Engineering Cowpea Mosaic Virus RNA-2 into a Vector to Express Heterologous Proteins in Plants

AbstractA series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP and L proteins was achieved by creating artificial processing sites each side of the insert, either by duplicating the MP-L cleavage site or by introducing a sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. Eight amino acids derived from the C-terminus of the MP and 14–19 amino acids from the N-terminus of the L coat protein were necessary for efficient processing of the artificial Gln/Met sites. Insertion of the FMDV 2A sequence at the C-terminus of the GFP resulted in a genetically stable construct, which produced particles containing about 10 GFP-2A-L fusion proteins. Immunocapture experiments indicated that some of the GFP is present on the virion surface. Direct fusion of GFP to the C-terminus of the S coat protein resulted in a virus which was barely viable. However, when the sequence of GFP was linked to the C-terminus by an active FMDV 2A sequence, a highly infectious construct was obtained

Determination of the Proteolytic Processing Sites in the Polyprotein Encoded by the Bottom-Component RNA of Cowpea Mosaic Virus

The bottom-component RNA (B-RNA) of cowpea mosaic virus is expressed by the production of a ∼200,000-dalton polyprotein (200K polyprotein), from which the functional proteins are formed by specific proteolytic cleavages. Partial amino-terminal sequences of the various B-RNA-encoded proteins have now been determined. Comparison of the information obtained with the B-RNA sequence allowed the localization of the coding regions for these proteins on B-RNA, the calculation of their precise molecular weights, and the determination of the cleavage sites at which they are released from the polyprotein precursor. Sequence analysis of the 32K protein, which is derived from the amino-terminal end of the 200K polyprotein, indicated that the AUG codon at nucleotide position 207 of the RNA sequence is the translation initiation codon. Sequence analysis of the 170K, 110K, 87K, 84K, 60K, and 58K proteins revealed the existence of three types of cleavage site in the 200K polyprotein: glutamine-serine (two sites), glutamine-methionine (one site), and glutamine-glycine (one site) amino acid pairs. The nature of these cleavage sites suggested that two different viral proteases are involved in the processing of the B-RNA-encoded polyprotein

Cowpea Mosaic Virus 32- and 60-Kilodalton Replication Proteins Target and Change the Morphology of Endoplasmic Reticulum Membranes

Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis

A web-based adaptive tutor to teach PCR primer design

When students have varying prior knowledge, personalized instruction is desirable. One way to personalize instruction is by using adaptive e-learning to offer training of varying complexity. In this study, we developed a web-based adaptive tutor to teach PCR primer design: the PCR Tutor. We used part of the Taxonomy of Educational Objectives (the three cognitive processes: remember, understand, and apply) to design exercises of varying complexity. Using this method, we demonstrated that we were able to systematically categorize exercises. There was also a good learning effect and a positive student perception when using the PCR Tutor