Reverse Transcriptase Activity of Hepatitis B Virus Polymerase in Eukaryotic Cell Extracts in Vitro

Reverse Transcriptase Activity of Hepatitis B Virus Polymerase in Eukaryotic Cell Extracts In Vitro Daniel Favre Helpatitis, Lausanne, Switzerland Summary Zusammenfassung: In vitro reverse Transkriptase Aktivität der In hepadnaviruses, reverse transcription is primed by the viral Hepatitis B Virus Polymerase in eukariotischen Zellextrakten reverse transcriptase (RT) and requires the specific interaction In Hepadnaviren wird die reverse Transkription von der viralen between the RT and the viral RNA encapsidation signal termed reversen Transkriptase (RT) ausgeführt. Hier findet eine spezifi- ε. To study the activity of the RT in vitro, the current procedure sche Interaktion zwischen der RT und dem viralen Verpackungs- uses in vitro translated duck hepatitis B virus polymerase, but signal ε statt. Um die Aktivität der RT in vitro zu untersuchen, not the hepatitis B virus polymerase itself, in the rabbit reticulo- wird im aktuellen Protokoll in vitro translatierte Entenhepatitis cyte lysate expression system. B Virus Polymerase, aber nicht die Hepatitis B Virus Polyme- Here, the hepatitis B virus (HBV) polymerase has been suc- rase selbst, in einem Kaninchen Retikulozytenlysat Expressions- cessfully expressed in a translational extract that was obtained system eingesetzt. from monolayer human hepatocyte cells HuH-7. The translated In dieser Arbeit wurde die Hepatitis B Virus (HBV) Polymerase polypeptide retained the RNA-directed polymerase (reverse erfolgreich in einem translationalen Extrakt aus einer einlagigen transcriptase) activity on the viral RNA template containing the Kultur der humanen Hepatozytenzellen HuH-7 exprimiert. Das ε signal. We suggest that the reverse transcription event of the translatierte Polypeptid behielt die RNA-gerichtete Polymerase viral RNA coding for the polymerase and containing an ε struc- (reverse Transkriptase) Aktivität auf dem viralen RNA Template ture is concomitant to the translation of the viral polymerase to mit einem ε Signal. Wir schlagen vor, dass die reverse Transkrip- the messenger RNA. In contrast to the duck polymerase, only a tion der viralen RNA, welche die Polymerase kodiert und eine fraction of the reverse transcribed complementary DNA (cDNA) ε Struktur besitzt, gleichzeitig mit der Translation der viralen was covalently bound to the HBV polymerase in this system. Polymerase in die Boten-RNA (mRNA) abläuft. Im Gegensatz When the ε signal was missing on the mRNA, the translated zur Entenpolymerase war nur ein geringer Anteil der revers full-length HBV polymerase could not reverse transcribe the vi- transkribierten komplementär DNA (cDNA) in diesem System ral RNA template. A truncated HBV polymerase that was lack- kovalent an die HBV Polymerase gebunden. Wenn die mRNA ing the YMDD catalytic active site for the initiation of reverse kein ε Signal enthielt, konnte die translatierte HBV Polymera- transcription was unable to reverse transcribe the viral mRNA se das virale RNA Template nicht revers transkribieren. Eine template containing the ε signal. The reverse transcription ac- verkürzte HBV Polymerase, welcher die katalytische Aktiv- tivity could also be partially inhibited by employing nucleoside domäne YMDD zur Initiation der reversen Transkription analogues, such as 2’-3’-dideoxy-3’-thiacytidine (3TC; lamivu- fehlte, konnte das virale mRNA Template trotz ε Signal nicht dine) in the expression system. revers transkribieren. Die reverse Transkriptionsaktivität The procedure described here provides a method for the in vitro konnte auch partiell durch den Einsatz von Nukleosidanaloga, screening of new anti-HBV compounds directed against wild- wie 2’-3’-Dideoxi-3’-thiacytidin (3TC; Lamivudin) im Expressi- type and mutants of this crucial viral protein, the HBV polymer- onssystem inhibiert werden. ase, without the use of animals (ducks) or animal extracts (rab- Diese Arbeit beschreibt eine Methodik mit der in vitro bit reticulocyte lysate). neue anti-HBV Wirkstoffen gegen Wildtyp und Mutanten dieses zentralen viralen Proteins, der HBV Polymerase, ohne den Ein- satz von Tieren (Enten) oder tierischen Extrakten (Kaninchen Retikulozytenlysat) gesucht werden können. Keywords: hepatitis B virus, HBV, viral polymerase, reverse transcriptase, hepatocyte, cell extract, HuH-7, translation, RNA, reverse transcription, inhibitors Received 3rd March 2008; received in final form and accepted for publication 28th June 2008 ALTEX 25, 3/08 197 FAVRE 1 Introduction polymerase is translated in vitro in a first of the RT reaction, was unable to reverse step and thereafter the reverse transcrip- transcribe the template RNA despite the Hepatitis B virus (HBV) is a major pub- tion activity is analysed in a subsequent ε signal. The reverse transcriptase