Systemic sclerostin antibody treatment increases osseointegration and biomechanical competence of zoledronic-acid-coated dental implants in a rat osteoporosis model.

Osseointegration of dental implants can be promoted by implant-surface modifications using bisphosphonate coatings. In addition, it is of clinical interest to promote peri-implant bone formation and to restore bony structure in low bone-mass patients. The present study evaluated a combination of an anti-resorptive zoledronic acid (ZOL) implant-coating and a systemically applied sclerostin antibody, a known bone anabolic treatment principle, versus sole sclerostin antibody treatment or ZOL implant-coating in a rat osteoporosis model. Uncoated reference surface implants or ZOL-coated implants (n = 64/group) were inserted into the proximal tibia of aged osteoporotic rats three months following ovariectomy. 32 animals of each group received once weekly sclerostin antibody therapy. Osseointegration was assessed 2 or 4 weeks post-implantation by ex vivo µCT, histology and biomechanical testing. Overall implant survival rate was 97 %. Histomorphology revealed pronounced bone formation along the entire implant length of ZOL-coated implants. At 4 weeks following implant insertion, bone-implant contact, cancellous bone mineral density and bone volume/tissue volume were significantly increased for the combination of ZOL and sclerostin antibody as compared to sclerostin antibody or ZOL implant-coating alone. Removal torque was also significantly increased in the combination therapy group relative to animals receiving only sclerostin antibody therapy or ZOL-coated implants. In an osteoporotic rat model, the combination of anti-resorptive ZOL implant-coating and systemically applied sclerostin antibody led to significantly increased peri-implant bone formation. Therefore, the combination of ZOL and the osteoanabolic sclerostin antibody was more effective than either agent alone.

Hepatitis C: The beginning of the end-key elements for successful European and national strategies to eliminate HCV in Europe.

Hepatitis C virus (HCV) infection is a major public health problem in the European Union (EU). An estimated 5.6 million Europeans are chronically infected with a wide range of variation in prevalence across European Union countries. Although HCV continues to spread as a largely "silent pandemic," its elimination is made possible through the availability of the new antiviral drugs and the implementation of prevention practices. On 17 February 2016, the Hepatitis B & C Public Policy Association held the first EU HCV Policy Summit in Brussels. This summit was an historic event as it was the first high-level conference focusing on the elimination of HCV at the European Union level. The meeting brought together the main stakeholders in the field of HCV: clinicians, patient advocacy groups, representatives of key institutions and regional bodies from across European Union; it served as a platform for one of the most significant disease elimination campaigns in Europe and culminated in the presentation of the HCV Elimination Manifesto, calling for the elimination of HCV in Europe by 2030. The launch of the Elimination Manifesto provides a starting point for action in order to make HCV and its elimination in Europe an explicit public health priority, to ensure that patients, civil society groups and other relevant stakeholders will be directly involved in developing and implementing HCV elimination strategies, to pay particular attention to the links between hepatitis C and social marginalization and to introduce a European Hepatitis Awareness Week.

Addressing barriers to the prevention, diagnosis and treatment of hepatitis B and C in the face of persisting fiscal constraints in Europe: report from a high level conference.

In the WHO-EURO region, around 28 million people are currently living with chronic viral hepatitis, and 120,000 people die every year because of it. Lack of awareness and understanding combined with the social stigma and discrimination exacerbate barriers related to access to prevention, diagnosis and treatment services for those most in need. In addition, the persisting economic crisis has impacted on public health spending, thus posing challenges on the sustainable investment in promotion, primary and secondary prevention, diagnosis and treatment of viral hepatitis across European countries. The Hepatitis B and C Public Policy Association in cooperation with the Hellenic Center for Disease Prevention and Control together with 10 partner organizations discussed at the Athens High Level Meeting held in June 2014 recent policy developments, persisting and emerging challenges related to the prevention and management of viral hepatitis and the need for a de minimis framework of urgent priorities for action, reflected in a Call to Action (Appendix S1). The discussion confirmed that persisting barriers do not allow the full realisation of the public health potential of diagnosing and preventing hepatitis B and C, treating hepatitis B and curing hepatitis C. Such barriers are related to (a) lack of evidence-based knowledge of hepatitis B and C, (b) limited access to prevention, diagnosis and treatment services with poor patient pathways, (c) declining resources and (d) the presence of social stigma and discrimination. The discussion also confirmed the emerging importance of fiscal constraints on the ability of policymakers to adequately address viral hepatitis challenges, particularly through increasing coverage of newer therapies. In Europe, it is critical that public policy bodies urgently agree on a conceptual framework for addressing the existing and emerging barriers to managing viral hepatitis. Such a framework would ensure all health systems share a common understanding of definitions and indicators and look to integrate their responses to manage policy spillovers in the most cost-effective manner, while forging wide partnerships to sustainably and successfully address viral hepatitis.

Cholinergic nerve fibers in bone defects of a rat osteoporosis model and their regulation by implantation of bone substitution materials.

OBJECTIVES: Bone is innervated by autonomic nervous system that consists of sympathetic and parasympathetic nerves that were recently identified in bone. Thus we asked whether parasympathetic nerves occur in bone defects and at the interface of substitution materials that were implanted for stabilization and improvement of healing in an osteoporosis animal model. METHODS: Osteoporosis was induced in rats by ovariectomy and deficiency diet. A wedge-shaped osteotomy was performed in the metaphyseal area of femur. Eight different implants were inserted that were based on calcium phosphate cement, iron, silica-mineralized collagen, and modifications with strontium. Nerves were identified by immunohistochemistry with antibodies against vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and protein gene product 9.5 (PGP 9.5) as neuronal marker. RESULTS: Cholinergic nerves identified with VAChT immunostaining were detected in defects filled with granulation tissue and in surrounding mast cells. No immunolabeling of cholinergic nerves was found after implantation. The general presence of nerves was reduced after implantation as shown by PGP 9.5. Sympathetic nerves identified by TH immunolabeling were increased in strontium functionalized materials. CONCLUSION: Since cholinergic innervation was diminished after implantation a further increase in the compatibility of substitution materials to nerves could improve defect healing especially in osteoporotic bone.

Historical epidemiology of hepatitis C virus (HCV) in selected countries.

Chronic infection with hepatitis C virus (HCV) is a leading indicator for liver disease. New treatment options are becoming available, and there is a need to characterize the epidemiology and disease burden of HCV. Data for prevalence, viremia, genotype, diagnosis and treatment were obtained through literature searches and expert consensus for 16 countries. For some countries, data from centralized registries were used to estimate diagnosis and treatment rates. Data for the number of liver transplants and the proportion attributable to HCV were obtained from centralized databases. Viremic prevalence estimates varied widely between countries, ranging from 0.3% in Austria, England and Germany to 8.5% in Egypt. The largest viremic populations were in Egypt, with 6,358,000 cases in 2008 and Brazil with 2,106,000 cases in 2007. The age distribution of cases differed between countries. In most countries, prevalence rates were higher among males, reflecting higher rates of injection drug use. Diagnosis, treatment and transplant levels also differed considerably between countries. Reliable estimates characterizing HCV-infected populations are critical for addressing HCV-related morbidity and mortality. There is a need to quantify the burden of chronic HCV infection at the national level.

Strategies to manage hepatitis C virus (HCV) disease burden.

The number of hepatitis C virus (HCV) infections is projected to decline while those with advanced liver disease will increase. A modeling approach was used to forecast two treatment scenarios: (i) the impact of increased treatment efficacy while keeping the number of treated patients constant and (ii) increasing efficacy and treatment rate. This analysis suggests that successful diagnosis and treatment of a small proportion of patients can contribute significantly to the reduction of disease burden in the countries studied. The largest reduction in HCV-related morbidity and mortality occurs when increased treatment is combined with higher efficacy therapies, generally in combination with increased diagnosis. With a treatment rate of approximately 10%, this analysis suggests it is possible to achieve elimination of HCV (defined as a >90% decline in total infections by 2030). However, for most countries presented, this will require a 3-5 fold increase in diagnosis and/or treatment. Thus, building the public health and clinical provider capacity for improved diagnosis and treatment will be critical.

The present and future disease burden of hepatitis C virus (HCV) infection with today's treatment paradigm.

The disease burden of hepatitis C virus (HCV) is expected to increase as the infected population ages. A modelling approach was used to estimate the total number of viremic infections, diagnosed, treated and new infections in 2013. In addition, the model was used to estimate the change in the total number of HCV infections, the disease progression and mortality in 2013-2030. Finally, expert panel consensus was used to capture current treatment practices in each country. Using today's treatment paradigm, the total number of HCV infections is projected to decline or remain flat in all countries studied. However, in the same time period, the number of individuals with late-stage liver disease is projected to increase. This study concluded that the current treatment rate and efficacy are not sufficient to manage the disease burden of HCV. Thus, alternative strategies are required to keep the number of HCV individuals with advanced liver disease and liver-related deaths from increasing.

The state of hepatitis B and C in the Mediterranean and Balkan countries: report from a summit conference.

