Use of vascular endothelial cell growth factor gene transfer to enhance implantable sensor function in vivo.

In the current study, we developed and validated a simple, rapid and safe in vivo model to test gene transfer and sensor function in vivo. Using the model, we tested the specific hypothesis that in vivo gene transfer of angiogenic factors at sites of biosensor implantation would induce neovascularization surrounding the sensor and thereby enhance biosensor function in vivo. As the in vivo site for testing of our gene transfer cell and biosensor function systems, the developing chorioallantoic membrane (CAM) of the embryo was utilized. Vascular endothelial cell growth factor (VEGF) was used as a prototype for angiogenic factor gene transfer. A helper-independent retroviral vector derived from Rous sarcoma virus (RSV), designated RCAS, was used for gene transfer of the murine VEGF (mVEGF) gene (mVEGF:RCAS) into the DF-1 chicken cell line (designated mVEGF:DF-1). Initially, the ability of VEGF:DF-1 cells to produce VEGF and RCAS viral vectors containing the mVEGF gene (mVEGF:RCAS) was validated in vitro and in vivo, as was the ability of the mVEGF:DF-1 cells to induce neovascularization in the ex ova CAM model. Using the system, we determined the ability of mVEGF:DF-1 cells to enhance acetaminophen sensor function in vivo, by inducing neovascularization at sites of sensor implantation in the ex ova CAM model. For these studies, acetaminophen sensors were placed on 8-day-old ex ova CAMs, followed by addition of media or cells (mVEGF:DF-1 cells or GFP:DF-1 cells) at the sites of biosensor implantation on the CAM. At 4 to 10 days after sensor placement, the biosensor function was determined by measuring sensor response to an intravenous injection of acetaminophen. Sensors implanted on CAMs with buffer or control cells (GFP:DF-1 cells) displayed no induced neovascularization around the sensor and had minimal/baseline sensor responses to intravenous acetaminophen injection (media, 133.33 +/- 27.64 nA; GFP:DF-1, 187.50 +/- 55.43 nA). Alternatively, the sensors implanted with mVEGF:DF-1 cells displayed massive neovascularization and equally massive sensor response to intravenous injection of acetaminophen (VEGF:DF-1, 1387.50 +/- 276.42 nA). These data clearly demonstrate that enhancing vessel density (i.e., neovascularization) around an implanted sensor dramatically enhances sensor function in vivo.

Efficacy of clarithromycin in preventing viridans streptococcal bacteremia following autologous stem cell transplantation.

BACKGROUND: In a study involving 200 patients, we previously found that 17.5% of patients developed viridans streptococcal (VS) bacteremia following autologous peripheral blood stem cell transplantation (aPBSCT) when ciprofloxacin or ciprofloxacin plus ampicillin was used for prophylaxis. PATIENTS AND METHODS: A retrospective evaluation of 100 consecutive recipients of aPBSCT was conducted to ascertain the incidence and outcome of VS bacteremia when a combination of ciprofLoxacin and clarithromycin was utilized for antimicrobiaL prophylaxis following transplantation. The 200 patients from our previous study, in which ciprofloxacin alone or ciprofloxacin with ampicillin was used for prophylaxis, were combined with the current group for the purpose of statistical analysis. RESULTS: Streptococcus mitis was isolated from the blood of five individuals at a median of 5 days following stem cell infusion. Each of these patients was neutropenic and presented with fever. Three isolates demonstrated intermediate resistance to macrolides in vitro. However, all episodes of bacteremia were treated successfully with systemic antibiotic therapy. CONCLUSION: Age, duration of neutropenia, type of underlying malignancy and type of conditioning chemotherapy regimen failed to have a significant impact on subsequent VS bacteremia. Only female sex and use of ciprofloxacin without clarithromycin as antimicrobiaL prophyLaxis predicted a significantly increased risk of VS bacteremia in both univariate and Logistic regression analyses.

Incidence and outcome of vancomycin-resistant enterococcal bacteremia following autologous peripheral blood stem cell transplantation.

A retrospective evaluation of 321 consecutive recipients of high-dose chemotherapy (HDC) and autologous peripheral blood stem cell transplantation (PBSCT) was conducted to ascertain the incidence and outcome of vancomycin-resistant enterococcal (VRE) bacteremia. Ten patients developed VRE bacteremia at a median of 6 days following PBSCT. Nine isolates were Enterococcus faecium and one was E. faecalis. The median duration of bacteremia was 5 days. The central venous catheter was removed in seven individuals. Nine patients were treated with a variety of antimicrobial agents including quinupristin-dalfopristin, chloramphenicol, doxycycline, oral bacitracin, co-trimoxazole, and nitrofurantoin. Bacteremia resolved without adverse sequelae in seven patients. Two individuals who died of other causes had persistent or relapsed bacteremia at the time of death. An additional patient suffered multiple relapses of VRE bacteremia and died as a result of VRE endocarditis 605 days following PBSCT. Mortality as a direct result of VRE bacteremia was 10% in this series. The optimal type and duration of treatment of VRE bacteremia has not been clearly defined. Therefore, we perform weekly stool surveillance cultures for VRE in our hospitalized transplant population and apply strict barrier precautions in those individuals in whom stool colonization has been identified. Furthermore, the empiric use of vancomycin has been restricted. Bone Marrow Transplantation (2000) 25, 147-152.

Cytomegalovirus viremia, viruria and disease after autologous peripheral blood stem cell transplantation: no need for surveillance.

A retrospective evaluation of 200 consecutive recipients of autologous peripheral blood stem cell transplantation (PBSCT) was conducted to ascertain the incidence, risk factors, clinical features, complications, and outcome of cytomegalovirus (CMV) infection. A total of 26 patients (13%) developed CMV viremia (n = 5), DNAemia (n = 3), viruria (n = 18) and/or disease (n = 3) at a median of 45 days following stem cell infusion. None of the patients underwent surveillance testing for CMV. A diagnosis was established by culture and polymerase chain reaction of blood, urine or other tissue samples submitted when patients exhibited clinical features suggestive of CMV infection. Cytomegalovirus seropositivity prior to transplantation was the only statistically significant risk factor predicting subsequent identification of CMV (P < 0.001). The symptoms were severe enough in 23 patients to warrant treatment with intravenous ganciclovir. Three patients developed CMV disease; two developed fatal CMV pneumonia and one developed CMV gastritis which responded to antiviral treatment. Clinical signs and symptoms as well as viremia and viruria resolved with (20 patients) and without (three patients) treatment in the remaining individuals. All instances of CMV viremia, DNAemia, viruria and disease occurred within 3 months of stem cell infusion. These results demonstrate that CMV is a common pathogen after autologous PBSCT and may result in fatality in rare instances. Surveillance programs appear to be neither useful nor cost-effective. Diagnostic evaluation should be performed only in patients exhibiting suspicious clinical features and antiviral chemotherapy should be administered for persistent and severe signs and symptoms.

Incidence and outcome of Clostridium difficile infection following autologous peripheral blood stem cell transplantation.

A retrospective evaluation of 200 consecutive recipients of autologous peripheral blood stem cell transplantation (PBSCT) was conducted to ascertain the incidence and outcome of infection with Clostridium difficile. The diagnosis was confirmed in 14 patients with diarrhea (15 episodes) at a median of 33 days after stem cell infusion. Five patients were neutropenic at the time of diagnosis. Every individual had adverse known risk factors such as recent or current use of antibiotic, corticosteroid and antiviral therapy, recent administration of myeloablative chemotherapy and numerous, prolonged periods of hospitalization. Diarrhea, frequently hemorrhagic, was the most common presenting feature along with fever, abdominal cramps and abdominal distention. Diagnosis was established by the stool-cytotoxin test. Response to standard treatment with oral vancomycin or metronidazole was prompt despite the presence of several adverse prognostic features in these patients. There was only one instance of relapse which was also treated successfully. Several transplant-related variables such as age, sex, underlying malignancy, myelo-ablative regimen, duration of neutropenia, and prophylactic use of oral ampicillin underwent statistical analysis but failed to be predictive of C. difficile infection in such a setting. Finally, C. difficile is not uncommon after autologous PBSCT and must be included in the differential diagnosis in any such patient with diarrhea.

Varicella zoster virus infection associated with high-dose chemotherapy and autologous stem-cell rescue.

