Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites

Baum, Ellen Z.; Bebernitz, Geraldine; Hulmes, Jeffrey D.; Muzithras, V P; Jones, Thomas R.; Gluzman, Y

doi:10.1128/jvi.67.1.497-506.1993

Cited by 85 publications

(101 citation statements)

References 32 publications

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“…Several reports have suggested that the HCMV protease belongs to either the serine or cysteine class of proteases [5,8,101 because the HCMV protease is sensitive to reagents which modify serine or cysteine residues [ S , 8, 10, 111. To further characterize these modifications biochemically, purified HCMV protease was incubated with inactivators of serine and cysteine proteases and then tested in assays with either protein or peptide substrates.…”

Section: Hsvl L Q a S E K F K M W G A L V N A S S A A H V D V Hcmv S mentioning

confidence: 99%

“…encoded by the UL80 open reading frame, which is 3' coterminal with the gene encoding the preassembly protein [5, 8,91. Consequently, the 373 amino acids of the preassembly protein are identical to the carboxy-terminal 373 amino acids of the protease [9] and the protease is capable of autoproteolytic processing its own carboxy terminus at a site identical to that of its substrate (maturational site) between Ala643 and Ser644.…”

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confidence: 99%

“…The HCMV protease also undergoes autoproteolytic processing at a site (release) closer to its amino-terminus [ 5 , 81 between Ala256 and Ser257. Studies have demonstrated that the catalytic domain of the protease is contained in the first 256 amino acids of the protease [5,8,10,111, and is released by the release cleavage. A third autoprocessing site has been identified in the catalytic domain of HCMV protease, at Ala143-Ala144 and is proposed to inactivate the enzyme [8,11,121.…”

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confidence: 99%

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In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

Stevens

Mapelli

Tsao

et al. 1994

European Journal of Biochemistry

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Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein. Two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). Identification of rP5-P4' and mP4-P6' as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV. Kinetic parameters determined for release-site-derived and maturation-site-derived peptides revealed a 10-fold increase in k,,JK,, for a maturational peptide (mP4-PS') over release-site peptide (rP5-P5'). Experimental results with a panel of class-specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases. Further investigation revealed that the HCMV protease activity decreased with incorporation of ['4C]iodoacetic acid, but when approximately 4.5 mol I4C were incorporatedmol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile. Analysis of diisopropyl-fluorophosphate-inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile. The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 'H-labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation. This residue and the region in which it is found is highly conserved among the herpes virus proteases. These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active-site nucleophile.Members of the herpes virus family, including human cytomegalovirus (HCMV) and HSV-1, encode an assembly protein which is a major component of intermediate B-capsids. The assembly protein is only transiently associated with virus particles, and is absent from mature virions [l -31. As a consequence, the assembly protein is proposed to play a role in virus maturation analogous to that of the scaffolding protein of bacteriophages [4]. During infection, the HCMV preassembly protein undergoes proteolytic processing to form the assembly protein, a lower molecular-mass species that lacks 64 amino acids at the carboxy terminus [5]. Mutants of HSV-1, defective in processing the HSV-1 assembly protein, form aberrant empty capsids and fail to package DNA, indicating that proteolytic processing of the virus assembly protein is critical for herpes virus particle maturation [6, 71.The protease responsible for processing the preassembly protein during HCMV infection consists of 708 amino acids Abbreviations. HCMV, human cytomegalovirus; iPr,PF, diisopropylfluorophosphate; ESI, electrospray ionization; GST, glutathione S-transferase; HSV-1. herpes simplex virus type 1; SCMV, simian cytomegalovirus. encoded by the UL80 open reading frame, which is 3' co...

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Section: Hsvl L Q a S E K F K M W G A L V N A S S A A H V D V Hcmv S mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

Stevens

Mapelli

Tsao

et al. 1994

European Journal of Biochemistry

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“…Although genetic and functional analyses clearly demonstrate that N o , consisting of the first 247 aa of Pra, contains the catalytic domain (18)(19)(20)(21), the enzymatic activity of the protease can be independent of capsid assembly (5, 6, 20-22, 24, 43). Autoprocessing and trans cleavage of cytomegalovirus protease have been extensively studied elsewhere (2,45,46). An additional autoproteolytic site has been identified in the catalytic domain of the cytomegalovirus protease (2,31,45).…”

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confidence: 99%

Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth

Matusick-Kumar

McCann

Robertson

et al. 1995

J Virol

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The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain N o (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain N o from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.

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“…Cleavage at this sequence would result in products of the appropriate size and immunogenicity. A similar autoprocessing site has been identified within the N-terminal domain of the cytomegalovirus protease at the sequence Val-141-Glu-142-Ala-143/Ala-144 and was postulated to have a role in regulation of protease activity (1).…”

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confidence: 74%

Investigation of the specificity of the herpes simplex virus type 1 protease by point mutagenesis of the autoproteolysis sites

McCann

O’Boyle

Deckman

1994

J Virol

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The herpes simplex virus type 1 (HSV-1) protease is cleaved at two autoprocessing sites during viral maturation, one of which shares amino acid identity with its substrate, ICP35. Similar autoprocessing sites have been observed within other members of the Herpesviridae. Introduction of point mutations within the autoprocessing sites of the HSV-1 protease indicated that specificity resides within the P4-Pl' region of the cleavage sites.Assembly of herpes simplex virus type 1 (HSV-1) begins late during the infectious cycle (2). Prior to packaging of genomic DNA, the interior of the immature particle (B capsid) is filled primarily with infected cell protein 35 (ICP35) (13). Similar to the gp22 protein of bacteriophage T4 (9), ICP35 is thought to function as a scaffold (14) for the assembly of capsid proteins. A temperature-sensitive mutant which demonstrated that posttranslational processing of ICP35 is required for production of infectious virions has been described (15). Overlapping open reading frames, UL26 and UL26.5, were shown to encode the HSV-1 protease and its substrate, ICP35, respectively (11,12,16). We previously described the expression of the HSV-1 protease and substrate in Escherichia coli and demonstrated cleavage of two autoprocessing sites, depicted in Fig. 1 (3). These two processing sites, first alluded by Welch et al. (20), have been mapped by amino acid sequencing to positions A-247/S-248 and A-610/S-611 (5). To investigate the sequence specificity of the HSV-1 protease, we examined the effects of cleavage site point mutations on autoprocessing of the E. coli-expressed protein.Point mutations were introduced into the P5-P4' positions of the C-terminal cleavage site and the P4-Pl' positions of the N-terminal cleavage site, using degenerate oligonucleotides and PCR amplification. Purified PCR fragments containing C-terminal point mutants were digested with AflIII and PstI, and the 364-bp band was gel purified again prior to ligating into the unique AfllII-PstI sites of pT7635C, a full-length HSV-1 protease construct derived from pT7635A (3), and pLysS (Novagen). Mutagenic oligonucleotides (Genosys; National Biosciences) were 40 bp in length and spanned the cleavage sites. Individual point mutants were identified by DNA sequencing and transformed into E. coli BL21(DE3) for expression. Bacterial expression was induced with isopropylthiogalactopyranoside (IPTG; 0.5 mM) at t = 0, and HSV-1 protease products were detected by Western blot (immunoblot) analysis with monoclonal antibody MCA406 (Serotec, Inc.) and a polyclonal antibody to the C-terminal 25 amino acids (aa) (data not shown) as previously described (3). The four proteins revealed by MCA406 ( Fig. 2a, lanes 13 to 16; Fig. 2c, lanes 9 to 12) correspond to full-length (Pra; aa 1 to 635),

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Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites

Cited by 85 publications

References 32 publications

In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth

Investigation of the specificity of the herpes simplex virus type 1 protease by point mutagenesis of the autoproteolysis sites

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