The multifunctional capsid proteins of plant RNA viruses.

This article summarizes studies of viral coat (capsid) proteins (CPs) of RNA plant viruses. In addition, we discuss and seek to interpret the knowledge accumulated to data. CPs are named for their primary function; to encapsidate viral genomic nucleic acids. However, encapsidation is only one feature of an extremely diverse array of structural, functional, and ecological roles played during viral infection and spread. Herein, we consider the evolution of viral CPs and their multitude of interactions with factors encoded by the virus, host plant, or viral vector (biological transmission agent) that influence the infection and epidemiological facets of plant disease. In addition, applications of today's understanding of CPs in the protection of crops from viral infection and use in the manufacture of valuable compounds are considered.

A single amino acid mutation in the carnation ringspot virus capsid protein allows virion formation but prevents systemic infection.

A Carnation ringspot virus (CRSV) variant (1.26) was identified that accumulates virions but is incapable of forming a systemic infection. The 1.26 capsid protein gene possesses a Ser-->Pro mutation at amino acid 282. Conversion of 1.26 amino acid 282 to Ser restored systemic infection, while the reciprocal mutation in wild-type CRSV abolished systemic infection. Similar mutations introduced into the related Red clover necrotic mosaic virus capsid protein gene failed to induce the packaging but nonsystemic movement phenotype. These results provide additional support for the theory that virion formation is necessary but not sufficient for systemic movement with the dianthoviruses.

Spontaneous deletion enhances movement of a cucumber necrosis virus based chimera expressing the red clover necrotic mosaic virus movement protein genedagger.

Summary The 35-kDa movement protein (MP) gene of red clover necrotic mosaic virus (RCNMV) and 3' flanking sequence were inserted in a cucumber necrosis virus (CNV) deletion mutant lacking a large portion of the coding region for the MP. Nicotiana benthamiana plants inoculated with chimeric synthetic transcripts of the resulting hybrid cDNA clone (M5/RM2) developed both local and systemic symptoms and accumulated high levels of chimeric viral RNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and sequence analysis of viral RNA extracted from systemically infected leaves of four different plants revealed that in each plant a large portion (305, 308, 315 or 127 nts) of the 3' terminus of the inserted sequence spontaneously deleted during infection. In three of the deletion derivatives, the truncated RCNMV MP open reading frame (ORF) was fused in-frame with the remaining portion of the 3' terminal region of CNV MP ORF. The movement efficiencies of M5/RM2, a cloned copy of one of the deletion derivatives (ClM5/RM2dd1), and a stop codon mutant of ClM5/RM2dd1 (ClM5/RM2dd1stop), which prevents translational fusion to the CNV MP, were compared and it was determined that deletion of RCNMV MP sequences in conjunction with fusion to CNV MP sequences increases the movement efficiency of the chimeric virus genome. Absence of the C-terminal region of the RCNMV MP in RCNMV RNA-2 abolished RCNMV movement. However, movement could be complemented in trans if cells were coinoculated with ClM5/RM2dd1. Complementation of RCNMV movement did not occur using ClM5/RM2dd1stop, suggesting a role for appended CNV MP sequences in movement of the RCNMV genome. The ability of the CNV replicase to delete unnecessary or deleterious RCNMV sequences and to append the required CNV MP sequences reinforces the role of RNA recombination in the adaptation and evolution of viral genomes.

RNA-mediated trans-activation of transcription from a viral RNA.

The red clover necrotic mosaic virus genome is composed of two single-stranded RNA components, RNA-1 and RNA-2. The viral capsid protein is translated from a subgenomic RNA (sgRNA) that is transcribed from genomic RNA-1. Here, a 34-nucleotide sequence in RNA-2 is shown to be required for transcription of sgRNA. Mutations that prevent base-pairing between the RNA-1 subgenomic promoter and the 34-nucleotide trans-activator prevent expression of a reporter gene. A model is proposed in which direct binding of RNA-2 to RNA-1 trans-activates sgRNA synthesis. This RNA-mediated regulation of transcription is unusual among RNA viruses, which typically rely on protein regulators.

Mutational analysis of the cucumber necrosis virus coat protein gene.

A series of frameshift, deletion, and inversion mutations were made in the coat protein (CP) gene of the icosahedral cucumber necrosis tombusvirus (CNV) to investigate the role of the CP protruding (P) domain in the production of virus particles and, also, to investigate the basis for the accumulation of CP deletion derivatives previously reported in plants inoculated with PD(-), a P-domainless CNV CP mutant. P-domainless coat protein subunit could be detected in extracts of CP mutant-infected plants; however, virus-like particles could not, suggesting that the P domain is essential for tombusvirus particle assembly and/or stability. In addition, each of the P-domain mutants analyzed invariably produced coat protein deletion derivatives in infected plants whereas shell domain mutants rarely produced deletion derivatives. Finally, P-domain inversion and deletion mutants accumulated deletion derivatives very rapidly in comparison to P-domain frameshift mutants. Protoplast studies show that PD(-) RNA inoculum does not undergo further deletion in infected protoplasts, suggesting that PD(-) CP deletion derivatives preferentially accumulate in plants because they have a greater capacity for cell-to-cell movement.

Coat protein of cucumber necrosis virus is not required for efficient generation or accumulation of defective interfering RNAs.