A retrospective evaluation of 215 consecutive recipients of high-dose chemotherapy (HDC) and autologous stem cell rescue (ASCR) was conducted to ascertain the incidence, temporal course, and outcome of varicella zoster virus (VZV) infection. Herpes zoster was identified in 40 individuals at a median of 69 days following ASCR. Six of these cases occurred at a median of 33 days prior to ASCR but following the initiation of high doses of stem cell mobilization chemotherapy. Twenty-five percent of patients demonstrated cutaneous or systemic dissemination and 32.5% required medical intervention for post-herpetic neuralgia. All except two individuals received antiviral chemotherapy. One patient with active VZV infection died of multiorgan failure 39 days after ASCR. Multivariate analysis of risk factors disclosed the significance of prophylactic acyclovir use in Herpes simplex virus seropositive individuals in reducing the risk of VZV infection. Moreover, the use of busulfan, thiotepa and carboplatin as the conditioning chemotherapy regimen was associated with an increased risk of subsequent VZV infection. The incidence of VZV reactivation after HDC and ASCR is similar to that observed following bone marrow transplantation but has an earlier onset. This may be related to an earlier induction of immunosuppression by stem cell mobilization chemotherapy administered prior to ASCR. We demonstrated a marked reduction in the proliferative and synthetic capacities of peripheral blood mononuclear cells obtained prior to and following stem cell mobilizing chemotherapy. Moreover, greater than 80% of VZV infections occurred within 6 months following ASCR and late cases were seldom observed compared to allogeneic and autologous bone marrow transplantation. The role of antiviral chemoprophylaxis during the period of maximum immunocompromise needs to be studied further in the HDC-ASCR setting.

Identification of transactivator and nuclear localization domains in the Epstein-Barr virus DNA polymerase accessory protein, BMRF1.

The Epstein-Barr virus (EBV) BMRF1 gene product is an essential component of the viral DNA polymerase and is absolutely required for lytic virus replication. In addition to its polymerase accessory protein function, we recently demonstrated that BMRF1 is a transactivator, inducing expression of the essential oriLyt promoter, BHLF1. However, the regions of BMRF1 required for transactivation of BHLF1 are unknown. Here we demonstrate that the carboxy-terminal portion of the BMRF1 protein (amino acids 378404), although not required for DNA binding or polymerase processivity function, is required for transactivator function as well as nuclear localization. Site-directed mutagenesis of this region allowed us to separate the transactivator and nuclear localization motifs of BMRF1. The two DNA-binding domains of BMRF1 are also required for efficient transactivation of the BHLF1 promoter.

An indicator cell assay for T-cell tropic, macrophage-tropic, and primary isolates of HIV-1 based on green fluorescent protein.

Quantitation of HIV-1 in blood is now widely used by clinicians to manage antiviral therapy. Current methods to detect viral RNA are expensive, have slow turnaround times, and do not directly quantitate infectious particles. Indicator cell assay (ICA) methods for titering HIV-1 rely on the activation of HIV-1 long terminal repeat (LTR)-driven expression of a reporter gene by the viral tat gene product, which is expressed early in the course of infection. The Aequorea victoriana green fluorescent protein (GFP) has proven to be a useful reporter gene for detecting tat-mediated HIV-LTR activation. A general approach to developing a clinically useful ICA required a method of introducing the LTR-GFP expression cassette into various HIV1-infectable cell lines. The LTR-GFP expression cassette was inserted into the LXSN retrovector in a reverse orientation with respect to transcription from the 5' LTR. In cells transduced by the RH5 retrovector, GFP expression was tightly dependent on expression of HIV-1 tat. The PM1 human T-cell line was transduced with RH5 and was further engineered to express the CCR5 HIV-1 CD4 coreceptor constitutively. The resulting cell line, D5-R5, was susceptible to infection by primary HIV-1 strains, macrophage-tropic (M-tropic) and T-cell tropic (T-tropic) laboratory strains, and syncytium-inducing (SI) and and non-SI (NSI) variants. Four days after HIV-1 infection of the indicator cells, GFP expression was detected and quantitated by fluorescence activated cell sorter (FACS), without any false-positive signals. This GFP-based ICA method is of potential use in clinical management of HIV-1, especially in the detection and recovery of drug-resistant virus and the direct determination of antiviral drug sensitivities.

Streptococcus viridans bacteremia following autologous peripheral blood stem cell transplantation.

A retrospective evaluation of 200 consecutive recipients of autologous peripheral blood stem cell transplantation was conducted to ascertain the incidence and outcome of Streptococcus viridans bacteremia as well as to determine the role of prophylactic ampicillin therapy in the peri-transplant setting. Viridans streptococci were isolated from the blood of 35 individuals at a median of 6 days (range 2-8 days) following stem cell infusion. The most common isolates were S. sanguis and S. mitis. All patients received ciprofloxacin orally during the peri-transplant period. Additionally, 79 patients received oral ampicillin prophylactically against gram-positive cocci. Although none of the patients suffered a fatal outcome, three individuals developed respiratory compromise requiring mechanical ventilation. Female sex proved to be the only independent risk factor for viridans streptococcal bacteremia (P=0.04). The shorter duration of neutropenia observed after stem cell transplantation did not impact on the incidence of S. viridans infections. Moreover, the prophylactic use of ampicillin failed to decrease the incidence of viridans sepsis and selected out organisms that were resistant to beta-lactam antibiotics.

