The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8).

A genomic clone encoding the protease (Pr) and the assembly protein (AP) of Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) has been isolated and sequenced. As with other herpesviruses, the Pr and AP coding regions are present within a single long open reading frame. The mature KSHV Pr and AP polypeptides are predicted to contain 230 and 283 residues, respectively. The amino acid sequence of KSHV Pr has 56% identity with that of herpesvirus salmiri, the most similar virus by phylogenetic comparison. Pr is expressed in infected human cells as a late viral gene product, as suggested by RNA analysis of KSHV-infected BCBL-1 cells. Expression of the Pr domain in Escherichia coli yields an enzymatically active species, as determined by cleavage of synthetic peptide substrates, while an active-site mutant of this same domain yields minimal proteolytic activity. Sequence comparisons with human cytomegalovirus (HCMV) Pr permitted the identification of the catalytic residues, Ser114, His46, and His134, based on the known structure of the HCMV enzyme. The amino acid sequences of the release site of KSHV Pr (Tyr-Leu-Lys-Ala*Ser-Leu-Ile-Pro) and the maturation site (Arg-Leu-Glu-Ala*Ser-Ser-Arg-Ser) show that the extended substrate binding pocket differs from that of other members of the family. The conservation of amino acids known to be involved in the dimer interface region of HCMV Pr suggests that KSHV Pr assembles in a similar fashion. These features of the viral protease provide opportunities to develop specific inhibitors of its enzymatic activity.     

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.     

Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.     

HCV丝氨酸蛋白酶小分子底物构建及GST融合表达

丝氨酸蛋白酶是丙型肝炎病毒重要的功能蛋白和药物作用靶点,其通过分子内（cis）和分子间（trans）方式催化水解前体蛋白,释放病毒功能蛋白。目的：为深入研究病毒蛋白酶活性和抑制剂鉴定需要,实验研究参照丙型肝炎病毒1a亚型菌株蛋白酶天然底物的氨基酸序列特点,设计了一段包含两个天然底物酶切位点的小分子多肽2S,并进行了原核表达。方法：利用PCR方法,合成2S小分子多肽基因,目的基因两端引入BamH I和EcoR I两个限制性酶切位点,双酶切后将基因与表达载体pGEX-4T-2重组,转化大肠杆菌DH5α,经化学诱导进行GST融合蛋白表达,通过亲和层析柱纯化目的蛋白。纯化的GST 2S融合蛋白在体外反应系统进行酶切鉴定,SDS-PAGE和ELISA鉴定酶切结果。结果：PCR合成的小分子底物多肽2S基因,经与表达载体重组后测序,证实基因序列正确。采用0.5mmol/L浓度的IPTG诱导工程菌过夜,获得表达的目的蛋白,经分离纯化得到融合蛋白GST-2S。GST-2S在体外磷酸盐缓冲系统中与丝氨酸蛋白酶反应,15%SDS-PAGE鉴定酶切产物,证实融合蛋白底物条带明显消失,ELISA结果同样说明融合蛋白的底物活性。结论：含有两个天然底物酶切位点的小分子多肽可以替代病毒天然底物,实验结果为丙型肝炎病毒丝氨酸蛋白酶活性研究和酶抑制剂研究奠定了方法学基础。     

Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.

An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1' position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme's dimerization dissociation constant of 1.7 (+/-0.9) microM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease.     

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.     

Human immunodeficiency virus protease. Bacterial expression and characterization of the purified aspartic protease

The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 128-135. The enzyme expressed as a nonfusion protein exhibits proteolytic activity with a pH optimum of 5.5 and is inhibited by the aspartic protease inhibitor pepstatin with a Ki of 1.1 microM. Replacement of the conserved residue Asp-25 with an Asn residue eliminates proteolytic activity. Analysis of the minimal peptide substrate size indicates that 7 amino acids are required for efficient peptide cleavage. Size exclusion chromatography is consistent with a dimeric enzyme and circular dichroism spectra of the purified enzyme are consistent with a proposed structure of the protease (Pearl, L.H., and Taylor, W.R. (1987) Nature 329, 351-354). These data support the classification of the human immunodeficiency virus protease as an aspartic protease, likely to be structurally homologous with the well characterized family that includes pepsin and renin.     

Herpesvirus protease inhibition by dimer disruption

Kaposi's sarcoma-associated herpesvirus (KSHV), like all herpesviruses, encodes a protease (KSHV Pr), which is necessary for the viral lytic cycle. Herpesvirus proteases function as obligate dimers; however, each monomer has an intact, complete active site which does not interact directly with the other monomer across the dimer interface. Protein grafting of an interfacial KSHV Pr alpha-helix onto a small stable protein, avian pancreatic polypeptide, generated a helical 30-amino-acid peptide designed to disrupt the dimerization of KSHV Pr. The chimeric peptide was optimized through protein modeling of the KSHV Pr-peptide complex. Circular dichroism analysis and gel filtration chromatography revealed that the rationally designed peptide adopts a helical conformation and is capable of disrupting KSHV Pr dimerization, respectively. Additionally, the optimized peptide inhibits KSHV Pr activity by 50% at a approximately 200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 microM. Mutagenesis of the interfacial residue M197 to a leucine resulted in an inhibitory concentration which was twofold higher for KSHV Pr M197L than for KSHV Pr, in agreement with the model that the dimer interface is involved in peptide binding. These results indicate that the dimer interface, as well as the active sites, of herpesvirus proteases is a viable target for inhibiting enzyme activity.     

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.     

