Discovery and development of VX-950, a novel, covalent, and reversible inhibitor of hepatitis C virus NS3.4A serine protease.

The hepatitis C virus (HCV) NS3.4A protease, which is essential for viral replication, is considered one of the most attractive targets for developing novel anti-HCV therapies. However, discovery of potent and selective small-molecule inhibitors of HCV NS3.4A protease as oral drug candidates has been hampered by the shallow substrate-binding groove of the protease. Serine trap warheads have been used to covalently anchor inhibitor scaffolds and to increase their affinity to the protease. This review will examine the evolution of covalent inhibitors of the HCV NS3.4A protease from early aldehyde molecules to alpha-ketoamide inhibitors. Kinetic and structural studies of alpha-ketoacid and alpha-ketoamide inhibitors revealed an unusual mechanism of binding in the catalytic site. Optimization of alpha-ketoamide scaffolds by scientists at Vertex and Eli Lilly led to the discovery of VX-950, a novel, potent, selective inhibitor of HCV NS3.4A protease. VX-950 possesses excellent antiviral activity in both HCV replicon cells and human fetal hepatocytes infected with HCV-positive patient sera. In addition, VX-950 exhibits a favorable pharmacokinetic profile in several animal species and demonstrates potent inhibition of the HCV NS3.4A protease activity in a mouse model. In a recent phase 1b clinical trial, VX-950 was able to rapidly reduce the plasma viral load of patients chronically infected with genotype 1 HCV by a mean approximately 3 log(10) in 2 days. The median viral load reduction was 4.4 log(10) for the best dose group after 14 days of dosing. The pre-clinical profile and early clinical data of VX-950 will be discussed in this review.

Broad-spectrum antiviral activity of the IMP dehydrogenase inhibitor VX-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon.

The enzyme IMP dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, namely, the conversion of IMP to XMP. The major event occurring in cells exposed to competitive IMPDH inhibitors such as ribavirin or uncompetitive inhibitors such as mycophenolic acid (MPA) is a depletion of the intracellular GTP and dGTP pools. Ribavirin is approved as an inhaled antiviral agent for treatment of respiratory syncytial virus (RSV) infection and orally, in combination with alpha interferon (IFN-alpha), for the treatment of chronic hepatitis C virus (HCV) infection. VX-497 is a potent, reversible uncompetitive IMPDH inhibitor which is structurally unrelated to other known IMPDH inhibitors. Studies were performed to compare VX-497 and ribavirin in terms of their cytotoxicities and their efficacies against a variety of viruses. They included DNA viruses (hepatitis B virus [HBV], human cytomegalovirus [HCMV], and herpes simplex virus type 1 [HSV-1]) and RNA viruses (respiratory syncytial virus [RSV], parainfluenza-3 virus, bovine viral diarrhea virus, Venezuelan equine encephalomyelitis virus [VEEV], dengue virus, yellow fever virus, coxsackie B3 virus, encephalomyocarditis virus [EMCV], and influenza A virus). VX-497 was 17- to 186-fold more potent than ribavirin against HBV, HCMV, RSV, HSV-1, parainfluenza-3 virus, EMCV, and VEEV infections in cultured cells. The therapeutic index of VX-497 was significantly better than that of ribavirin for HBV and HCMV (14- and 39-fold, respectively). Finally, the antiviral effect of VX-497 in combination with IFN-alpha was compared to that of ribavirin with IFN-alpha in the EMCV replication system. Both VX-497 and ribavirin demonstrated additivity when coapplied with IFN-alpha, with VX-497 again being the more potent in this combination. These data are supportive of the hypothesis that VX-497, like ribavirin, is a broad-spectrum antiviral agent.

Structure and function of hepatitis C virus NS3 helicase.

Hepatitis C Virus helicase activity has been mapped to the COOH-terminal 450 residues of the NS3 protein. Due to its complexity and presumed essentiality for viral replication, the helicase is an attractive target for drug discovery. The elucidation of the atomic structure of the HCV NS3 helicase in complex with oligonucleotide and with ADP has helped clarify our understanding of potential sites for inhibitor binding. Molecular details of the mechanism of this enzyme, and in particular, a better understanding of the mechanism by which ATP hydrolysis is coupled to unwinding of double-stranded substrate may facilitate more efficient structure-based drug design.

Development of a hepatitis C virus RNA helicase high throughput assay.

In this chapter, the development of a 96-well plate increasing signal helicase assay will be described. The authors have used this assay to detect inhibitors of hepatitis C virus (HCV) NS3/4A RNA helicase.

Hepatitis C virus NS3/4A protease.

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.

Generation of recombinant herpes simplex virus ampiicons.