The burden of disease due to chronic viral hepatitis constitutes a global threat. In many Balkan and Mediterranean countries, the disease burden due to viral hepatitis remains largely unrecognized, including in high-risk groups and migrants, because of a lack of reliable epidemiological data, suggesting the need for better and targeted surveillance for public health gains. In many countries, the burden of chronic liver disease due to hepatitis B and C is increasing due to ageing of unvaccinated populations and migration, and a probable increase in drug injecting. Targeted vaccination strategies for hepatitis B virus (HBV) among risk groups and harm reduction interventions at adequate scale and coverage for injecting drug users are needed. Transmission of HBV and hepatitis C virus (HCV) in healthcare settings and a higher prevalence of HBV and HCV among recipients of blood and blood products in the Balkan and North African countries highlight the need to implement and monitor universal precautions in these settings and use voluntary, nonremunerated, repeat donors. Progress in drug discovery has improved outcomes of treatment for both HBV and HCV, although access is limited by the high costs of these drugs and resources available for health care. Egypt, with the highest burden of hepatitis C in the world, provides treatment through its National Control Strategy. Addressing the burden of viral hepatitis in the Balkan and Mediterranean regions will require national commitments in the form of strategic plans, financial and human resources, normative guidance and technical support from regional agencies and research.

Osseointegration of biochemically modified implants in an osteoporosis rodent model.

The present study examined the impact of implant surface modifications on osseointegration in an osteoporotic rodent model. Sandblasted, acid-etched titanium implants were either used directly (control) or were further modified by surface conditioning with NaOH or by coating with one of the following active agents: collagen/chondroitin sulphate, simvastatin, or zoledronic acid. Control and modified implants were inserted into the proximal tibia of aged ovariectomised (OVX) osteoporotic rats (n = 32/group). In addition, aged oestrogen competent animals received either control or NaOH conditioned implants. Animals were sacrificed 2 and 4 weeks post-implantation. The excised tibiae were utilised for biomechanical and morphometric readouts (n = 8/group/readout). Biomechanical testing revealed at both time points dramatically reduced osseointegration in the tibia of oestrogen deprived osteoporotic animals compared to intact controls irrespective of NaOH exposure. Consistently, histomorphometric and microCT analyses demonstrated diminished bone-implant contact (BIC), peri-implant bone area (BA), bone volume/tissue volume (BV/TV) and bone-mineral density (BMD) in OVX animals. Surface coating with collagen/chondroitin sulphate had no detectable impact on osseointegration. Interestingly, statin coating resulted in a transient increase in BIC 2 weeks post-implantation; which, however, did not correspond to improvement of biomechanical readouts. Local exposure to zoledronic acid increased BIC, BA, BV/TV and BMD at 4 weeks. Yet this translated only into a non-significant improvement of biomechanical properties. In conclusion, this study presents a rodent model mimicking severely osteoporotic bone. Contrary to the other bioactive agents, locally released zoledronic acid had a positive impact on osseointegration albeit to a lesser extent than reported in less challenging models.

[Waiting time for and length of physicianc's consultation define the patient-physician interaction: results of a prospective study in 478 patients with chronic viral hepatitis]. / Wartezeiten und Länge des Arztgesprächs bestimmen aus Patientensicht die Qualität der Arzt-Patienten-Beziehung bei chronischer Hepatitis B und C: Ergebnisse einer prospektiven Studie bei 478 Patienten mit chronischer Virushepatitis.

BACKGROUND: Little is known about the physician-patient interaction in hepatitis B and C. METHODS: This study by the federal competence network hepatitis analysed the physician-patient interaction using the validated FAPI questionnaire. The questionnaire also contained questions concerning demography and disease characteristics. Of the total 1500 questionnaires sent out, 478 were returned (32 %) (20 % HBV vs. 80 % HCV). RESULTS: The FAPI index of patients with HBV/HCV infection (3.10 +/- 0,99) was lower than that in patients with other internal medicine diseases (3.61 +/- 0.92; n = 148). Women had lower values than men (2.98 vs. 3.25; p = 0.005). Patients with active HBV infection showed higher values than those with HCV infection (3.27 vs. 2.97; p < 0.05). Patients with successfully treated hepatitis B/C had higher values than those with active disease (3.36 vs. 3.02; p = 0.004). The index was lower in patients who waited > 2 weeks for the consultation when compared to those with a shorter wait (2.92 vs. 3.31; p < 0.001) and was also lower in patients who waited > 30 min at the consultation day when compared to those with a shorter wait (2.81 vs. 3.39; p < 0.001). The index gradually increased with the consultation length (2.47 < 10 min vs. 2.79 10 - 15 min vs. 3.21 15 - 30 min vs. 3.82 > 30 min; p < 0.001). The index was higher in patients seen by gastroenterologists (3.43) when compared with general practioners (3.10), internists (3.02) and clinical settings (3.13) (p < 0.05). A good information status was associated with higher FAPI values when compared with a bad information status (3.43 vs. 2.76; p < 0.001). Fibrosis, health insurance and age were not associated with FAPI index (p > 0.2). By multivariate analysis a long consultation, a good information status, patients' patience, short waiting times, and providing contact to a patient support group were independently associated with a high index. CONCLUSIONS: The physician-patient interaction in chronic viral hepatitis is worse than in other internal medicine diseases with problems being more pronounced in HCV infection and women when compared to men and HBV infection. Short waiting times and patients' patience ameliorated the interaction as well as the consultation length, the information status of the patient and providing contact to a patient support group. Type of health insurance did not affect either waiting times or physician-patient interaction; thus there is no hint for a two-class medicine in this part of hepatology.

[Changes in socio-economics, quality of life and knowledge of patients with chronic hepatitis C during the Hepatitis Competence Net Project]. / Entwicklung von sozioökonomischen Charakteristika, Lebensqualität und Wissensstand bei Patienten mit Hepatitis C während des Projektes Kompetenznetz Hepatitis.

AIMS AND METHODS: The study of the Patient Support Group Deutsche Leberhilfe e.V. and the Federal Hepatitis Competence Net prospectively analysed questionnaires about quality of life (SF12) and socio-economical data of patients with chronic hepatitis C (CHC). A 1st questionnaire 3 1/2 years ago gathered information in 714 CHC patients all of whom now received a 2nd questionnaire which was sent back by 503 subjects (71%). RESULTS: Both mental and physical SF 12 scores remained markedly decreased compared with scores for the general population which approximate 50 points (p<0.001). When compared to values obtained 3S years ago, mental scores increased from 41.2 to 42.5 and physical scores from 41.9 to 42.9 (changes not significant by analysis of variance; p>0.05). However, in patients with negative HCV-RNA, physical and mental scores significantly increased by 6.9 and 4.0 points (p<0.01). In contrast, mental and physical scores decreased by 2.4 and 2.5 points during follow-up in patients with fibrosis (Metavir F2-4) (p<0.05). SF 12-scores closely correlated with degrees of inflammation and fibrosis (p<0.001, respectively). Quality of life was associated with socio-economical data and gradually increased with higher school graduation. A high percentage of patients with CHC were unemployed (18%); of patients aged 65 years, 25% were already retired; 60% of the retired subjects received a disability pension, 37% because of CHC. Unemployed patients had lower SF 12 scores than patients with a job. Subjects with public employers had significantly lower SF 12 scores than privately or self-employed subjects. Complications of CHC markedly reduced SF 12 scores. Just 37% of subjects had a life insurance and only 9% had an insurance of occupational invalidity (values 2-3-times lower than those in the general German population). Insurance applications for life and occupational invalidity had been denied in 17 and 53%, respectively; the denial values were even higher than in the 1st questionnaire. CONCLUSIONS: The prospective follow-up shows that quality of life continuously improves after elimination of HCV, whereas mental and physical health get increasingly worse with ongoing fibrosis. The analysis demonstrates that many CHC subjects have severe socio-economical problems leading to unemployment, early retirement and lack of appropriate insurances. These problems further reduce the quality of life in CHC.

[Socio-economical aspects, quality of life and state of knowledge in hepatitis B patients. Socio-economical aspects in hepatitis B]. / Sozioökonomische Charakteristika, Lebensqualität und Wissensstand bei Patienten mit Hepatitis-B-Virusinfektion in Deutschland. Sozioökonomische Aspekte bei Hepatitis B.

