Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad.

The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.     

Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.     

Herpesvirus proteinase: site-directed mutagenesis used to study maturational, release, and inactivation cleavage sites of precursor and to identify a possible catalytic site serine and histidine.

The cytomegalovirus maturational proteinase is synthesized as a precursor that undergoes at least three processing cleavages. Two of these were predicted to be at highly conserved consensus sequences--one near the carboxyl end of the precursor, called the maturational (M) site, and the other near the middle of the precursor, called the release (R) site. A third less-well-conserved cleavage site, called the inactivation (I) site, was also identified near the middle of the human cytomegalovirus 28-kDa assemblin homolog. We have used site-directed mutagenesis to verify all three predicted sequences in the simian cytomegalovirus proteinase, and have shown that the proteinase precursor is active without cleavage at these sites. We have also shown that the P4 tyrosine and the P2 lysine of the R site were more sensitive to substitution than the other R- and M-site residues tested: substitution of alanine for P4 tyrosine at the R site severely reduced cleavage at that site but not at the M site, and substitution of asparagine for lysine at P2 of the R site reduced M-site cleavage and nearly eliminated I-site cleavage but had little effect on R-site cleavage. With the exception of P1' serine, all R-site mutations hindered I-site cleavage, suggesting a role for the carboxyl end of assemblin in I-site cleavage. Pulse-chase radiolabeling and site-directed mutagenesis indicated that assemblin is metabolically unstable and is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, the enzymatic amino half of the proteinase precursor. Among those tested, only 2 amino acids were identified as essential for activity: the single absolutely conserved serine and one of the two absolutely conserved histidines. When the highly conserved glutamic acid (Glu22) was substituted, the proteinase was able to cleave at the M and I sites but not at the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., protein conformation) role for this residue in determining substrate specificity.     

Cytomegalovirus assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells.

The cytomegalovirus (CMV) serine proteinase assemblin is synthesized as a precursor that undergoes three principal autoproteolytic cleavages. Two of these are common to the assemblin homologs of all herpes group viruses: one at the maturational site near the carboxyl end of the precursor and another at the release site near the midpoint of the precursor. Release-site cleavage frees the proteolytic amino domain, assemblin, from the nonproteolytic carboxyl domain of the precursor. In CMV, a third autoproteolytic cleavage at an internal site divides assemblin into an amino subunit (An) and a carboxyl subunit (Ac) of approximately the same size that remain associated as an active "two-chain" enzyme. We have cloned the sequences encoding An and Ac as separate genes and expressed them by transfecting human cells with recombinant plasmids and by infecting insect cells with recombinant baculoviruses. When An and Ac from either simian CMV or human CMV were coexpressed in human or insect cells, active two-chain assemblin was formed. This finding demonstrates that An and Ac do not require synthesis as single-chain assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the independently expressed An and Ac subunits was demonstrated by coimmunoprecipitation experiments, and efforts to disrupt the complex indicate that the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain assemblin formed from independently expressed An and Ac also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain assemblin, (ii) R-site cleavage is not essential for the activity of two-chain recombinant assemblin, and (iii) the human CMV and simian CMV An and Ac recombinant subunits are functionally interchangeable.     

Cytomegalovirus assemblin (pUL80a): cleavage at internal site not essential for virus growth; proteinase absent from virions

The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called assemblin. I-site cleavage cuts assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of assemblin nor the presence of assemblin in the mature virion.     

Cytomegalovirus capsid protease: biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin)

We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na(2)SO(4) reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.     

Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains.

We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP. The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs. Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay. This finding indicates that the pAP self-interaction influences the pAP-MCP interaction. Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP. We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus.     

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.     

Enzymatic activities of human cytomegalovirus maturational protease assemblin and its precursor (pPR, pUL80a) are comparable: [corrected] maximal activity of pPR requires self-interaction through its scaffolding domain

Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered "tail" (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.     

Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation.

Herpesviruses encode an essential serine proteinase called assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of assemblin mutants. I-site cleavage of the mutated assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV assemblin). These results indicate that assemblin is able to form active multimeric structures that may be of functional importance.     

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.     

RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a ‘disabled’ ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.     

Cryomicroscopy of human cytomegalovirus virions reveals more densely packed genomic DNA than in herpes simplex virus type 1

All members of the herpesvirus family have a characteristic virion structure, comprising a DNA containing, icosahedral capsid, embedded in a proteinaceous layer (tegument) and surrounded by a lipid envelope. Human cytomegalovirus (HCMV, the prototypic beta-herpesvirus) has a genome that is significantly larger (>50 %) than that of the alpha-herpesvirus HSV-1. Although the internal volume of the HCMV capsid is approximately 17 % larger than that of HSV-1, this slight increase in volume does not provide adequate space to encapsidate the full length HCMV genome at the same packing density as HSV-1.We have investigated the nature of DNA packing in HCMV and HSV-1 virions by electron-cryomicroscopy and image processing. Radial density profiles calculated from projection images of HCMV and HSV-1 capsids suggest that there is no increase in the volume of the HCMV capsid upon DNA packaging. Packing density of the viral DNA was assessed for both HCMV and HSV-1 by image analysis of both full and empty particles. Our results for packing density in HSV-1 are in good agreement with previously published measurements, showing an average inter-layer spacing of approximately 26 A. Measurements taken from our HCMV images, however, suggest that the viral genomic DNA is more densely packed, with an average inter-layer spacing of approximately 23 A. We propose therefore, that the combination of greater volume in HCMV capsids and increased packing density of viral DNA accounts for its ability to encapsidate a large genome.     

Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third

The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule--the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites.     

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines,a novel class of herpesvirus antiviral agents

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.     

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.     

The effect of substrate on the production of infectious virus by cells in culture

Herpes simplex virus type I (HSV-1), infectious bovine rhinotracheitis virus (IBR) and turkey herpesvirus were examined for growth in cells cultured on three different substrates. The substrates were glass, DEAE-dextran and collagen gel. With two of the viruses, HSV-1 and IBR, there were no apparent differences in production as a function of substrate. In contrast, the amount of the turkey herpesvirus which was recovered varied greatly with the substrate. Titers were highest on glass, followed by DEAE-dextran and then collagen gel. Our previous studies have indicated that the substrate on which anchorage-dependent cells are grown in vitro has an affect on a number of biological and biochemical properties. The present study indicates that the production of commercially important biologicals can be affected by the substrate.     

Guanosine analogues as anti-herpesvirus agents

Several guanosine analogues, i.e. acyclovir (and its oral prodrug valaciclovir), penciclovir (in its oral prodrug form, famciclovir) and ganciclovir, are widely used for the treatment of herpesvirus (i.e. HSV-1, HSV-2, VZV and HCMV) infections. In recent years, several new guanosine analogues have been developed, including the 3-membered (cyclopropyl) sugar derivative A-5021 and the 6-membered D- and L-cyclohexenyl derivatives. Prominent features shared by all guanosine analogues are the following. They depend for their phosphorylation on the virus-encoded thymidine kinase (TK), which makes them particularly effective against those viruses (HSV-1, HSV-2 and VZV) that encoded for such TK. They are also active against HCMV, whether or not they are subject of phosphorylation by the HCMV-induced UL97 protein kinase. Their antiviral activity can be markedly potentiated by mycophenolic acid, an IMP dehydrogenase inhibitor, and they hold great promise, not only as antiviral agents for the treatment of herpesvirus infections, but also as antitumor agents for the combined gene therapy/chemotherapy of cancer, provided that (part of) the tumor cells have been transfected by the viral TK gene.