activ- lic health problem with over 350 million reaction involving radiolabelled ribonucle- ity was tributary to the new translation of chronically infected people worldwide. otides. To date, all attempts at producing the HBV polymerase, since no RT activ- HBV, as a member of the hepadnavirus the biologically active HBV polymerase ity was detected in the presence of both family, is an enveloped virus with par- itself in the reticulocyte lysate system have the mRNA and an inhibitor of the initia- tially double-stranded DNA. The viral in- not been fully convincing (Jeong et al., tion of the protein synthesis. As expected, fection is associated with the development 1996; Kim and Jung, 1999). It was hypoth- the use of various dideoxynucleotides for of hepatocellular carcinoma and liver cir- esised that this might be due to the lack the inhibition of the reverse transcriptase rhosis. Although HBV is a DNA virus, the of additional protein factors or divalent activity revealed that the incorporation of mode of replication involves reverse tran- cations (Jeong et al., 1996; Li and Tyrrell, the first nucleotides was a T followed by scription of an RNA pregenome, a process 1999). Finally, several attempts have also G and A, respectively. The additional use that occurs in intracellular viral cores. been made to produce the HBV polymer- of the nucleoside analogue 2’-3’-dideoxy- The unique virus-encoded reverse tran- ase in insect cells by employing the bacu- 3’-thiacytidine triphosphate (3TC; lami- scriptase (RT) is able to initiate DNA lovirus expression system (in which only a vudine) inhibited the RT reaction by about synthesis de novo, using the RT itself as a very minor percentage of the polymerase is 40% in this system. The RT activity of protein primer (Hu and Seeger, 1996). The biologically active) (Lanford et al., 1997), the HBV polymerase was also obtained protein priming requires the specific inter- in E. coli (Jeong et al., 1996) or in yeast in the rabbit reticulocyte lysate in which action between the RT and a short RNA (Tavis and Ganem, 1993) in order to pro- the translation reaction was concomitantly signal, termed ε, located at the 5’ end of the duce large amounts for further biochemi- performed with the RT reaction; however pregenomic RNA (pgRNA) that serves as a cal and structural studies. the efficiency was lower than that obtained template for the reverse transcription (Nas- A novel and simple in vitro system for in the translational extracts prepared from sal and Rieger, 1996). For the polymerase the rapid analysis of the HBV polymerase eukaryotic cells grown as monolayers. protein of DHBV, but not that of HBV, the is thus needed. Recently, we developed The above results support the conclu- priming reaction for reverse transcription an efficient cell-free translational system sion that, in addition to the presence of could be efficiently reconstituted by in using eukaryotic cells grown as monolay- the very conserved YMDD catalytic mo- vitro translation of the protein in the rab- ers (Favre and Trépo, 2001). This expres- tif in the viral polypeptide and the pres- bit reticulocyte lysate (Wang and Seeger, sion system is particularly suited for the ence of an ε signal on the template RNA, 1992). This event required the presence translation of exogenous viral mRNAs the reverse transcriptase activity of the of a functional ε signal on the mRNA originating from various sources. By em- HBV polymerase is only detected dur- (Beck and Nassal, 1998). In the case of ploying an in vitro transcribed viral RNA ing a reaction in which the translation HBV, some reports show that the puri- coding for the full length HBV polymer- and the reverse transcriptase reactions are fied polymerase protein alone efficiently ase, which also comprised an ε structure concomitantly performed. The generation reverse transcribes the mRNA containing at the 5’ end, the HBV polypeptides with of an in vitro assay for the study of the the ε signal (Jeong et al., 1996; Lanford the expected sizes of 94 and 81 kDa were reverse transcriptase activity of the HBV et al., 1997). The catalytic site is located successfully translated in a cytoplasmic polymerase will undoubtedly allow broad in the YMDD nucleus of the polymerase extract that was obtained from the hepa- in vitro screening for new antiviral mol- (Jeong et al., 1996) and is susceptible to tocyte cell line HuH-7. When the trans- ecules directed against this important pro- mutation during antiviral therapy. In in- lation of the HBV polymerase was con- tein. Moreover, it might be employed for fected patients, the HBV polymerase is comitantly performed with the reverse the ad hoc in vitro screening of antiviral expressed by internal initiation and acts transcription reaction, the incorporation molecules directed against mutants of

Load more