Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

Cidofovir Diphosphate Inhibits Adenovirus 5 DNA Polymerase via both Nonobligate Chain Termination and Direct Inhibition, and Polymerase Mutations Confer Cidofovir Resistance on Intact Virus.

Human adenovirus (AdV) can cause fatal disease in immune-suppressed individuals, but treatment options are limited, in part because the antiviral cytidine analog cidofovir (CDV) is nephrotoxic. The investigational agent brincidofovir (BCV) is orally bioavailable, nonnephrotoxic, and generates the same active metabolite, cidofovir diphosphate (CDVpp). However, its mechanism of action against AdV is poorly understood. Therefore, we have examined the effect of CDVpp on DNA synthesis by a purified adenovirus 5 (AdV5) DNA polymerase (Pol). CDVpp was incorporated into nascent DNA strands and promoted a nonobligate form of chain termination (i.e., AdV5 Pol can extend, albeit inefficiently, a DNA chain even after the incorporation of a first CDVpp molecule). Moreover, unlike a conventional mismatched base pair, misincorporated CDVpp was not readily excised by the AdV5 Pol. At elevated concentrations, CDVpp inhibited AdV5 Pol in a manner consistent with both chain termination and direct inhibition of Pol activity. Finally, a recombinant AdV5 was constructed, containing Pol mutations (V303I and T87I) that were selected following an extended passage of wild-type AdV5 in the presence of BCV. This virus had a 2.1-fold elevated 50% effective concentration (EC50) for BCV and a 1.9-fold increased EC50 for CDV; thus, these results confirmed that viral resistance to BCV and CDV can be attributed to mutations in the viral Pol. These findings show that the anti-AdV5 activity of CDV and BCV is mediated through the viral DNA Pol and that their antiviral activity may occur via both (nonobligate) chain termination and (at high concentration) direct inhibition of AdV5 Pol activity.

Genetic diversity of the human adenovirus species C DNA polymerase.

BACKGROUND: Human Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. METHODS: We assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. RESULTS: We identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (±6.2) and 5.2 (±2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. CONCLUSIONS: Our study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments.

[Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

Poor growth of human adenovirus-12 compared to adenovirus-2 correlates with a failure to impair PKR activation during the late phase of infection.

Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cultured human cells, which is manifested by premature death of HAdV-12-infected cells. Whereas HAdV-2 induction of IFN-ß expression was transient, HAdV-12-infected cells maintained high levels of IFN-ß expression, protein kinase R (PKR) activation and eIF-2α phosphorylation throughout the infectious cycle. The importance of the IFN-inducible PKR kinase in restriction of HAdV-12 was supported by the enhanced growth of the virus following PKR knockdown in HeLa cells. Ectopic expression of HAdV-2 VA RNAI increased HAdV-12 hexon protein expression, suggesting that insufficient VA RNA expression contributes to the restricted growth of HAdV-12. Although some adenovirus species are known to persist in human lymphoid tissues, HAdV12 has so far not been found. Thus, it is possible that the inability of HAdV12 to evade the INF response may have implications for the virus to establish long-lasting or persistent infections.

Processing of the l1 52/55k protein by the adenovirus protease: a new substrate and new insights into virion maturation.

Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: II. adenovirus proteinase is activated in an unusual one-dimensional biochemical reaction.

Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors, the viral DNA and pVIc, an 11-amino acid peptide originating from the C terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence-independent DNA-binding proteins with nm equilibrium dissociation constants such that in the virus particle, they are predicted to be essentially irreversibly bound to the viral DNA. Here, we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D(1) = 1.45 × 10(6) bp(2)/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc, which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: IV. viral proteinase slides along DNA to locate and process its substrates.

Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 10(6) bp(2)/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: III. atomic resolution structure of the nascent form of the adenovirus proteinase.

The adenovirus proteinase (AVP), the first member of a new class of cysteine proteinases, is essential for the production of infectious virus, and here we report its structure at 0.98 Å resolution. AVP, initially synthesized as an inactive enzyme, requires two cofactors for maximal activity: pVIc, an 11-amino acid peptide, and the viral DNA. Comparison of the structure of AVP with that of an active form, the AVP-pVIc complex, reveals why AVP is inactive. Both forms have an α + ß fold; the major structural differences between them lie in the ß-sheet domain. In AVP-pVIc, the general base His-54 Nδ1 is 3.9 Å away from the Cys-122 Sγ, thereby rendering it nucleophilic. In AVP, however, His-54 Nδ1 is 7.0 Å away from Cys-122 Sγ, too far away to be able to abstract the proton from Cys-122. In AVP-pVIc, Tyr-84 forms a cation-π interaction with His-54 that should raise the pK(a) of His-54 and freeze the imidazole ring in the place optimal for forming an ion pair with Cys-122. In AVP, however, Tyr-84 is more than 11 Å away from its position in AVP-pVIc. Based on the structural differences between AVP and AVP-pVIc, we present a model that postulates that activation of AVP by pVIc occurs via a 62-amino acid-long activation pathway in which the binding of pVIc initiates contiguous conformational changes, analogous to falling dominos. There is a common pathway that branches into a pathway that leads to the repositioning of His-54 and another pathway that leads to the repositioning of Tyr-84.

Functionally distinct monomers and trimers produced by a viral oncoprotein.

While the process of homo-oligomer formation and disassembly into subunits represents a common strategy to regulate protein activity, reports of proteins in which the subunit and homo-oligomer perform independent functions are scarce. Tumorigenesis induced by the adenovirus E4-ORF1 oncoprotein depends on its binding to a select group of cellular PDZ proteins, including MUPP1, MAGI-1, ZO-2 and Dlg1. We report here that in cells E4-ORF1 exists as both a monomer and trimer and that monomers specifically bind and sequester MUPP1, MAGI-1 and ZO-2 within insoluble complexes whereas trimers specifically bind Dlg1 and promote its translocation to the plasma membrane. This work exposes a novel strategy wherein the oligomerization state of a protein not only determines the capacity to bind separate related targets but also couples the interactions to different functional consequences.

The adenovirus E4orf6 E3 ubiquitin ligase complex assembles in a novel fashion.

The human adenovirus E4orf6 and E1B55K proteins are part of an E3 ubiquitin ligase complex that degrades p53, Mre11 and probably other cellular polypeptides. Our group has demonstrated previously that this complex contains Cul5, Rbx1 and Elongin B and C and is formed through interactions of these cellular proteins with E4orf6. Although this E4orf6 complex is similar in many ways to the cellular SCF and VBC E3 ligase complexes, our previous work indicated that unlike all known Cullin-containing complexes, E4orf6 contains two functional BC-box motifs that permit interactions with Elongin B and C. Here we show that a third BC-box exists that also appears to be fully functional. In addition, we attempted to identify a region in E4orf6 responsible for the specific selection of Cul5, which we show herein by knocking down Cul5 protein levels, is essential for p53 degradation. One sequence within E4orf6 shares limited homology with the 'Cul5 box motif', a recently identified sequence found to be responsible for selection of Cul5 in some cellular Cullin-containing E3 ligase complexes; however, genetic analysis indicated that this motif is not involved in Cullin binding or p53 degradation. Thus E4orf6 appears to utilize a different mechanism for Cul5 selection, and, both in terms of interactions with Elongin B and C and with Cul5, assembles the E3 ligase complex in a highly novel fashion.

Adenovirus ubiquitin-protein ligase stimulates viral late mRNA nuclear export.

Theadenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular proteins to form an E3 ubiquitin-protein ligase that polyubiquitinates p53 and probably one or more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their proteasomal degradation. The MRN complex is required for cellular DNA double-strand break repair and induction of the DNA damage response by adenovirus infection. To determine if the ability of E1B-55K and E4orf6 to stimulate viral late mRNA nuclear export requires the ubiquitin-protein ligase activity of this viral ubiquitin-protein ligase complex, we designed and expressed a dominant-negative mutant form of cullin 5 in HeLa cells before infection with wild-type Ad5 or the E1B-55K null mutant dl1520. The dominant-negative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitin-protein ligase but had no effect on viral early mRNA synthesis, early protein synthesis, or viral DNA replication. However, expression of the dominant-negative cullin 5 protein caused a decrease in viral late protein synthesis and viral nuclear mRNA export similar to the phenotype produced by mutations in E1B-55K. We conclude that the stimulation of adenovirus late mRNA nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein ligase activity of the adenovirus ubiquitin-protein ligase complex.

Interaction of the adenovirus proteinase with protein cofactors with high negative charge densities.

The interactions of the human adenovirus proteinase (AVP) with polymers with high negative charge densities were characterized. AVP utilizes two viral cofactors for maximal enzyme activity (k(cat)/K(m)), the 11-amino acid peptide from the C-terminus of virion precursor protein pVI (pVIc) and the viral DNA. The viral DNA stimulates covalent AVP-pVIc complexes (AVP-pVIc) as a polyanion with a high negative charge density. Here, the interactions of AVP-pVIc with different polymers with high negative charge densities, polymers of glutamic acid (polyE), were characterized. The rate of substrate hydrolysis by AVP-pVIc increased with increasing concentrations of polyE. At higher concentrations of polyE, the increase in the rate of substrate hydrolysis approached saturation. Although glutamic acid did not stimulate enzyme activity, glutamic acid and NaCl could displace DNA from AVP-pVIc.(DNA) complexes; the K(i) values were 230 and 329 nM, respectively. PolyE binds to the DNA binding site on AVP-pVIc as polyE and DNA compete for binding to AVP-pVIc. The equilibrium dissociation constant for 1.3 kDa polyE binding to AVP-pVIc was 56 nM. On average, one molecule of AVP-pVIc binds to 12 residues in polyE. Comparison of polyE and 12-mer single-stranded DNA interacting with AVP-pVIc revealed the binding constants are similar, as are the Michaelis-Menten constants for substrate hydrolysis. The number of ion pairs formed upon the binding of 1.3 kDa polyE to AVP-pVIc was 2, and the nonelectrostatic change in free energy upon binding was -6.5 kcal. These observations may be physiologically relevant as they infer that AVP may bind to proteins that have regions of negative charge density. This would restrict activation of the enzyme to the locus of the cofactor within the cell.

DNA binding provides a molecular strap activating the adenovirus proteinase.

Human adenovirus proteinase (AVP) requires two cofactors for maximal activity: pVIc, a peptide derived from the C terminus of adenovirus precursor protein pVI, and the viral DNA. Synchrotron protein footprinting was used to map the solvent accessible cofactor binding sites and to identify conformational changes associated with the binding of cofactors to AVP. The binding of pVIc alone or pVIc and DNA together to AVP triggered significant conformational changes adjacent to the active site cleft sandwiched between the two AVP subdomains. In addition, upon binding of DNA to AVP, it was observed that specific residues on each of the two major subdomains were significantly protected from hydroxyl radicals. Based on the locations of these protected side-chain residues and conserved aromatic and positively charged residues within AVP, a three-dimensional model of DNA binding was constructed. The model indicated that DNA binding can alter the relative orientation of the two AVP domains leading to the partial activation of AVP by DNA. In addition, both pVIc and DNA may independently alter the active site conformation as well as drive it cooperatively to fully activate AVP.

Adenain, the adenovirus endoprotease (a review).

With the possible exception of very simple viruses, most viruses appear to encode at least one virus specific endopeptidase. In addition to facilitating the orchestrated fragmentation of polyproteins of RNA viruses, these proteolytic enzymes may also be involved in the suppression of host protein synthesis, the regulation of virus assembly, the egress and subsequent uncoating in another cycle of infection of both RNA and DNA viruses. The endopeptidase encoded by adenoviruses (AVP or adenain) appears to be involved in several of these functions. Most of the literature concerns the protease of human adenovirus type 2, but there are good reasons to believe that the proteases of other adenovirus serotypes will be very similar. For a review see Weber [1,2].