It is generally assumed that defective interfering (DI) forms of viruses are encapsidated in structural proteins encoded by the helper virus. Virion RNA extracts from cucumber necrosis virus (CNV) infections showing high levels of cellular DI RNAs contain barely detectable levels of DI RNAs, suggesting that DI RNAs are encapsidated very inefficiently. In addition, accumulation of CNV DI RNAs occurs at equal efficiency in coinoculated plants using either synthetic wild-type CNV genomic RNA as helper or a mutant of CNV which lacks the coat protein-coding sequence. Together this shows that the CNV coat protein is not required for efficient accumulation of CNV DI RNA in plants. Factors that could account for the high level of CNV DI RNAs in plants are discussed.

The minimal 5' sequence for in vitro initiation of papaya mosaic potexvirus assembly.

The minimal 5' initiation of assembly sequence for papaya mosaic virus (PMV) has been determined by the generation of successive 3' deletion clones from a full-length cDNA copy of the PMV genome followed by in vitro assembly of RNA transcripts. Sequences closest to the 5' terminus were cloned into plasmid vectors with synthetic complementary oligonucleotides representing viral sequences. The in vitro initiation sequence has been narrowed down to 38-47 nucleotides at the 5' terminus which are capable of initiation alone or in conjunction with homologous or heterologous sequences. The minimum initiation sequence is rich in adenosine and cytidine and very poor in uridine nucleotides and lacks any discernible secondary structure.

Infectious RNA transcripts derived from cloned cDNA of papaya mosaic virus: effect of mutations to the capsid and polymerase proteins.

Genomic length cDNAs of papaya mosaic virus (PMV) RNA were generated utilizing reverse transcriptase (RNase H-) for first strand synthesis, Sequenase for second strand synthesis and primers specific for the 5' and 3' termini of the viral genome. These cDNAs were cloned into plasmid pUC18 and infectious RNA transcripts were synthesized in vitro from a bacteriophage T7 RNA polymerase promoter incorporated into the 5' specific primer. The infectivity of transcripts was 16% that of native PMV RNA. Increasing the poly(A) tail length from A24 to A71 produced a 43% increase in infectivity. Transcripts synthesized with or without an m7GpppG cap structure were biologically active although uncapped transcripts were much less infectious. The addition of up to 2434 non-viral nucleotides at the 3' end of transcripts decreased but did not abolish infectivity. Insertions of two amino acid residues within the polymerase coding region inactivated viral transcripts. A single amino acid deletion within the capsid protein (CP) produced local lesions of a reduced size as compared to native PMV RNA. Viral particles could not be observed in crude extracts from lesions produced by this deletion mutant suggesting that it exists as a naked RNA species within the host. Mutations to the CP suggest that it is required not only for viral assembly but also for some other unidentified function(s) during the replication cycle.

Cloning and sequence determination of the valS gene, encoding valyl-tRNA synthetase in Lactobacillus casei.

The DNA sequence of the valS gene from Lactobacillus casei and the predicted amino acid sequence of its valyl-tRNA synthetase product have been determined. An open reading frame coding for a protein of 901 amino acids was found. A clone containing the intact L. casei valS gene functionally complemented the temperature-sensitive growth of the valS mutant strain 236c of Escherichia coli. The valS gene and the downstream folylpolyglutamate synthetase gene are transcribed in the same direction but are separated by a putative transcription terminator.

Complete nucleotide sequence of clover yellow mosaic virus RNA.

The entire genomic RNA of clover yellow mosaic virus was sequenced from cDNA clones and run-off cDNA transcripts. The genomic RNA is 7015 nucleotides in length [excluding a 3' poly(A) tail], with six open reading frames (ORFs) greater than 150 nucleotides in length. The first five ORFs encode proteins of Mr 191K, 26K, 12K, 6.5K and 28K, respectively. The sixth ORF lies completely within ORF1 and codes for a protein of Mr 14K. The capsid protein coding region (Mr 23K) is found within ORF5 which encodes the Mr 28K protein. Proteins encoded by ORFs 1 to 3 and ORF5 show strong homology with proteins of other potexviruses, especially papaya mosaic virus.

Highly sensitive and specific non-radioactive biotinylated probes for dot-blot, Southern and colony hybridizations.

We describe a simple method for the incorporation of biotin into nucleic acid probes. This method has been improved and optimized to produce biotinylated DNA probes for the detection of DNA by dot-blot, Southern and colony hybridization techniques. The sensitivity of this method has been particularly improved to allow detection of DNA quantities under one femtogram. Probes prepared by this method are highly specific for target DNA even in crude bacterial lysates.

Nucleotide sequence of papaya mosaic virus RNA.

The RNA genome of papaya mosaic virus is 6656 nucleotides long [excluding the poly(A) tail] with six open reading frames (ORFs) more than 200 nucleotides long. The four nearest the 5' end each overlap with adjacent ORFs and could code for proteins with Mr 176307, 26248, 11949 and 7224 (ORFs 1 to 4). The fifth ORF produces the capsid protein of Mr 23043 and the sixth ORF, located completely within ORF1, could code for a protein with Mr 14113. The translation products of ORFs 1 to 3 show strong similarity with those of other potexviruses but the ORF 4 protein has only limited similarity with the other potexvirus ORF 4 proteins of 7K to 11K.