Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein.
The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f. To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli. The expressed protease underwent autoproteolytic processing at two independent sites. The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease. The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus. A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria. The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria. This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease. ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E. coli. This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus.
Deckman
I C
IC
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Hagen
M
M
McCann
P J
PJ
D015153:Blotting, Western; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D007700:Kinetics; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013997:Time Factors; D014764:Viral Proteins / Q000378:metabolism*
C054582:ICP35 protein, Human herpesvirus 1; D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases