Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

Indices of water deficit in susceptible and resistant cucumbers in response to infection by cucumber mosaic virus

Indices of water deficit were determined under conditions of non-limited water supply in cucumber mosaic virus (CMV)-infected seedlings of the susceptible cv. Bet Alpha. An increase in the concentration of soluble solids, decrease of water and osmotic potentials, and increase of proline concentration were found in the CMV-infected cotyledons. In the cv. Shimshon, which is resistant to CMV, virus infection caused only a slight change in the concentration of the soluble solids and in the osmotic potential; water potential and proline content were not affected. Concomitantly, infectivity of cotyledons by CMV was much lower in the tolerant cv. Shimshon than in the susceptible cv. Bet Alpha. The possible association of water deficit with virus-induced growth retardation is discussed.     

First novel synthesis of triazole thioglycosides as ribavirin analogues

This study reports a novel and efficient method for the synthesis of the first reported novel class of triazole thioglycosides. These series of compounds were designed through the reaction of potassium cyanocarbonimidodithioate 2 with hydrazine derivatives 3a-d in EtOH at room temperature to give the corresponding potassium 5-amino-1H-1,2,4-triazole-3-thiolates 4a-d. The latter compounds were treated with tetra-O-acetyl-α-D-glucopyranosyl bromide 6a and tetra-O-acetyl-α-D-galactopyranosyl bromide 6b in DMF at room temperature to give in high yields the corresponding triazole thioglycosides 7a-h. Treatment of triazole salts 4a–d with hydrochloric acid afforded the corresponding 3-mercaptotriazoles 5a-d. Compounds 5a-d were then reacted with bromoperacetylated sugars 6a,b in sodium hydride-DMF at ambient temperature to afford the thioglycosyl compounds 7a-h. Ammonolysis of the triazole thioglycosides 7a-h afforded the corresponding free thioglycosides 8a-h. The scope and limitation of the method is demonstrated. The structure of the reaction products was confirmed on the basis of their elemental analysis and spectral data (IR, ¹H NMR, MS and ¹³C NMR).     

Preparation of an isomorphous heavy-atom derivative of tobacco mosaic virus by chemical modification with 4-sulpho-phenylisothiocyanate

The reaction of the vulgare and U2 strains of tobacco mosaic virus with 4-sulpho-phenylisothiocyanate has been investigated. The coat protein of the U2 strain has a proline residue at its N-terminus and a lysine residue at position 53. Whereas both residues could be reacted with 4-sulpho-phenylisothiocyanate in the isolated coat protein, only proline-1 was modified during treatment of the intact virus with the same reagent, thereby showing that the loss of reactivity of the ?-amino group of lysine-53 is a consequence of the virus structure. The 4-sulpho-phenylthiocarbamoyl derivative of amino groups shows considerable tautomerism and, as a consequence, it proved possible to prepare a heavy-atom derivative of the intact U2 strain in which methyl mercury nitrate was bound by the modified N-terminal residue of the coat protein.On the other hand, when the intact vulgare strain was treated with 4-sulphophenylisothiocyanate, little or no modification of the ?-amino groups of the two lysine residues (positions 53 and 68) per polypeptide chain was observed. Taking into account previous studies on the reactivity of the amino groups of the coat protein in tobacco mosaic virus vulgare and assuming that all strains and mutants have closely similar three-dimensional structures, these experiments suggest that the N-terminal residue is more exposed (i.e. probably nearer the virus “surface”) than the side-chain of lysine-68, which in turn is more accessible than the side-chain of lysine-53. This interpretation is readily compatible with the results of X-ray diffraction analysis carried out on these chemically modified viruses (Mandelkow &; Holmes, 1974) and lends support to the hope that such methods of preparing heavy-atom derivatives of proteins will be of general use.     

Structural principles for the propeller assembly of beta-sheets: the preference for seven-fold symmetry.

Twisted beta-sheets, packed face to face, may be arranged in circular formation like blades of a propeller or turbine. This beta-propeller fold has been found in three proteins: that in neuraminidase consists of six beta-sheets while those in methylamine dehydrogenase and galactose oxidase are composed of seven beta-sheets. A model for multisheet packing in the beta-propeller fold is proposed. This model gives both geometrical parameters of the beta-propellers composed of different numbers of sheets and patterns of residue packing at their sheet-to-sheet interfaces. All the known beta-propeller structures have been analyzed, and the observed geometries and residue packing are found to be in good agreement with those predicted by models. It is shown that unusual seven-fold symmetry is preferable to six- or eight-fold symmetry for propeller-like multi-sheet assembly. According to the model, a six-beta-sheet propeller has to have predominantly small residues in the beta-strands closed to its six-fold axis, but no strong sequence constraints are necessary for a seven-fold beta-propeller.     

Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence.These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.