Herod, Morgan R., Jones, Daniel M., McLauchlan, John and McCormick, Christopher J.
Increasing the rate of cleavage at the boundary between non-structural protein 4B and 5A inhibits replication of hepatitis C virus
The Journal of Biological Chemistry, 287, . (doi:10.1074/jbc.M111.311407).
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In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. While proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However it is not known whether slow cleavage is important for viral replication, or a consequence of restrictions on sequences which can be tolerated at the cleaved ends of non-structural
proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary and their effect on replication and polyprotein processing examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon
degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall the data indicate that slow cleavage of the NS4B5A boundary is important, and identify a previously unrecognised role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.
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