Abstracts Poster Abstracts - Dr Falk

98Increased expression of geranylgeranyl pyrophosphate synthaseis associated with hepatitis C virus RNA persistence in peripheralblood mononuclear cellsSidorkiewicz M., Józwiak B., Majda-Stanislawska E., Piekarska A., Bartkowiak J.Department of Medical Biochemistry, Medical University of Lodz, Mazowiecka 6/8,PL-92-215 Lodz, Poland, Tel./Fax: 48 42 678 24 65, E-mail: msidor@zdn.am.lodz.plIntroduction: Although the liver is the main place of HCV replication, HCV can persistin peripheral blood mononuclear cells (PBMC) long after apparently complete hepatitisC resolution [Pham (2004) JVI 78]. Our previous studies have demonstrated that insome patients, who responded to IFN therapy with HCV RNA elimination from sera,HCV genome in PBMC was still detectable. From the other hand recent papers haveshown that HCV replication seems to be regulated by geranylgeranylation process[Kapadia (2005) PNAS 102] which in turn requires geranylgeranyl pyrophosphate(GGPP) presence in host cell and that HCV replication could be disrupted by theinhibition of geranylgeranylation process [Ye (2003) PNAS 26].As geranylgeranyl pyrophosphate synthase is an enzyme critical for GGPP synthesis,the aim of our study was to investigate the relationship between expression level ofGGPP synthase and persistence of HCV RNA in PBMC after elimination HCV RNA fromsera.Methods: HCV RNA was determined in "one-tube" RT-PCR reaction in PBMCoriginated from the patients that eliminated HCV RNA from sera after IFN-alpha therapy.PBMC were isolated from venous blood by centrifugation on Histopaque-1077 gradient.GGPP synthase -specific PCR and beta-actin-specific (control) PCR were performedon 100-times diluted cDNA that was synthesized in RT reaction (Improm, Promega)from 6 microgram of RNA isolated from each PBMC sample in the presence of randomprimers.Results: HCV RNA determinations in PBMC originated from 27 patients that have lostHCV RNA as a result of IFN therapy, let us select two groups of PBMC samples: firstgroup, where HCV RNA was eliminated (15 samples) and the second, where HCVgenome was still detectable (12 samples). In both group stable expression of beta-actingene was confirmed by RT-PCR. The semiquantitative RT-PCR analysis showed thatGGPP synthase expression was markedly altered depending on the selected group.cDNA originated from the PBMC samples that maintained HCV genome revealed over10-times higher GGPP sythase gene expression then cDNA originated from PBMCsamples, where HCV RNA was eliminated.Discussion/Conclusion: Despite of the elimination HCV RNA from sera, HCV genomecan persist in PBMC which is associated with significant elevation of GGPP synthase.GGPP synthesis seems to be essential for sustained HCV replication in PBMC.