Substrate specificity of adenovirus protease.

The adenovirus protease, adenain is functionally required for virion uncoating and virion maturation and release from the infected cell. In addition to hydrolysis of precursor proteins at specific consensus sites, adenain has also been observed to cleave viral proteins at other sites. Here we re-examine the sequences in the consensus sites and also the phenomena of cleavage at other sites on viral proteins II, 100K, V, VI and VII. An examination of the eight residues flanking the scissile bond in 274 consensus sites from 36 different adenovirus serotypes in the DNA sequence databanks provided the following main conclusions: (1) two types of consensus sites, type 1, (M,I,L)XGX-G and type 2, (M,I,L)XGG-X, (2) the variant positions P(3) and P(1) never contained C,P,D,H,W,Y and C,P,G,M amino acids, respectively in type 1, (3) the variant positions P(3) and P(1)' never contained C,D,L,W and C,P,D,Q,H,Y,W amino acids, respectively in type 2, and (4) the thiol forming C residue occurred only twice within the eight residues flanking the scissile bond and that in the P(4)' position. Six unusual serotypes had (M,L,I)XAT-G as the PVII consensus site. Adenain has been proposed to cleave protein VI at an unknown site in the course of virion uncoating. The cleavage of capsid protein VI in the absence of a consensus site is confirmed here in vitro using recombinant adenain. Virion proteins II, V and VII and the nonstructural protein 100K were also digested in vitro into discrete fragments by recombinant adenain. We conclude that adenain preferentially cleaves viral proteins at their consensus sites, but is capable, in vitro of cleavages at other discrete sites which resemble the consensus cleavage sites.

Sequence changes in the human adenovirus type 5 DNA polymerase associated with resistance to the broad spectrum antiviral cidofovir.

Although there is currently no FDA approved antiviral treatment for adenovirus (Ad) infections, the broad spectrum antiviral cidofovir (CDV) has demonstrated potent inhibitory activity against many Ad serotypes in vitro and in an in vivo ocular replication model. The clinical potential of CDV prompted the assessment for the emergence of CDV resistance in Ad5. Serial passage of Ad5 in increasing concentrations of CDV resulted in derivation of four different Ad5 variants with increased resistance to CDV. CDV resistance was demonstrated by ability to replicate viral DNA in infected cells at CDV concentrations that inhibit the parental virus, by ability to form plaques in CDV concentrations of >20 microg/ml and by increased progeny release following infection and growth in media containing CDV. Using marker rescue, the loci for CDV resistance in variant R1 was shown to be mediated by one residue change L741S, one of two mutations within the R1 encoded DNA polymerase. The CDV-resistant variants R4, R5 and R6 also contained mutations in their respective DNA polymerase sequences, but these were different from R1; variant R4 contained two changes (F740I and V180I), whereas both R5 and R6 variants contained the non-conserved mutation A359E. R6 contained additional alterations L554F and V817L. The location of the R1 change is close to a region of the DNA polymerase which is conserved with other polymerases that is predicted to involve nucleotide binding.

Deubiquitinating function of adenovirus proteinase.

The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.

Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.

The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc.

Discovery of a new inhibitor lead of adenovirus proteinase: steps toward selective, irreversible inhibitors of cysteine proteinases.

Using the computer docking program EUDOC, in silico screening of a chemical database for inhibitors of human adenovirus cysteine proteinase (hAVCP) identified 2,4,5,7-tetranitro-9-fluorenone that selectively and irreversibly inhibits hAVCP in a two-step reaction: reversible binding (Ki = 3.09 microM) followed by irreversible inhibition (ki = 0.006 s(-1)). The reversible binding is due to molecular complementarity between the inhibitor and the active site of hAVCP, which confers the selectivity of the inhibitor. The irreversible inhibition is due to substitution of a nitro group of the inhibitor by the nearby Cys122 in the active site of hAVCP. These findings suggest a new approach to selective, irreversible inhibitors of cysteine proteinases involved in normal and abnormal physiological processes ranging from embryogenesis to apoptosis and pathogen invasions.