The herpes simplex virus type 1 (HSV-1) amphcon has been developed as a novel eukaryotic expression vector, which contains an HSV-1 ori for DNA replication and a pac signal for cleaving/packaging genomes into viral capsids (1-4). As shown in Fig. 1, amplicon vector can be amplified into head-to-tail concatemers and then packaged into defective HSV-1 viral particles up to one genome size (~150 kb) in the presence of HSV-1 helper viruses. The helper viruses provide all necessary proteins and enzymes for amplicon DNA replication/packaging and for the assembly of defective amplicon viruses (1). One of the applications of this defective amplicon virus system is to transfer efficiently high copy numbers of foreign genes into a broad range of mammalian cells for high-level expression and gene therapy (4,5). We have utilized the amphcon system to produce high levels of HCV NS3/4A complexes in mammalian cells (6). Our results have demonstrated that the amplicon system provides a potential to study the expression of HCV proteins in a broadspectrum of mammalian cell lines, especially in those of human hepatocyte origin that may be biologically more relevant to HCV infection. In this chapter, methods for using the amphcon expression system will be described in three subsections: 1. Generation of high-titer defective HSV-1 amplicon virus stocks Fig. 1. Illustration of HSV-I amplicon expression system. 2. Determination of the titers of amphcon viruses. 3. Expression of HCV NS3/4A complexes using the defective amphcon viruses.

Hepatitis C virus NS3/4A protease.

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.

In situ PCR for in vivo detection of foreign genes transferred into rat brain.

Here we describe the use of in situ PCR to detect a viral transgene in rat brain. Previously, we have reported in vivo gene transfer by using a defective herpes simplex viral vector in mammalian brain (Kaplitt, M.G., Pfaus, J.G., Kleopoulos, S.P., Hanlon, B.A., Rabkin, S.D., Pfaff, D.W., Mol. Cell. Neurosci. 2 (1991) 320-330). For detection of the LacZ transgene, we have used histochemical staining for the protein product, beta-galactosidase, and in situ hybridization for its mRNA, but the DNA itself cannot be reliably detected with conventional methods. Therefore we have adapted the technique of in situ PCR, so that we may detect minute quantities of transgenic vector DNA following in vivo gene. The brain sections, prefixed, were treated with PBS-detergent before PCR amplification to increase permeability for peptides and oligonucleotides across cellular barriers in brain tissue. Pretreatment with detergent retained better brain morphology than the more widely used proteinase treatment. The PCR mixture containing dNTPs, primers, digoxigenin-dUTP (Dig-dUTP) and buffer was loaded onto each brain section. Slides containing brain sections were placed in an aluminum boat and then on the block of the thermal cycler. Temperature was brought to 82 degrees C before adding Taq polymerase ('hot start' method). Dig-labeled PCR amplified fragments were then detected by alkaline-phosphatase-linked anti-digoxigenin-antibody. Positive signals were seen within the nucleus of transduced neurons, indicating presence of viral DNA. Enhanced specificity was observed with the use of Dig-labeled primers which eliminates the possibility of non-specific viral DNA detection through primer-independent reactions. Overall, this technique can serve not only as an internal control for transgene presence during comparisons of experimental groups of animals, but may also have clinical applications including the detection of viral infection in human brain such as HIV in pathology specimens.

Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex.

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus-insect cell expression system.

A gene expression system using recombinant Autographa california nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O2 mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6-10 x 10(6) cells ml-1) in shake flasks in serum-free media. With these procedures, cell densities of 5 x 10(6) cells ml-1 were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell-1, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11-38 mg L-1 of recombinant protein and > or = 90% purity. Maximum production of this protein was observed at a cell density of 5.0 x 10(6) cells ml-1.

SCH 43478 and analogs: in vitro activity and in vivo efficacy of novel agents for herpesvirus type 2.

SCH 43478 and analogs are a class of non-nucleoside antiviral agents that have potent and selective activity against herpes simplex virus type 2 (HSV-2). The IC50 for these compounds in plaque reduction analysis using Vero cells ranges from 0.8 to 2.0 microg/ml. All compounds have a LC50 > 100 microg/ml in cytotoxicity analysis. Mechanism of action studies suggest that these molecules have an effect on the transactivation of viral immediate early (alpha) gene expression. Time of addition studies indicate that antiviral activity of these analogs is limited to the initial 2-3 h after infection and is not due to inhibition of viral adsorption or penetration. Analysis of HSV protein expression demonstrates that SCH 49286 inhibits the accumulation of viral immediate early (alpha) gene products. SCH 43478 demonstrates statistically significant efficacy (P < 0.05) in the guinea pig genital model of HSV infection. Following subcutaneous administration in a therapeutic treatment regimen, SCH 43478 (90 mg/kg/day) is efficacious in reducing the number and severity of lesions and the neurological complications of acute HSV infection. Thus, SCH 43478 and analogs are anti-herpesvirus agents with a unique mechanism of action.

