Actividades de Control Gerencial

The site-directed mutagenesis of the FMDV 2A region was designed to test which

residues might be important for 2A-mediated proteolysis. The activities of mutated 2A regions however were not consistent with a proteolytic activity and the results of these experiments posed more questions than answers.

The site-directed mutations were inserted in two distinct contexts. Firstly, most mutations were inserted into the 2A sequence of pMD2, which encoded the wild-type sequence (-PFFF-) immediately after the 2A cleavage site. Upon translation all of these mutations resulted largely in unprocessed [CAT2AGUS] poly protein, but also in the cleavage

product CAT2A. Only a few showed evidence of the accompanying GUS cleavage

product. A similar effect was also seen in some of the EMCV 2A mutation experiments

of Hahn and Palmenberg (1996). In both cases at least four wild-type residues followed the cleavage site. These data were, therefore, not consistent with a co-translational proteolytic cleavage hypothesis, for which both cleavage products would have been produced in equal amounts. Secondly, some mutations were made within the 2A sequence of plasmid pCAT2AGUS(3), which encoded the methionine of GUS immediately following the PT-proline of the 2A cleavage site. The polyproteins, thus encoded, yielded reduced levels of both cleavage products compared to the wild-type sequence, and as with the wild-type sequence the CAT2A product was produced in excess.

The differences between the two plasmid systems may again indicate that the context

following the 2A region is important for the "cleavage" reaction in determining the ratio of products. Since the sequence following the 2A region may not have been translated at the moment of "cleavage", either the following protein and / or RNA sequence may be implicated in this effect. Three mutated 2A sequences were made in both plasmid systems: the mutations of P5-glutamate to aspartate, and P4-serine to phenylalanine, and

the inseition of an alanine within the -KLAG- motif to give -KLAAG. The mutation of P5-glutamate to aspartate and the insertion of alanine in the 2A sequence of pMD2 both

resulted in a 2A region which was largely unprocessed but also made a small quantity of CAT2A. When made in the context of pCAT2AGUS(3) the alanine insertional mutant was completely inactive resulting in uncleaved poly protein alone, whereas the glutamate to aspartate mutant "cleaved" very poorly, also yielding an excess of CAT2A. The observation that the CAT2A "cleavage" product of the insertional mutation in pMD2 was abolished on being placed in the context of pCAT2AGUS(3) strongly supports the theory that the sequence C-terminal to 2A is important for termination or pausing of translation at the C-terminus of 2A. The mutation of P4-serine to phenylalanine showed moderate activity in both systems, although the excess of CAT2A was greater in the translation of the pMD2 derived plasmid.

The FMDV 2A region appears either to be capable of two separate activities - termination

of translation and protein cleavage - or to have a single activity which has two alternative consequences - protein termination or protein cleavage. The small size of the 2A region perhaps makes it more likely that it possesses a single activity which can result in either termination of translation or an appaient proteolytic cleavage.

The mutated sequences which were obtained are shown in Figure 4.3.1. Their "cleavage"

activities are indicated and compared with the mutations of the EMCV 2A sequence made by Hahn and Palmenberg (1996). To consider the activity of each 2A region only the

production of both CAT2A and GUS was considered as cleavage activity.

Significant cleavage activity was seen for the mutations of P4-serine to isoleucine encoded by pCAT2AGUS(3) and P4-serine to phenylalanine encoded by both

pCAT2AGUS(3) and pMD2. Hence, it can be concluded that P4-serine, (conesponding to methionine or threonine in the cardioviruses) is neither a catalytic residue nor important for formation of the active structure.

•o bû • «-H W Pui M W O Û _{W Q} JD H O T3 •O a -H •H •H •H _M T3 rH •H •H CN <N 73 .S

i/I

Mutations of the P3-asparagine residue to histidine and glutamate both resulted in 2A regions which retained a significant amount of activity. This was interesting since this

residue is entirely conserved throughout cardio- and aphthoviruses. The proposed structural function of the asparagine in its interaction with the P5-glutamate during folding (see Section 1,6) is not supported by this data, since mutation to glutamate would surely abolish this interaction. However, this data does not disprove such a theory, it merely shows that if an interaction occurs it is not a requirement for 2A activity.

Mutations of the P5-glutamate residue were also fairly active when encoded in the

plasmid pCAT2AGUS(3). Surprisingly the mutation to aspartate was less efficient than the mutation to glutamine, suggesting that the side-chain length was important for the function of this group.

The P7-aspartate residue appeared to be essential for activity with mutations to both glutamate and glutamine producing inactive 2A regions, suggesting that this residue plays a critical role, either structurally or directly in catalysis. In the mutational studies of Hahn and Palmenberg this residue was also essential for activity with neither mutation to histidine nor asparagine being active (1996).

The insertion of residues towards the N-terminus of 2A all resulted in inactive 2A regions. The insertion of proline residues, within the sequence -LLKLAG-, would have disrupted the proposed a-helical nature of the 2A region and both proline and alanine residues could have disrupted possible stabilising side-chain / side-chain interactions of P 14-aspartate and lysine or lysine and P7-aspartate. Inactivation of the 2A region by replacement of the -KLAG- motif with the cardiovims motif, -IH-, and the mutants, -HI- and -IR-, may also have prevented the side-chain interactions stabilising the proposed a-

helix. The side-chain interactions which might be possible within the FMDV 2A sequence and these mutated sequences are shown in Figure 4.3.2.