Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.
The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.
Robertson
B J
BJ
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492-7660, USA.
McCann
P J
PJ
Matusick-Kumar
L
L
Newcomb
W W
WW
Brown
J C
JC
Colonno
R J
RJ
Gao
M
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D005816:Genetic Complementation Test; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D014764:Viral Proteins; D014779:Virus Replication
D017931:DNA Primers; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D012697:Serine Endopeptidases