Structure of the hepatitis C virus RNA helicase domain.

Helicases are nucleotide triphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication. The 2.1 A resolution structure of the HCV helicase from the positive-stranded RNA hepatitis C virus reveals a molecule with distinct NTPase and RNA binding domains. The structure supports a mechanism of helicase activity involving initial recognition of the requisite 3' single-stranded region on the nucleic acid substrate by a conserved arginine-rich sequence on the RNA binding domain. Comparison of crystallographically independent molecules shows that rotation of the RNA binding domain involves conformational changes within a conserved TATPP sequence and untwisting of an extended antiparallel beta-sheet. Location of the TATPP sequence at the end of an NTPase domain beta-strand structurally homologous to the 'switch region' of many NTP-dependent enzymes offers the possibility that domain rotation is coupled to NTP hydrolysis in the helicase catalytic cycle.

Virus-specific interaction between the human cytomegalovirus major capsid protein and the C terminus of the assembly protein precursor.

We previously identified a minimal 12-amino-acid domain in the C terminus of the herpes simplex virus type 1 (HSV-1) scaffolding protein which is required for interaction with the HSV-1 major capsid protein. An alpha-helical structure which maximizes the hydropathicity of the minimal domain is required for the interaction. To address whether cytomegalovirus (CMV) utilizes the same strategy for capsid assembly, several glutathione S-transferase fusion proteins to the C terminus of the CMV assembly protein precursor were produced and purified from bacterial cells. The study showed that the glutathione S-transferase fusion containing 16 amino acids near the C-terminal end was sufficient to interact with the major capsid protein. Interestingly, no cross-interaction between HSV-1 and CMV could be detected. Mutation analysis revealed that a three-amino-acid region at the N-terminal side of the central Phe residue of the CMV interaction domain played a role in determining the viral specificity of the interaction. When this region was converted so as to correspond to that of HSV-1, the CMV assembly protein domain lost its ability to interact with the CMV major capsid protein but gained full interaction with the HSV-1 major capsid protein. To address whether the minimal interaction domain of the CMV assembly protein forms an alpha-helical structure similar to that in HSV-1, peptide competition experiments were carried out. The results showed that a cyclic peptide derived from the interaction domain with a constrained (alpha-helical structure competed for interaction with the major capsid protein much more efficiently than the unconstrained linear peptide. In contrast, a cyclic peptide containing an Ala substitution for the critical Phe residue did not compete for the interaction at all. The results of this study suggest that (i) CMV may have developed a strategy similar to that of HSV-1 for capsid assembly; (ii) the minimal interaction motif in the CMV assembly protein requires an alpha-helix for efficient interaction with the major capsid protein; and (iii) the Phe residue in the CMV minimal interaction domain is critical for interaction with the major capsid protein.

Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system.

The hepatitis C virus (HCV) NS3 protein possesses three enzymatic activities: an N-terminal serine protease activity, a C-terminal RNA-stimulated NTPase activity, and an RNA helicase activity. To characterize them, the full-length NS3(631)/4A and three C-terminal truncated proteases (NS3(201)/4A, NS3(181)/4A, and NS3(155)/4A were expressed in mammalian cells with HSV amplicon-defective viruses. Our results revealed that all of the NS3/4A proteins produced in mammalian cells (except NS3(155)/4A) are active in processing both cis and trans cleavage sites. Temperature optimization studies revealed that the protease is more active at temperatures ranging from 4 to 25 degrees C and is completely inactive at 42 degrees C. The RNA-stimulated ATPase activity was characterized with a partially purified NS3(631)/4A fraction and has a higher optimal temperature at 37 to 42 degrees C. The effects of detergents on both NS3 protease and RNA-stimulated ATPase were similar. Nonionic detergents such as Triton X-100, Nonidet P-40 and Tween 20 did not affect the activities, while anionic detergents such as sodium dodecyl sulfate and deoxycholic acid were inhibitory. Zwitterionic detergent such as 3-[(3-cholamidopropyl)- dimethyl-ammoniol-1-propanesulfonate (CHAPS) inhibited protease activity at a concentration of 0.5% (8 mM), which had no effect on ATPase activity. Finally, RNA-unwinding activity was demonstrated in the NS3(631)/4A fraction but not in the similarly purified NS3(181)/4A and NS3(201)/4A fractions. NS(363)/4A unwinds RNA duplexes with 3' but not 5' single-stranded overhangs, suggesting that the NS3 RNA helicase functions in a 3'-to-5' direction.

Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein.

Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fusion proteins were then incubated with 35S-labeled herpes simplex virus type 1-infected cell lysates containing VP5. The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads. Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C-terminal domain alone is sufficient for interaction with VP5. Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif. Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one. A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong alpha-helix structure. Most interestingly, the alpha-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the alpha-helix. Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction. Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5. In addition, a cyclic analog of the minimal peptide which is designed to stabilize an alpha-helical structure competed more efficiently than the minimal peptide. The evidence suggests that the C-terminal end of ICP35 forms an alpha-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5.

Preproenkephalin promoter yields region-specific and long-term expression in adult brain after direct in vivo gene transfer via a defective herpes simplex viral vector.

We have previously used a defective herpes simplex virus vector to express a foreign gene in the adult rat brain. One application of this technology would be the in vivo analysis of promoter function in brain after de novo transfer, which would allow the rapid generation of vectors with localized application in a broad range of mammalian species while avoiding influences of other nearby promoters. A 2.7-kb fragment of the rat preproenkephalin promoter was placed upstream of the bacterial lacZ gene in our herpes simplex virus amplicon. A restricted pattern of lacZ expression was observed in vivo, which follows previously observed patterns of endogenous preproenkephalin expression. These results, from the direct gene transfer into an adult animal brain for in vivo promoter analysis, demonstrate that sequence information that influences restricted expression of preproenkephalin is located within 2.7 kb upstream of transcriptional initiation. lacZ expression was also observed in rat brain for 2 months after direct transfer, and PCR analysis confirmed the continued presence of amplicon DNA in lacZ-positive sections. Restricted and long-term expression observed with an endogenous promoter has important implications for gene therapy using viral vectors.

Minireview: the herpes simplex virus amplicon--a versatile defective virus vector.

The Herpes simplex virus (HSV) amplicon was designed in our laboratory, as a defective virus vector, capable of shuttle delivery of DNA sequences and genes from prokaryotic to eukaryotic cells, tissues or organs. The HSV vector was termed "amplicon" to delineate the fact that it carried the cloned sequences of interest, within amplified concatemeric defective genomes, packaged in HSV virions. Employing the replication functions of their helper virus, the amplicons are wide tropic vectors, capable of entry, replication, and gene expression in varied types of cells, both in vitro, and in vivo. In this brief review, the amplicons will be revisited, beginning with studies of the naturally occurring defective genomes, where many of the virologic aspects of the system were established. Properties of the amplicon system and its components, will be enlightened. The cis acting functions required for amplicon propagation will be briefly described. This will follow with examples of our studies of prokaryotic, viral and eukaryotic DNA sequences, which were amplified and expressed within HSV defective genomes. Studies designed to examine the involvement of helper viruses will be described, including our analyses of host shutoff mutant helper viruses. Finally, recent studies of other laboratories will be reviewed, with emphasis on what appears to be the use of amplicons as neurotropic vectors, towards potential gene therapy.

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.

Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta.

Activator 1 (A1) is a multiprotein complex which is essential for proliferating cell nuclear antigen (PCNA)-dependent DNA polymerase delta (pol delta) activity and efficient in vitro DNA synthesis in the SV40 dipolymerase replication system. In this report, we describe the isolation of A1 from HeLa cytosolic extracts. A1 stimulated pol delta activity in singly primed phi X174 DNA or (dA)4500.oligo(dT)12-18 in reactions containing PCNA, single-stranded DNA binding protein (SSB), and ATP. Using this assay, A1 has been extensively purified. Purified preparations contained five discrete subunits of 145, 40, 38, 37, and 36.5 kDa. ATP hydrolysis to ADP and Pi is essential for A1-dependent pol delta activity, and we have shown that A1 contains an intrinsic ATPase which is stimulated by DNA. The DNA-dependent hydrolysis of ATP can be stimulated by PCNA and further activated by PCNA plus the human single-stranded DNA binding protein. These stimulatory effects were observed with (dA)4500.oligo(dT)12-18, but were not detected with each poly-deoxynucleotide alone. Furthermore, A1 formed a complex with (dA)4500.oligo(dT)12-18 which could be measured by nitrocellulose binding. No complex with (dA)4500 or oligo(dT)12-18 alone was detected by this procedure. Data are also presented which indicate that A1, in conjunction with PCNA, functions as a primer-recognition factor for pol delta, increasing its ability to utilize low levels of primer ends, but it does not increase the size of the DNA products. A1 also markedly reduced the amount of PCNA required for pol delta activity on a multiply primed DNA suggesting that PCNA interacts with A1 at the primer end. These multiple effects of A1 closely resemble the properties of the multisubunit protein RF-C described by Tsurimoto and Stillman (Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1023-1027).