Pierre Pfeiffer

Involvement of reverse transcription in the replication of cauliflower mosaic virus: A detailed model and test of some aspects

A model is presented according to which cauliflower mosaic virus (CaMV) DNA is replicated via an RNA intermediate. The model explains the typical S1 nuclease-sensitive sites in mature CaMV DNA, the occurrence of the large, terminally redundant transcript, the local separation of CaMV transcription and CaMV assembly, the abundance of knotted CaMV DNA forms, and the high recombination frequency. A site of perfect homology to plant tRNA was detected. Extracts from a mixture of nuclei and inclusion bodies can be separated into fast-sedimenting complexes elongating endogenous CaMV RNA, and slow-sedimenting ones elongating endogenous CaMV DNA. The CaMV DNA synthesis can be partly inhibited both by RNAase and actinomycin D, suggesting the presence of a mixture of RNA- and DNA-templates.

A virus specified protein produced upon infection by cauliflower mosaic virus (CaMV)

A virus specific protein produced during infection by the cauliflower mosaic virus (CaMV) is characterized: it is a 62 Kd protein. It is constitutive of inclusion bodies or ⪡ viroplasms ⪢. This protein is coded by the viral genome: its polyadenylated mRNA hybridizes with the viral DNA. Its gene is located between the EcoRI-B and EcoRI-E sites on the restriction map.

The structure of cauliflower mosaic virus. II. Identity and location of the viral polypeptides.

Abstract The major structural protein in cauliflower mosaic virus (CaMV) is a 42,000-dalton species. We demonstrate that the multiplicity of CaMV polypeptides reported by previous authors is an artifact that arises from partial proteolysis of the 42,000-dalton species into lighter chains and also from partial polymerization of the various protein monomers. Besides the expected sequence homologies in proteins derived from the major coat protein, further sequence homologies were detected between the 42,000-dalton species and two minor proteins, MW, 56,000 and 49,000 daltons, also present in the virus.

The double-stranded RNA associated with the ‘447’ cytoplasmic male sterility in Vicia faba is packaged together with its replicase in cytoplasmic membranous vesicles

The 447 male sterility trait in Vicia faba is strictly correlated with the presence of well-defined membranous vesicles or ‘cytoplasmic spherical bodies’ not found in fertile isogenic maintainer plants, and by the occurrence of a discrete high molecular weight double-stranded RNA. We have purified these cytoplasmic membranous vesicles and find that they contain the dsRNA together with an RNA-dependent RNA polymerase whose activity depends upon the presence of Mg2+, requires the four-nucleoside triphosphates and is unaffected by inhibitors of cellular transcriptases, e.g. α-amanitin and Actinomycin D. The dsRNA can be labelled in vitro by incubating the cytoplasmic vesicles with radioactive NTPs, and the RNA synthesized in vitro is also in a double-stranded form as judged by its resistance to RNase digestion at high salt and its behaviour upon CF-11 chromatography. Treatment of the vesicles with a non-ionic detergent releases the dsRNA in the form of a complex with the RNA-dependent RNA polymerase. The enzyme can still carry out the specific synthesis of dsRNA in these solubilized complexes. The cytoplasmic vesicles therefore isolate this vertically transmitted, self-replicating dsRNA from the cellular milieu: the possible mode of action and relevance of this novel genetic element to the 447 cytoplasmic male sterility trait are discussed.

Structural polymorphism of bromegrass mosaic virus: A neutron small angle scattering investigation

Abstract Neutron small angle scattering has been used to investigate the architecture of bromegrass mosaic virus (BMV) in solution as a function of pH, ionic strength, and nature of the polyvalent ions at room temperature: The radial extensions of RNA and protein were determined from measurements performed in buffers containing suitable amounts of heavy water (D20) to match the scattering of either component. Our results confirm the polymorphism of BMV known from the variation of its overall size ( N. L. Incardona and P. Kaesberg, 1964 , Biophys. J. 4, 11–21; M. Zulauf, 1977 , J. Mol. Biol. 114, 259–266) and extend it to the RNA. Indeed, we found that although at pH 5.5 the overall size of compact BMV remains constant, measurable changes in the radial extension of RNA take place under the compacting action of high ionic strength or spermine. At neutral pH both the overall size of BMV and the extension of RNA vary in a similar way and depend strongly upon the environment: Swelling is maximal in the absence of polyvalent cations (spermine, Ca2+, Mg2+), and the compacting action of at least Mg2+ has been shown to be partially reversed by increasing the concentration of K+. The thickness of the protein shell does not change significantly upon swelling. Back-titration of swollen BMV is reversible only in the presence of divalent ions. The lack of reversibility extends to both RNA and overall sizes and is accompanied by an increase of the thickness of the capsid. The scattering curve of empty capsids obtained by self-assembly of dissociated BMV protein differs qualitatively from that of the capsid of the virions, implying not only that the size is intermediate between those of compact and swollen BMV but also that the clustering of the protein subunits is probably different at the periphery of the particle. Implications on the relative importance of protein-protein and protein-RNA interactions as well as on the action of ions on the stability of BMV are discussed, and mechanism is proposed for the lack of reversibility of BMV upon back-titration.

Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader

The effect of the 600 nucleotide-long CaMV 35S RNA 5′ leader sequence on the expression of downstream genes was analyzed both in plant protoplasts and in vitro. For transient expression studies in protoplasts derived from host and nonhost plants, the bacterial chloramphenicol acetyl transferase (CAT) gene was fused to the initiation codon of ORF VII. The leader sequence reduced CAT expression two- to four-fold in protoplasts derived from three host species, but 10- to 50-fold in protoplasts derived from three different nonhost species. For in-vitro studies the 35S promoter was replaced by the SP6 promoter. The leader reduced in-vitro translation of SP6 transcripts approximately six-fold, indicating that at least part of the inhibition observed in protoplasts is directly due to the interference of the leader sequence with translation. Other steps in gene expression that may also be affected are discussed.

The cation binding associated with structural transitions in bromegrass mosaic virus

Abstract Hydrogen ion titration curves are described for bromegrass mosaic virus under various conditions of temperature, ionic strength, and Ca 2+, Mg2+, or polyamine concentration, with particular reference to the hysteresis region near neutral pH. The curves are correlated with the sedimentation properties of the virus. There are two relatively nonspecific cation-binding sites per protein subunit, present in the virion but not in the protein or RNA alone, which control the virions swelling. The swelling is reversible in the presence of enough cations; without them, swollen BMV can contract to an incorrect structure with RNA partially exposed. We interpret these results qualitatively and structurally in terms of binding sites formed in a region of high electronegativity contributed by RNA phosphates and protein carboxylates.

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.

Cauliflower Mosaic Virus replication complexes: characterization of the associated enzymes and of the polarity of the DNA synthesized in vitro

The synthesis of both strands of CaMV-DNA has been studied in vitro using viral replication complexes obtained by hypotonic extraction of infected plant organelles. Hybridization of the DNA synthesized in vitro to single stranded CaMV DNA probes cloned in bacteriophage M 13 confirmed that the 35 S RNA served as a template for the synthesis of the (−) DNA strand. The response of CaMV DNA synthesis to various inhibitors suggests that a single enzyme directs both steps of the replication cycle. A comparative activity gel analysis of the DNA polymerases present in nuclear extracts from healthy and CaMV-infected turnips revealed an increase of a DNA polymerase species migrating in the 75 Kd range in infected tissue. When the enzyme activity associated with the isolated replicative complexes was similarly analyzed, the 75 Kd polymerase was markedly predominant, confirming that DNA polymerases of the α-type (MW in the 110 Kd range) are not involved in the aphidicolin-insensitive CaMV DNA replication. It seems therefore increasingly probable that CaMV codes for its own polymerase.

Changes in the organization of bromegrass mosaic virus in response to cation binding as probed by changes in susceptibility to degradative enzymes

The swelling at alkaline pH and the sensitivity of bromegrass mosaic virus to degradative enzymes are less in the presence of moderate concentrations of KC1 than at low salt concentration. The virus can be efficiently stabilized above neutral pH by divalent metal cations, especially Ca2+, but this effect is antagonized by KC1. Polyamines also can stabilize the virus, but the salt concentrations for optimal protection of the viral RNA and protein moieties are then different, as judged by their susceptibility to degradative enzymes. These changes in the structural organization of the virus revealed by proteases and pancreatic ribonuclease as accessibility probes complement the data from neutron-scattering studies. On the basis of these studies, a tentative model is proposed to account for the polymorphism of BMV in various ionic environments.