BACKGROUND: Little is known about socio-economical consequences and information status of patients with chronic hepatitis B virus (HBV) infection. AIMS AND METHODS: The present study prospectively analyzed questionnaires about socio-economical consequences and information status including the SF12 quality-of-life analysis in HBV-infected subjects. Overall 1500 questionnaires were distributed by clinics, practioners, patient support groups and internet; 255 questionnaires were sent back. Results were compared with a recent study in 714 HCV infected patients (Z Gastroenterol 2006; 44: 305-317). RESULTS: HBV-infected patients were younger (mean 46 vs. 52 years), more likely to be male (62 vs. 44%) and to come from abroad (30 vs. 9%) when compared with HCV-infected subjects. Only 1 and 4% of HBV- and HCV-infected subjects, respectively, considered the public information about hepatitis as good or very good, 73 and 77%, however, as bad or very bad. Mental and physical quality-of-life (SF12) was better in HBV- than in HCV-infected subjects, but reduced when compared with a sex- and age-matched general population (p < 0.001). Quality-of-life decreased with increases in HBV-DNA, fibrosis and inflammation. In both HBV- and HCV-infected subjects there were information deficits concerning the risks for infection; some of these were more pronounced in HBV-infected subjects when compared to HCV-infected ones. German subjects with HBV and HCV infection are in general well informed about their infection (73-87% knew ALT and histology results); however, HBV-infected subjects are less well informed in particular about viral load and HBeAg (59 and 30%) when compared with HCV infected subjects who knew HCV-RNA and genotype in 80-85%. CONCLUSIONS: The information deficits about viral load are of concern for HBV-infected subjects because these data are more important in HBV than in HCV infection. This lack of information likely reflects a lack of attentiveness towards HBV-DNA levels by the patients' physician. Both HBV- and HCV-infected subjects have problems at work and with various insurances; both have a reduced quality-of-life which correlates with viral load and degree of inflammation and fibrosis. Both populations consider the public information status about viral hepatitis to be bad.

[Socioeconomic characteristics, quality of life, and state of knowledge of patients with hepatitis C viral infection in Germany--socioeconomic aspects in hepatitis C]. / Sozio-ökonomische Charakteristika, Lebensqualität und Wissensstand bei Patienten mit Hepatitis-C-Virusinfektion in Deutschland--Sozio-ökonomische Aspekte bei Hepatitis C.

BACKGROUND: Little is known as yet about the socio-economic consequences for patients with hepatitis C in Germany. AIMS AND METHODS: The study of the Deutsche Leberhilfe e. V., supported by the federal hepatitis competence net, prospectively analyzed questionnaires about quality-of-life, education and work situation, insurance, and various other socio-economical aspects of patients with chronic hepatitis C. The questionnaire included questions about the information status of patients concerning hepatitis C in general and their individual disease. Overall, 1500 questionnaires were distributed by clinics, general practitioners, patient-support groups and via the internet; 714 were sent back and analyzed. RESULTS: Most of the 714 patients were born in Germany; 56 % were women and 44 % men, with a mean age of 52 years and a hepatitis duration of 18 years. More than 60 % of subjects younger than 65 years of age did not have a regular job, and 27 % were already retired. Only 47 % had a sufficient retirement insurance, whereas almost all had a health insurance. Only 12 % had an insurance covering work invalidity, and of those who had applied for the latter insurance, it was denied in 29 %. About 80 % of subjects reported that the hepatitis disturbed various aspects of their life. Only 4 % considered the public knowledge about hepatitis C as good or very good, but 80 % as bad or very bad. Of the subjects 40 % did not know how they had been infected; 37 % considered blood products as their infection mode, but only 10 % drug abuse. Almost all subjects knew that HCV cannot be transmitted via shaking hands, use of bathrooms, kisses or food (< 1 %, respectively). Surgery (17 %) and the dentist (15 %), however, were mentioned relatively often as a major risk for infection. About 80 % of subjects knew recent quantitative data on ALT and HCV-RNA, their genotype and the results of liver biopsy. Both mental and physical scores in the SF12 questionnaire were markedly reduced by about one standard deviation in subjects with HCV infection when compared with the general German population. Mental and physical scores deteriorated with increases in inflammatory and fibrosis scores. Subjects with negative HCV-RNA and normal ALT had the best quality of life, whereas subjects with high levels of HCV-RNA and ALT had the worst. CONCLUSIONS: The data show that the public opinion is wrong when pretending that hepatitis C today is just a disease of drug addicts. Our analysis demonstrates for the first time that many HCV-infected subjects in Germany have problems with their insurance and jobs. German subjects are well informed about their infection including genotype, liver histology, ALT and HCV-RNA; on the other hand, there are information deficits and fears concerning the mode of infection. The recent analysis clearly shows that HCV-infected subjects consider the public information about the HCV infection as catastrophically bad. The recent data in addition show that elimination of HCV decisively ameliorates quality of life, whereas mental and physical health get increasingly worse with progressive liver disease and unsuccessful antiviral therapies.

Amino acids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro.

Although p48 is the most conserved subunit of mammalian DNA polymerase alpha-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

Different regions of primase subunit p48 control mouse polyomavirus and simian virus 40 DNA replication in vitro.

DNA polymerase alpha-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N- and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.

Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit.

DNA polymerase alpha-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase alpha-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.

A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.