Stomatococcus mucilaginosus meningitis in a patient with multiple myeloma following autologous stem cell transplantation.

Bacterial meningitis is an unusual complication of bone marrow transplantation. We report a case of Stomatococcus mucilaginosus meningitis in a patient with multiple myeloma shortly after an autologous peripheral blood stem cell transplant. The infection resolved with a combination of intravenous penicillin G and chloramphenicol, and intrathecal vancomycin.

The Epstein-Barr virus (EBV) DNA polymerase accessory protein, BMRF1, activates the essential downstream component of the EBV oriLyt.

The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the "downstream component"), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various protions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from -588 to -592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF-1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication.

Detection of HIV-1 infection with a green fluorescent protein reporter system.

Several systems for the detection of HIV-1 have been described in which HIV-1-susceptible cells contain a reporter gene (chloramphenicol acetyltransferase, beta-galactosidase, or alkaline phosphatase) under the control of the HIV-1 long terminal repeat (LTR). Upon infection by HIV-1, the expression of the viral tat product increases transcription from the HIV-1 LTR promoter, leading to high-level expression of the reporter gene product. Previously described reporter systems require processing of the cells by lysis, fixation, or other steps following infection to detect the reporter gene product. In the present study, the Aequorea green fluorescent protein S65T variant (GFP-S65T) was used in a reporter system for detecting HIV-1. HeLa-CD4 cells transfected with the plasmid pRH1, which encodes GFP-S65T under the control of the HIV-1 LTR promoter, and either co-transfected with a plasmid encoding the HIV-1 tat product or superinfected with HIV-1, expressed high levels of GFP-S65T, which was readily detected by fluorescence microscopy and fluorescence-activated cell-sorting analysis. The advantages of this system include its simplicity, sensitivity, and ability to detect and sort live HIV-1-infected cells using readily available instruments. The construction of cell lines stably transfected with pRH1 will provide a tool for titering HIV-1 and sorting HIV-1-infected cells.

Functional and physical interactions between the Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1: Effects on EBV transcription and lytic replication.

The Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1 are both essential for lytic EBV replication. BZLF1 is a transcriptional activator which binds directly to the lytic origin of replication (oriLyt) and plays a critical role in the disruption of viral latency. The BMRF1 protein is required for viral polymerase processivity. Here we demonstrate that the BMRF1 gene product functions as a transcriptional activator and has direct (as well as indirect) interactions with the BZLF1 gene product. The BMRF1 gene product activates an essential oriLyt promoter, BHLF1, but does not activate two other early EBV promoters (BMRF1 and BHRF1). Direct interaction between the BMRF1 and BZLF1 gene products requires the first 45 amino acids of BMRF1 and the bZip domain of BZLF1. The effect of the BZLF1-BMRF1 interaction on early EBV transcription is complex and is promoter specific. The oriLyt BHLF1 promoter is activated by either the BZLF1 or BMRF1 gene product alone and is further activated by the combination of the BZLF1 and BMRF1 gene products. Enhanced activation of BHLF1 transcription by the BMRF1-BZLF1 combination does not require direct interaction between these proteins. In contrast, BZLF1-induced activation of the BMRF1 promoter is inhibited in the presence of the BMRF1 gene product. A point mutation in the BZLF1 protein (amino acid 200), which prevents in vitro interaction with the BMRF1 protein but which does not reduce BZLF1 transactivator function, allows the BZLF1 protein to activate the BMRF1 promoter equally well in the presence or absence of the BMRF1 gene product. Therefore, direct interaction between the BZLF1 and BMRF1 proteins may inhibit BZLF1-induced transcription of the BMRF1 promoter. BZLF1 mutated at amino acid 200 is as efficient as wild-type BZLF1 in promoting replication of an oriLyt plasmid. However, this mutation reduces the ability of BZLF1 to induce lytic replication of the endogenous viral genome in D98/HE-R-1 cells. Our results indicate that functional and physical interactions between the BMRF1 and BZLF1 proteins may modulate the efficiency of lytic EBV infection. The BMRF1 gene product clearly has a transcriptional, as well as replicative, role during lytic EBV infection.

