Parameters of controlled expression of potentially toxic recombinant proteins in<i>E. coli</i>with picornaviral 3ABC as a model

M, Uma; Mallavarapu, Showmeya; Nagalekshmi, Karthika; Rao, P.P.; Hegde, Nagendra R.

doi:10.5958/0974-0147.2016.00002.7

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“…During this process, we attempted to express the PV 3ABC protein in prokaryotic systems. Possibly due to the toxicity of 3C protease, we encountered either low or no expression of the protein (Uma et al, 2016a). However, these experiments led to the serendipitous discovery of a novel mutation adjacent to the catalytic domain of 3C, inactivating its enzymatic activity, and resulting in abortive infection of susceptible cells by PV.…”

Section: Introductionmentioning

confidence: 99%

A novel point mutation (L70P) inactivates poliovirus 3C protease

Uma¹,

Hegde²,

Rao³

et al. 2018

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Poliovirus (PV) contains a single-stranded positive-sense RNA genome, which is translated into a single polyprotein. Viral proteases process this polyprotein to produce several individual as well as fused proteins. The major viral protease 3C cleaves at nine of the eleven cleavage sites. During the process of expressing PV 3ABC protein in Escherichia coli, we identified a 3C mutant (L70P), which lost its protease activity. This loss of function was confirmed by generating recombinant adenoviruses expressing mutant and wild-type 3C. Further, infectious PV could not be recovered from PV full-length cDNA containing the L70P mutation. However, 3C L70P mutant cDNA could complement a PV cDNA containing a 1AB deletion, producing a viable virus population containing defective complementing genomes. Structural analysis of the mutant protein indicated that the L70P mutation resulted in the loss of a hydrogen bond between two residues located within a loop between two β-sheets, potentially leading to strain on the catalytic site. We conclude that L70P inactivates 3C protease because of its close proximity to the 3C catalytic site.

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Section: Introductionmentioning

confidence: 99%