Geneviève Lebeurier

Partial reconstitution of tobacco mosaic virus

Abstract TMV reconstitutes rapidly in vitro at 24°; a yield of 30% was obtained after 30 min of reconstitution. The maximum yield was around 60%, reached after 7 hours. Electron micrographs of reconstituted TMV suspensions show particles of predominant lengths 700 A and 3000 A. The two populations were separated by rate zonal centrifugation on a linear sucrose gradient: the bottom zone contained infective 3000 A particles; the top zone contained whole infective RNA molecules partially coated to form a nucleoprotein of mean length 700 A. These incompletely coated particles were able to produce infective 3000 A particles when they were mixed with native viral protein. These results support a polar reconstitution.

Effect of elevated temperatures on the development of two strains of tobacco mosaic virus

Abstract The optimal temperature for multiplication of a common strain TMV(C) in submerged leaf disks was close to 24 °C. At 28 ° virus concentration decreases around the ninth day after inoculation. At 35 ° multiplication of the common strain is very low and capsids accumulate, but there is no parallel accumulation of free viral RNA. This suggests that at 35 ° and to a lesser degree at other temperatures that are not optimal, replication of viral RNA is inhibited. A “thermophilic” strain, TMV(LB), which develops actively at 36 ° was isolated. At 24 ° the multiplication of this strain was accompanied by a significant accumulation of capsids. At this same temperature the plants ( Nicotiana tabacum var. Samsun) inoculated with the thermophilic strain did not develop symptoms.

Cauliflower mosaic virus gene I product detected in a cell-wall-enriched fraction

Gene I product of cauliflower mosaic virus was immunodetected in a cell-wall-enriched fraction from infected turnip leaves in addition to its detection in viroplasms and replication complexes. The immunoreaction was carried out with an antiserum raised against a 15 amino acid long synthetic peptide corresponding to the carboxy-terminus of potential gene I protein (P1). The presence of P1 in different subcellular fractions was investigated as a function of time during viral multiplication. At late infection times, P1 was found only in the cell-wall-enriched fraction.

The structure of cauliflower mosaic virus I. pH-Induced structural changes

Cauliflower mosaic virus (CaMV) sediments near 200 S between pH 6 and 9. At pH 10, the virus begins to swell slightly, and expose to pH 11.25 generates two components, sedimenting at 110 S and 65 S. Electron microscopy revealed that both components are swollen virus particles, but the 65 S particles have DNA dangling out. When the pH reached 12, the virus particles collapsed, but their complete dissociation required incubation in 0.1 M NaOH (pH 12.8). Some of these structural transitions are reversible. The structure of the components formed at pH 11.25 and above is discussed in connection with the putative structure of the virus.

Restriction map of native and cloned cauliflower mosaic virus DNA.

Cloned CaMV DNA replicates faithfully in Escherichia coli, since the restriction map of the cloned DNA can be superimposed over that of the native viral DNA. However, some short fragments were difficult to detect in the restricted native viral DNA, whereas they formed clear bands when derived from cauliflower mosaic virus (CaMV) DNA clones propagated in the E. coli host. Apparently, the small fragments that carry variable-length single-stranded gaps present only in native viral DNA, give rise to diffuse weak bands difficult to recognize in gels. Comparison of maps for several CaMV strains permits evaluation of their possible evolutionary relationship.

Polymerization of tobacco mosaic virus protein in Na pyrophosphate.

The role of Na pyrophosphate in the “in vitro” reconstitution of TMV is not yet understood. FraenkelConrat and Singer [ 1 ] maintain that the highest yield of reconstituted nucleoprotein particles is obtained ln Na pyrophosphate buffer. On the other hand Leberman [2] thinks that the attribution to this salt of some property which facilitates the reconstitution process must be treated with a certain amount of caution. We describe in this paper the polymerization of TMV protein subunits in Na pyrophosphate as a function of pH and ionic strength at 20” and will show that other polyphosphates (Na triphosphate and a long chain Na polyphosphate) influence the polymerization process in a similar way to that of pyrophosphate. Discrepancy between the present results and those published by other authors will be discussed.

Physical map of DNA from a new cauliflower mosaic virus strain

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.

Self-assembly of protein subunits from alfalfa mosaic virus

Abstract The self-assembly of alfalfa mosaic virus (AMV) protein subunits containing various amounts of residual RNA has been investigated. AMV protein, containing about 0.8% of residual RNA, formed capsids corresponding in appearance to small virus particles and sedimenting at 38 S. Best yields were observed between pH 7 and 8.0, in a buffer containing 0.1 M NaCl. With protein containing about 1.5% to 2% residual RNA, both small and double-shelled spheres occurred, particularly at pH 8.0 in 0.1 M NaCl; this protein also formed double-bacilliform particles. At a level of residual RNA of approximately 3%, the proportion of double-shelled to single-shelled partic les was high and varied with temperature and pH. Residual RNA extracted from contaminated AMV protein had a very low molecular weight. When 3% of this residual RNA was added to AMV protein, double shells occurred. When 3% of unfractioned AMV RNA was added to protein contaminated by 0.8% of residual RNA, no double-shelled spheres or double-bacilliform particles occurred; heavy components corresponding to those of AMV were formed only when the unfractioned AMV RNA: protein ratio reached 1:10. Sonicated Hemophilus influenzae DNA (3 × 10 6 daltons) gave rise to long flexuous particles, the number of which decreased as soon as extracted residual AMV RNA was added to the mixture.

Evidence for a Protein Kinase Activity Associated with Purified Particles of Cauliflower Mosaic Virus

Summary A cyclic nucleotide-independent protein kinase (PK) activity has been found to be associated with purified particles of cauliflower mosaic virus. The main acceptors of phosphorylation were proteins with mol. wt. of 42000 (the capsid protein), 58000 (which may be the capsid protein precursor) and 110000 (of unknown function). Acid hydrolysis and phosphoamino acid analysis of nucleocapsid proteins phosphorylated in vitro showed that the PK catalyses the transfer of phosphate to both serine and threonine residues. Activation of the PK made the DNA more accessible to DNase I, suggesting that a modification of the structure of the capsid had occurred.

How do viral reverse transcriptases recognize their RNA genome

Reverse transcription is not solely a retroviral mechanism. Hepadnaviruses and caulimoviruses have RNA intermediates that are reverse transcribed into DNA. Moreover non‐viral retroelements, retrotransposons, use reverse transcription in their transposition. All these retroelements encode reverse transcriptase but each group developed their own expression modes capable of assuring a specific and efficient replication of their genomes.