture and in vivo. Without doubt, this system provides new possibilities for molecular studies of the HCV life cycle and the development of novel antiviral concepts.     

Preliminary X-ray diffraction analysis of crystals of tomato aspermy virus (TAV)

Tomato aspermy virus (TAV) is a member of the T = 3 cucumovirus group, and the chrysanthemum strain (C-TAV) has been crystallized in a form suitable for X-ray structural analysis. The crystals, which grow in 14–17% ethanol at pH 8.5, are of orthorhombic space group I222 with unit cell dimensions of a = 295.1 Å, b = 320.5 Å, and c = 383.6 Å. There are two T = 3 virus particles in the unit cell, which means that they must be centered at 0,0,0 and 1/2, 1/2, 1/2 with icosahedral 222 symmetry elements coincident with crystallographic symmetry operators. The asymmetric unit of the crystals, therefore, contains one quarter of a virus particle, or 45 capsid subunits. Native diffraction data to 4 Å resolution have been collected using synchrotron radiation, though data appear to be present beyond that resolution. © 1995 Wiley-Liss, Inc.     

Multiple targets for suppression of RNA interference by tomato aspermy virus protein 2B

Viral suppressors of RNA interference (RNAi) appear to have evolved as a response to this innate genomic defense. We report the nucleic acid binding properties of the Cucumovirus RNAi suppressor tomato aspermy virus protein 2B (TAV 2B). Using total internal reflection fluorescence spectroscopy (TIRFS), we show that TAV 2B binds double-stranded RNA corresponding to siRNAs and miRNAs, as well as single-stranded RNA oligonucleotides. A number of positively charged residues between amino acids 20 and 30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B, causing it to form a primarily helical structure and a 4:2 protein-RNA complex.     

Generation and characterization of closely related epizootic and enzootic infectious cDNA clones for studying interferon sensitivity and emergence mechanisms of Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV) is a reemerging pathogen and a continuing threat to humans and equines in the Americas. Identification of the genetic determinants that enable epizootic VEEV strains to arise and exploit equines as amplification hosts to cause widespread human disease is pivotal to understanding VEE emergence. The sensitivity to murine alpha/beta interferon-mediated antiviral activity was previously correlated to the epizootic phenotype of several VEEV strains. Infectious cDNA clones were generated from an epizootic subtype IC VEEV strain (SH3) isolated during the 1992 Venezuelan outbreak and a closely related enzootic, sympatric subtype ID strain (ZPC738). These VEEV strains had low-cell-culture-passage histories and differed by only 12 amino acids in the nonstructural and structural proteins. Rescued viruses showed similar growth kinetics to their parent viruses in several cell lines, and murine infections resulted in comparable viremia and disease. Unlike what was found in other studies of epizootic and enzootic VEEV strains, the sensitivities to murine alpha/beta interferon did not differ appreciably between these epizootic versus enzootic strains, calling into question the reliability of interferon sensitivity as a marker of epizootic potential.     

Biological properties of pseudorecombinant and recombinant strains created with cucumber mosaic virus and tomato aspermy virus.

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) are closely related cucumoviruses. We have made pseudorecombinant viruses in which the RNAs 3 of these two viruses have been exchanged and recombinant viruses containing chimeric RNA 3 molecules, in which the coat proteins and the 3'-end regions of CMV and TAV have been exchanged, giving rise to recombinants designated RT3 and TR3. The replication properties and the cell-to-cell and long-distance movement patterns of these pseudorecombinant and recombinant viruses were examined in different hosts. All the viruses were able to replicate and accumulate RNA 4 in protoplasts. The pseudorecombinants and the R1R2RT3 recombinant infected tobacco systemically, but the R1R2TR3 recombinant was not detectable, even in the inoculated leaves. Comparison of the abilities of the viruses to replicate in protoplasts and intact cucumber plants suggests that cell-to-cell movement factors are also encoded by RNAs 1 and/or 2. Major determinants of symptom severity in Nicotiana glutinosa are localized on the 3' part of RNA 3, and in Nicotiana benthamiana, more severe symptoms were observed with the T1T2R3 strain than with the others tested.     

Development of a chimeric strain of porcine reproductive and respiratory syndrome virus with an infectious clone and a Korean dominant field strain

The K418 chimeric virus of porcine reproductive and respiratory syndrome virus (PRRSV) was engineered by replacing the genomic region containing structure protein genes of an infectious clone of PRRSV, FL12, with the same region obtained from a Korean dominant field strain, LMY. The K418 reached 10⁶ TCID₅₀/ml of viral titer with similar growth kinetics to those of parental strains and had a cross-reactive neutralizing antibody response to field serum from the entire country. The chimeric clone pK418 can be used as a practical tool for further studying the molecular characteristics of PRRSV proteins through genetic manipulation. Furthermore, successful construction of the K418 will allow for the development of customized vaccine candidates against PRRSV, which has evolved rapidly in Korea.     

