RESUMO
The site of cleavage of the small coat protein of cowpea mosaic virus has been precisely mapped and the proteolysis has been shown to result in the loss of 24 amino acids from the carboxyl-terminus of the protein. A series of premature termination and deletion mutants was constructed to investigate the role or roles of these carboxyl-terminal amino acids in the viral replication cycle. Mutants containing premature termination codons at or downstream of the cleavage site were viable but reverted to wild-type after a single passage through cowpea plants, indicating that the carboxyl-terminal amino acids are important. Mutants with the equivalent deletions were genetically stable and shown to be debilitated with respect to virus accumulation. The specific infectivity of preparations of a deletion mutant (DM4) lacking all 24 amino acids was 6-fold less than that of a wild-type preparation. This was shown to be a result of DM4 preparations containing a much increased percentage (73%) of empty (RNA-free) particles, a finding that implicates the cleavable carboxyl-terminal residues in the packaging of the virion RNAs.
Assuntos
Capsídeo/fisiologia , Comovirus/genética , Comovirus/fisiologia , RNA Viral , Montagem de Vírus , Sítios de Ligação , Capsídeo/genética , Fabaceae/virologia , Espectrometria de Massas , Mutagênese , Fenótipo , Plantas Medicinais , Processamento de Proteína Pós-TraducionalRESUMO
Epitopes from human rhinovirus 14 (HRV-14) and human immunodeficiency virus type (HIV-1) have been expressed on the surface of particles of the plant virus, cowpea mosaic virus (CPMV). The chimaeras retain their ability to grow in plants and large quantities of virions can be easily purified. Immunological studies have shown that purified particles have the antigenic properties of the insert, and, in the case of the HIV-1 chimaera, can elicit the production of neutralising antibodies in mice. The chimaera containing the epitope from HRV-14 has been crystallised and the crystals shown to diffract to atomic resolution.
Assuntos
Vacinas contra a AIDS/biossíntese , Comovirus/genética , Vetores Genéticos , HIV-1/imunologia , Rhinovirus/imunologia , Vacinas Sintéticas/biossíntese , Vacinas Virais/biossíntese , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Comovirus/imunologia , Comovirus/patogenicidade , Comovirus/ultraestrutura , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Pisum sativum/virologia , Rhinovirus/genética , Vacinas Sintéticas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/imunologiaRESUMO
It has recently been shown that cowpea plants can be infected with a cowpea mosaic virus (CPMV) chimera containing an antigenic site from foot-and-mouth disease virus (Usha et al., Virology 197, 366-374, 1993). Analysis of progeny RNA produced during such an infection has revealed that the inserted sequence is rapidly lost during serial passaging, probably by a process of homologous recombination. Using the information gained from this analysis, we have redesigned the chimeras in such a way that they are now genetically stable. The modified constructs have been used to obtain large quantities of purified virus particles expressing epitopes derived from human rhinovirus 14 (HRV-14) and human immunodeficiency virus type 1 (HIV-1). The chimeric virus particles possess the antigenic properties of the inserted sequence and, in the case of the HRV-14-derived construct, it has been shown that the inserted epitope is immunogenic in rabbits. These results demonstrate that CPMV can be used as a high-yielding system for the presentation of foreign peptide sequences.
Assuntos
Antígenos Virais/imunologia , Comovirus/imunologia , Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Aphthovirus/genética , Aphthovirus/imunologia , Sequência de Bases , Capsídeo/química , Capsídeo/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Dados de Sequência Molecular , Rhinovirus/imunologiaRESUMO
To investigate if cowpea mosaic virus (CPMV) particles can be used to express foreign protein sequences, oligonucleotides encoding an epitope derived from VP1 of foot-and-mouth disease virus (FMDV) were cloned into the region of the CPMV genome encoding the small (S) coat protein. The chimeras were designed so that the foreign sequence was expressed either as an insertion or as a replacement for part of the wild-type sequence. While RNA from both chimeras was able to replicate in cowpea protoplasts only the construct containing the FMDV sequence as an insertion was able to direct capsid formation and infect whole cowpea plants. The modified S protein produced in plants infected with the insertion derivative reacted with FMDV-specific antiserum. These results show that CPMV can be used as an antigen presentation system and raises the possibility of producing vaccines in plants using a RNA virus-based vector.
