Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles.
The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.
Weinheimer
S P
SP
Virology Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
McCann
P J
PJ
O'Boyle
D R
DR
Stevens
J T
JT
Boyd
B A
BA
Drier
D A
DA
Yamanaka
G A
GA
DiIanni
C L
CL
Deckman
I C
IC
Cordingley
M G
MG
D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002213:Capsid / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013917:Thrombin / Q000378:metabolism; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D012694:Serine; D010450:Endopeptidases; D013917:Thrombin; D000409:Alanine