Alteration of tomato microRNAs expression during fruit development upon Cucumber mosaic virus and Tomato aspermy virus infection

The economic importance of Solanaceae plant species is well documented, and tomato has become a model for fleshy fruit development and ripening studies. Plant microRNAs (miRNAs) are small endogenous RNAs that are involved in a variety of activities including plant development, signal transduction and protein degradation, as well as response to environment stress and pathogen invasion. Here in this study, we aimed at quantifying the expression alterations of nine miRNAs and target mRNAs in tomato flower and fruit development upon Cucumber mosaic virus (CMV) and Tomato aspermy virus infections. Three different CMV strains CMV-Fny, CMV-FnyΔ2b and CMV-Fny-satT1 were used in our investigation, and the miRNA/mRNA expression alterations were analyzed by real-time quantitative RT-PCR. The results shown the levels of several miRNA/mRNA pairs were increased upon virus infections. However, the increased level of individual miRNA differed for different virus strains, reflecting differences in severity of symptom phenotypes. The altered expression patterns of these miRNA/mRNA pairs and their predicted functions indicate the possible roles in flower and fruit development, and provide experimental data for understanding the miRNA-mediated phenotype alterations in tomato fruit.     

In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain.

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.     

Generation of infectious molecular clones of simian immunodeficiency virus from fecal consensus sequences of wild chimpanzees

Studies of simian immunodeficiency viruses (SIVs) in their endangered primate hosts are of obvious medical and public health importance, but technically challenging. Although SIV-specific antibodies and nucleic acids have been detected in primate fecal samples, recovery of replication-competent virus from such samples has not been achieved. Here, we report the construction of infectious molecular clones of SIVcpz from fecal viral consensus sequences. Subgenomic fragments comprising a complete provirus were amplified from fecal RNA of three wild-living chimpanzees and sequenced directly. One set of amplicons was concatenated using overlap extension PCR. The resulting clone (TAN1.24) contained intact genes and regulatory regions but was replication defective. It also differed from the fecal consensus sequence by 76 nucleotides. Stepwise elimination of all missense mutations generated several constructs with restored replication potential. The clone that yielded the most infectious virus (TAN1.910) was identical to the consensus sequence in both protein and long terminal repeat sequences. Two additional SIVcpz clones were constructed by direct synthesis of fecal consensus sequences. One of these (TAN3.1) yielded fully infectious virus, while the second one (TAN2.69) required modification at one ambiguous site in the viral pol gene for biological activity. All three reconstructed proviruses produced infectious virions that replicated in human and chimpanzee CD4(+) T cells, were CCR5 tropic, and resembled primary human immunodeficiency virus type 1 isolates in their neutralization phenotype. These results provide the first direct evidence that naturally occurring SIVcpz strains already have many of the biological properties required for persistent infection of humans, including CD4 and CCR5 dependence and neutralization resistance. Moreover, they outline a new strategy for obtaining medically important "SIV isolates" that have thus far eluded investigation. Such isolates are needed to identify viral determinants that contribute to cross-species transmission and host adaptation.     

Molecular characterisation of cDNA clones representing pectin esterase isozymes from tomato

Two pectin esterase cDNA clones representing different isozymes with ca. 95% homology were isolated from an early ripening tomato fruit cDNA library. Both clones were longer than previously published sequences, and the encoded proteins possessed extended (229–233 amino acid) putative N-terminal extensions. In addition, the mRNA species corresponding to the two clones showed differential levels of expression in fruit.     

Mapping of ripening-related or -specific cDNA clones of tomato (Lycopersicon esculentum)

Summary Nineteen ripening-related or -specific clones from Lycopersicon esculentum were mapped via RFLP analysis using an F₂ population from the cross L. esculentum x L. pennellii and cDNA or genomic clones of known map location. The map produced using cDNA and genomic clones of known map location corresponded well with previously published maps of tomato. The number of loci detected for each ripening-related or-specific clone varied from one to seven. These loci were located on all 12 chromosomes of the tomato genome. There was no significant clustering of ripening-related or-specific genes. Regions of very low recombination were observed. The clone for polygalacturonase (TOM6) mapped to a single region on chromosome 10, the same chromosome as the nor and alc ripening mutants. To fine map this chromosome, two backcross populations were produced from the cross of L. esculentum x L. pimpenillifolium, in which the esculentum parents used were homozygous for either the alc or the nor. The coding region for polygalacturonase is functionally unlinked to either of these two ripening mutants.     

Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences

A cDNA library was derived from the poly(A)⁺ RNA of young tomato leaves. The library was cloned in a gt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5 end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)⁺ RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.