Bipartite DNA-binding region of the Epstein-Barr virus BMRF1 product essential for DNA polymerase accessory function.

The Epstein-Barr virus (EBV) BMRF1 gene product is necessary for DNA polymerase catalytic subunit (BALF5) activity in 100 mM ammonium sulfate. To map regions of BMRF1 necessary for polymerase accessory function, linker insertion and deletion mutant BMRF1 polypeptides were expressed by in vitro transcription-translation and assayed for DNA polymerase elongation activity and binding to double-stranded DNA (dsDNA)-cellulose. Amino-terminal deletions up to residue 303 were defective for stimulation of elongation. Deletions between residues 44 and 194 and residues 238 and 303 abolished binding to dsDNA-cellulose. The region from residues 194 to 238, therefore, is necessary for stimulation of BALF5 elongation but dispensable for dsDNA-cellulose binding. Deletion analysis also localized reactive epitopes of two neutralizing monoclonal antibodies to BMRF1 to a carboxy-terminal region which is dispensable for activity. These data suggest that a bipartite DNA-binding region is an essential component of the DNA polymerase accessory function and that the two noncontiguous regions are separated by a region (residues 194 to 217) which is essential for stimulation; therefore, it may interact with the BALF5 catalytic subunit of EBV DNA polymerase. Both EBV BMRF1 and herpes simplex virus UL42 gene products are DNA polymerase accessory proteins which bind dsDNA and increase the processivity of their corresponding catalytic components. Outstanding similarities between their primary amino acid sequences are not evident. However, it appears that their structural organizations are similar.

Resistance to antiviral inhibitors caused by the mutation S889A in the highly-conserved 885-GDTDS motif of the herpes simplex virus type 1 DNA polymerase.

Most point substitutions in the highly-conserved 885-GDTDS motif of the HSV-1 DNA polymerase inactivate polymerase elongation activity. However, in an assay system based on expression by in vitro transcription-translation, the mutant GDTDA (S889A) possessed wild-type elongation activity which was highly resistant to phosphonoacetic acid and acyclovir triphosphate, but retained sensitivity to aphidicolin.

Cooperation of EBV DNA polymerase and EA-D(BMRF1) in vitro and colocalization in nuclei of infected cells.

Expression of the Epstein-Barr virus (EBV) DNA polymerase (EBVpol) open reading frame (BALF5) by in vitro transcription-translation yielded a 116-kDa primary translation product. Enzymatic DNA polymerase activity of the in vitro translated polypeptide required the presence of the 47-kDa BMRF1 (EA-D) gene product. Antiserum raised to the BALF5 gene product expressed in Escherichia coli specifically precipitated a 116-kDa polypeptide in extracts of latently infected lymphoblastoid cells induced for EBV replication. Immunofluorescence microscopy revealed colocalization of the EBVpol and EA-D(BMRF1) to discrete foci within the nuclei of induced cells; however, the blockade of viral DNA synthesis resulted in diffuse nuclear staining patterns for both antigens. Bromodeoxyuridine staining of these discrete foci colocalizing with EBVpol suggests that they are sites of early viral DNA synthesis. These observations suggest that EA-D(BMRF1) may be an accessory protein of the EBV DNA polymerase which colocalizes in vivo with EBVpol to sites of viral DNA replication and cooperates in vitro to form an active EBVpol holoenzyme.

Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis.

Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNA-binding protein (65KDBP), the major DNA-binding protein (infected cell polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA- ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication protein to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.

Site-specific mutagenesis of a highly conserved region of the herpes simplex virus type 1 DNA polymerase gene.

Seven point mutations were introduced into region I of the herpes simplex virus type 1 DNA polymerase, which is most highly conserved among DNA polymerases and has no drug sensitivity markers mapped to it. The functional consequences of these mutations were studied in an in vitro transcription-translation system in which T7 transcripts of cloned polymerase genes were used to generate enzymatically active polypeptides in reticulocyte lysate. Analysis of labeled polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to show any alterations of stability caused by these mutations. The mutations G885R, D886N, T887K, D888A, and G896V lacked polymerase activity and failed to be stimulated by cotranslation of the herpes simplex virus 65-kilodalton DNA-binding protein, whereas R881G and S889A retained both polymerase activity and the capacity to be stimulated by the 65-kilodalton DNA-binding protein.

The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase.

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown. By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol). When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost. However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold. The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant. Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli. The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated. The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity. These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol.