Andrew J. Maule

Remorin, a Solanaceae Protein Resident in Membrane Rafts and Plasmodesmata, Impairs Potato virus X Movement

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in ∼70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

Identification of the initiation codons for translation of cowpea mosaic virus middle component RNA using site-directed mutagenesis of an infectious cDNA clone

A full-length cDNA copy of CPMV M RNA has been cloned downstream of a phage lambda promoter in the plasmid pPMI. Transcripts obtained from this clone can be translated in vitro and replicated in cowpea mesophyll protoplasts in the presence of viral B RNA. We have constructed a series of site-directed mutants of this clone to investigate the mechanism of translation of CPMV M RNA. The results obtained confirm that the AUG at position 161 is used to direct the synthesis of a 105K protein in vitro and the detection of a 58K protein in infected cowpea protoplasts suggests that it is also used in vivo. The synthesis of the 95K protein can be initiated from either of the AUGs at positions 512 and 524, though synthesis of this protein does not appear to be essential for CPMV replication in protoplasts.

Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.

The pattern of accumulation of cauliflower mosaic virus-specific products in infected turnips

The concentrations of cauliflower mosaic virus (CaMV) DNA and protein products in the developing leaves of a host, turnip, have been measured and the results have been correlated with symptom production. Virus-specific products were limited to the symptomatic leaves. CaMV DNA was detected in the youngest foliar tissues showing full systemic symptoms and continued to accumulate as the leaf expanded, indicating that virus multiplication was not restricted to meristematic tissues of the host plant and that virus concentration was not a primary determinant for symptom production. Using specific antisera for Western blot analysis, the distribution of CaMV-specific proteins (P1-P6) in a range of subcellular fractions of infected tissue was determined. The protein products (P2-P6) of genes II-VI were all detected in fractions enriched for virus inclusion bodies, although P5 was present only at low levels. A high-speed pellet fraction enriched for virus replication complexes revealed P5 in higher concentrations, and also contained P4 and small amounts of P6 in proportions which indicated that replication complexes had been released from inclusion bodies. In the different leaves of the host, P2, 3, 4, 5, and 6 all increased in concentration in parallel with viral DNA, although there appeared to be a bias toward protein rather than DNA synthesis in the very young leaves. P1 showed a different pattern of accumulation; it was most concentrated in the very young and the oldest infected tissues, and showed a different spectrum of products between leaves. The experiments described provide a more complete picture of the relationship between CaMV multiplication and expression, and leaf development, and an increased understanding of how the disease syndrome is established.

Identification and subcellular localization of a putative cell-to-cell transport protein from red clover mottle virus

To investigate the mode of gene expression of red clover mottle virus (RCMV) middle component (M) RNA, we have synthesized an oligopeptide corresponding to the predicted carboxy-terminus of the RCMV counterparts of the cowpea mosaic virus (CPMV) 48K and 58K proteins. Using an antiserum raised against this synthetic oligopeptide, we have detected a 43-kDa protein in the 30,000 g pellet from extracts of RCMV-infected cowpea protoplasts. Immunogold cytochemistry further localized this protein to the plasmodesmata of RCMV-infected pea tissue. This subcellular location, taken together with other evidence, suggests that this 43-kDa protein has a role in the cell-to-cell spread of RCMV.

Cauliflower mosaic virus aphid transmission factor protein is expressed in cells infected with some aphid nontransmissible isolates

Abstract An antibody raised to the gel-purified product (p18) of cauliflower mosaic virus (CaMV) open reading frame (ORF) II was used in conjunction with sensitive serological techniques to detect p18 in inclusion body preparations from turnip tissue infected with the aphid transmissible (AT + ) isolate Cabb B-JI. p18 was not detected when tissue was infected with the aphid nontransmissible (AT − ) isolate CM4-184 that contains a large deletion in ORF II. However, inclusion body preparations from tissue infected with the AT − isolates Campbell and CM1841 contained p18. Hence, the AT phenotype is not directly correlated with the presence or absence of the ORF II product, as was previously indicated when less sensitive techniques were used for analysis.

Independent replication of cowpea mosaic virus bottom component RNA: In vivo instability of the viral RNAs

We compared the accumulation of cowpea mosaic virus (CPMV) RNAs after inoculation of cowpea and Chenopodium quinoa protoplasts with bottom (B) or middle (M) component virions alone, or with a mixture of the M and B components. RNA extracted from infected protoplasts was subjected to quantitative spot hybridization after electrophoresis and blotting. Compared to the complete M plus B mixture, the B inoculum induced (1) a reduced rate and extent of accumulation of B-RNA of both the (+) and (-) polarities concomitant with a balance between synthesis and degradation and (2) B-RNA that decreased in amount after further RNA synthesis was prevented by administration of cordycepin. In contrast, the (-) B-RNA apparently was stable, possibly sequestered in double-stranded form. M-component-inoculated cowpea protoplasts were subsequently inoculated with B component at intervals, and, after further incubation, RNA was analyzed by electrophoresis and hybridization. Rescue of the ability to form M-RNAs was lost when the interval between inoculations was greater than 17 hr, although the protoplasts remained sensitive to the complete inoculum. The data suggest that both of the genomic RNAs are unstable in protoplasts when inoculated alone, i.e., under conditions that leave viral RNA unprotected by coat protein.

Partial characterisation of different classes of viral DNA, and kinetics of DNA synthesis in turnip protoplasts infected with cauliflower mosaic virus

SummaryTurnip protoplasts infected with cauliflower mosaic virus (CaMV) have been used to examine the kinetics of CaMV DNA synthesis, and the different classes of CaMV DNA found in vivo partially characterised. Differential extraction techniques for DNA from infected protoplasts has identified several distinct classes of viral DNA. The same approach applied to virus preparations revealed that while the majority of virion DNA was stably encapsidated, some small DNAs and a heterogeneous population 3.8-ca. 5.0 Kb were not. The structural relationship of sa-DNA (3) with the particle is such that only its 5′ RNA moeity is susceptible to nuclease attack. Two-dimensional gel electrophoresis of total CaMV DNA from infected protoplasts revealed all the DNA species found in virion DNA, those species representing the ‘free’ DNA class and a further class of molecules, rich in DNA of (−) polarity (24), to which the role of reverse transcription intermediates has been ascribed. ‘Free’ DNA contains 8 Kb supercoiled DNA (Form I DNA), an 8 Kb open circle (Form II), an 8 Kb linear (Form III) and a truncated molecule with an extension of the (−) strand previously observed from infected plants (10). Kinetic experiments show that the accumulation of total CaMV-DNA parallels the accumulation of progeny virions to reach a maximum around 72 h post-inoculation and that there is not a separation of CaMV-DNA synthesis into clearly defined early and late stages.

The Stability of Cowpea Mosaic Virus VPg in Reticulocyte Lysates

SummarynThe ability of the genome-linked protein (VPg) of cowpea mosaic virus (CPMV) to survive incubation in rabbit reticulocyte lysates was investigated. In contrast to the results obtained with picornavirus RNAs, there was no evidence for the specific removal (‘unlinking’) of the VPg from CPMV RNA during incubation. While linked to RNA, CPMV VPg was protected from proteolytic degradation; if the RNA was first digested with nuclease P1, rapid degradation of the VPg occurred. However if as few as 17 nucleotides were left attached to the VPg, stability was retained.