Aphid transmission of cauliflower mosaic virus requires the viral PIII protein

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids.     

Expression of the cauliflower mosaic virus capsid gene in vivo

Antisera against the N-terminal and C-terminal parts of the potential ORF IV product were used to analyse extracts from CaMV-infected turnip leaves by immunoblotting. Polypeptides of 87, 83, 82, 60 and 57 kDa were detected. The origin of these proteins is discussed.     

Mutagenesis of cauliflower mosaic virus

A series of insertion mutants of cauliflower mosaic virus (CaMV) DNA has been constructed in vitro. These insertions consist of a short DNA sequence (10 or 22 bp) containing a restriction endonuclease site (SmaI) not represented on the viral DNA. Viral infectivity was analyzed by inoculating plants with the mutated cloned viral DNA and observing symptoms. Insertions within ORFVII, and in one site within the large intergenic region, did not interfere with viral infectivity, whilst insertions within ORFII and at the end of ORFIV retarded the development of viral symptoms. All other insertion mutants analyzed were lethal. CaMV with a deletion of 105 bp within ORFVII was viable. Such viable mutants can be used to construct additional deletions or to insert foreign DNA into the viral genome.     

Expression of cauliflower mosaic virus gene I in Saccharomyces cerevisiae

Cauliflower mosaic virus (CaMV) gene I encodes a 40-kDa protein, P1, which is thought to be involved in the cell-to-cell movement of the virus. In order to investigate its functioning, P1 was expressed in Saccharomyces cerevisiae transformed by an expression vector containing CaMV gene I. When produced in yeast, P1 was 40 kDa in size and not N-glycosylated.     

Insertional mutagenesis of the cauliflower mosaic virus genome

A series of small insertions has been introduced into the various translational reading frames of the DNA of a "severe" strain of cauliflower mosaic virus (CaMV). A selectable gene (the kanamycin phosphotransferase gene of Tn903), flanked by a series of symmetrically arranged cloning sites taken from M13mp7, was used to prepare the site-specific mutants. In-phase insertions of 12 or 30 bp, which introduced unique SalI sites into reading regions I, III, IV, V and into the amino-proximal portion of region VI, destroyed infectivity. Insertions in the amino-distal portion of region VI, in the large intergenic region, and in region II retained infectivity. The amino-distal insertions in region VI reduced the severity of symptoms in plants. The insertion in region II destroyed aphid transmissibility. Longer DNA segments when inserted into region II or into the amino-distal portion of region VI destroyed infectivity, but similar insertions in the intergenic region were without effect on virus infection or development.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

Nucleotide sequence of cauliflower mosaic virus DNA

The complete nucleotide sequence (8024 nucleotides) of the circular double-stranded DNA of cauli-flower mosaic virus has been established. The DNA molecule is known to possess three discrete single-stranded discontinuities, often referred to as “gaps”, two in one strand and one in the other. The sequence data indicate that gap 1, the single discontinuity in the α strand, corresponds to the absence of no more than one or two nucleotides with respect to the complementary β strand. The two discontinuities in the β strand, however, are not authentic gaps since no nucleotides are missing, but are instead regions of sequence overlap: a short sequence (19 residues for gap 2, at least 2 residues for gap 3) at one terminus of each discontinuity, probably the 5′ terminus, is displaced from the double helix by an identical sequence at the other boundary of the discontinuity. Analysis of the distribution of nonsense codons in the DNA sequence is consistent with other evidence that only the α strand is transcribed. The coding region extends around the circular molecule from 4 map units of gap 1, the map origin, to map position 91, and consists of six long open reading frames. Our findings suggest, but do not prove, that the DNA sequence of the open reading frames is colinear with viral protein sequences. The cistron for the viral coat protein, which is probably synthesized in the form of a precursor, has been situated in coding region IV on the basis of its unusual amino acid composition.     

The cauliflower mosaic virus virion-associated protein is dispensable for viral replication in single cells

Cauliflower mosaic virus (CaMV) open reading frame III (ORF III) codes for a virion-associated protein (Vap), which is one of two viral proteins essential for aphid transmission. However, unlike the aphid transmission factor encoded by CaMV ORF II, Vap is also essential for systemic infection, suggesting that it is a multifunctional protein. To elucidate the additional function or functions of Vap, we tested the replication of noninfectious ORF III-defective mutants in transfected turnip protoplasts. PCR and Western blot analyses revealed that CaMV replication had occurred with an efficiency similar to that of wild-type virus and without leading to reversions. Electron microscopic examination revealed that an ORF III frameshift mutant formed normally structured virions. These results demonstrate that Vap is dispensable for replication in single cells and is not essential for virion morphogenesis. Analysis of inoculated turnip leaves showed that the ORF III frameshift mutant does not cause any detectable local infection. These results are strongly indicative of a role for Vap in virus movement.     

A new strategy to improve a cauliflower mosaic virus vector

Co-infection of plants with non-overlapping deletion mutants of cauliflower mosaic virus usually leads to the production of the wild-type virus. To prevent this, a pair of mutants with overlapping deletions was constructed. In infected plants both mutant DNAs were stably maintained. Such mutants with overlapping deletions will be used as a vector to overcome the size limitation of genes to be cloned.     

Molecular dissection of the cauliflower mosaic virus translation transactivator.

The cauliflower mosaic virus (CaMV) transactivator (TAV) is a complex protein that appears to be involved in many aspects of the virus life cycle. One of its roles is to control translation from the polycistronic CaMV 35S RNA. Here we report a molecular dissection of TAV in relation to its ability to enhance dicistronic translation in transient expression experiments. We have identified a protein domain that is responsible and sufficient for that activity. This 'MiniTAV domain' consists of only 140 of the 520 amino acids in the full-length sequence. A further domain located outside the MiniTAV, and therefore dispensable for transactivation, is probably involved in interactions with other molecules. This was identified by its ability to compete with wild-type TAV and some of its deletion mutants. We found, furthermore, that the TAV protein binds RNA. Two regions needed for RNA-binding properties were defined outside the MiniTAV domain and RNA binding seems not to be directly involved in the transactivation mechanism.     

Studies on mutagenesis and cross-protection of cauliflower mosaic virus

Three strains of cauliflower mosaic virus (CaMV) designated NVRS, CM4-184 and PK caused respectively severe, moderate and mild reactions in turnip cv. Just Right plants and severe, mild and symptomless reactions in Brussels sprout cv. Fasolt plants. Chlorotic local lesions formed consistently in the leaves of young turnip plants when inoculated with each of the virus strains. Lesions were suitable for infectivity assay of crude and purified preparations of the virus. Three variants of the NVRS strain were isolated by single-lesion transfer after treatment of the virus with nitrous acid (pH 5.0) and two variants were obtained after treating the virus in acetate buffer at the same pH. One of the variants (designated V3) caused symptomless infection in turnip and Brussels sprout plants. In cross-protection tests, Brussels sprout plants infected symptomlessly with the PK, CM4-184 or the V3 strains, subsequently resisted infection by the severe NVRS strain.