Inhibiting viral proteases: challenges and opportunities

Inhibitor design against viral targets must take into account the peculiar characteristics of viral biology-in particular, the plasticity of their replicative machinery. This includes maturational cleavage of the polyprotein, which is mediated by virally encoded proteases. Designing against a movable target is particularly challenging, but at the same time it offers new opportunities. Here we describe our experience with the NS3/4A (NS: nonstructural) serine protease of human hepatitis C virus (HCV). By extensive use of combinatorial peptide libraries, various inhibitor types were generated, including product inhibitors, serine traps, P-P' inhibitors, and prime side inhibitors. The latter represent a first case for a serine protease. A key finding, derived from structural studies utilizing these inhibitors, was that NS3 is an induced-fit protease, requiring both the NS4A cofactor protein and the substrate to fully activate its catalytic machinery. In the absence of cofactor and/or substrate, NS3 exists in solution as a large conformational ensemble, which can be matched by a correspondingly large set of peptide inhibitors, each one stabilizing a given conformer. In the perspective of inhibiting viral proteases in general, we suggest that combinatorial ligand ensembles may be a powerful tool, to contrast the adaptive potential of the viral quasispecies.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Properties of the protein encoded by the UL32 open reading frame of herpes simplex virus 1.

The functions previously assigned to the essential herpes simplex virus 1 UL32 protein were in cleavage and/or packaging of viral DNA and in maturation and/or translocation of viral glycoproteins to the plasma membrane. The amino acid sequence predicts N-linked glycosylation sites and sequences conserved in aspartyl proteases and in zinc-binding proteins. We report the following. (i) The 596-amino-acid UL32 protein accumulated predominantly in the cytoplasm of infected cells but was not metabolically labeled with glucosamine and did not band with membranes containing a known glycoprotein in flotation sucrose density gradients. The UL32 protein does not, therefore, have the properties of an intrinsic membrane protein. (ii) Experiments designed to demonstrate aspartyl protease activity in a phage display system failed to reveal proteolytic activity. Moreover, substitution of Asp-110 with Gly in the sequence Asp-Thr-Gly, the hallmark of aspartyl proteases, had no effect on viral replication in Vero and SK-N-SH cell lines or in human foreskin fibroblasts. Therefore, if the UL32 protein functions as a protease, this function is not required in cells in culture. (iii) Both the native UL32 protein and a histidine-tagged UL32 protein made in recombinant baculovirus-infected insect cells bound zinc. The consensus sequence is conserved in the UL32 homologs from varicella-zoster virus and equine herpesvirus 1. UL32 protein is therefore a cysteine-rich, zinc-binding essential cytoplasmic protein whose function is not yet clear.     

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.     

Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom

The amino acid sequence of a protease, protein C activator, from Agkistrodon contortrix contortrix venom was determined. Peptide fragments obtained by chemical or enzymatic cleavage of the S-carboxymethylated protein were purified by gel filtration and reverse-phase high-performance liquid chromatography. The present study demonstrates that protein C activator from A. contortrix contortrix venom is a trypsin-type serine protease that is composed of 231 residues with a molecular weight of 25,095 for the polypeptide portion of the molecule. By analogy to the mammalian serine proteases, the catalytic triad in venom protein C activator consists of His-40, Asp-85, and Ser-177. The protein also contains three N-linked glycosylation sites at Asn-21, Asn-78, and Asn-129. The amino acid sequence of protein C activator exhibits a high degree of sequence identity with other snake venom proteases: 73% with batroxobin, 68% with flavoxobin, and 55% with Russell's viper venom factor V activator.     

Rearrangement of terminal amino acid residues in peptides by protease-catalyzed intramolecular transpeptidation

Protease-catalyzed rearrangements of amino acid residues in peptides are observed during enzymatic digestion of proteins. When two enzyme-specific cleavage sites are within one or two residues of each other in the protein sequence, only one of the two sites usually is hydrolyzed by the protease, resulting in a peptide that contains an extra cleavage site near one of its termini. It is observed that in this type of peptide, the residues between the two cleavage sites often rearrange from one terminus of the peptide to the other terminus, catalyzed by the protease that created the peptide. It is proposed that the rearrangement is caused by protease-catalyzed intramolecular transpeptidation through a cyclic peptide intermediate. Several cases of this type of rearrangement were observed for different peptides generated by different proteases, indicating that this type of rearrangement is a general phenomenon occurring during enzymatic digestion of proteins.     

Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.     

Molecular cloning and nucleotide sequence of the 90k serine protease gene,hspK, from Bacillus subtilis (natto) No. 16

We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.     

The ubiquitin-specific protease USP7 modulates the replication of Kaposi's sarcoma-associated herpesvirus latent episomal DNA

Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987-29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins.     

Studies on Mold Proteases

The substrate specificity of the crystalline acid protease obtained from Rhizopus chinensis was determined using B-chain of oxidized beef insulin and numerous synthetic peptides, comparing with that of several acid proteases from various sources. The peptide bonds susceptible to the action of Rhiz. acid protease were found to be mainly those involving the amino group of bulky amino acids. The enzyme split the B-chain of oxidized insulin at twelve sites of the peptide linkages and a certain similarity in the specificity was observed among the three acid proteases, Rhiz. protease, rennin and pepsin, all of which were known to show potent milk clotting activities.     

Conserved herpesvirus kinases target the DNA damage response pathway and TIP60 histone acetyltransferase to promote virus replication

Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α, β, and γ herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase that is critical for virus replication and spread. The extent of substrate conservation and the key common cell-signaling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach, we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched, and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective antiviral strategies.