Comparison of Translatability of the HBV Encapsidation Sequence of preC RNA and pregenomic RNA

• Main Authors: Siou-Ying Chiang, 姜秀穎
• Other Authors: Tsung-Sheng Su
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/40868282673311949782

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 93 === Hepatitis B virus (HBV) is a partially double-stranded DNA virus. An encapsidation process is involved in HBV life cycle. Along with viral polymerase, 3.5-kb pregenomic RNA (pg RNA) produced by HBV is enclosed into capsid structure which composed of viral core protein molecules. The cis-element needed for HBV encapisidation is the ε stem-loop RNA structure located on pg RNA, while viral core protein (C) and polymerase (P) serves as trans-factors. Polymerase recognizes and binds to the ε secondary structure near the 5’end of pg RNA, and core protein polymerizes to P-ε complex then forms virus core particle. Precore RNA (preC RNA) is also classified to 3.5-kb genomic RNA and its RNA initiation site is located about 30 nt upstream of pg RNA. Though preC RNA and pg RNA both harbor the encapsidation signal ε, only the pg RNA serves as the template for virus encapsidation. Why is preC RNA not encapsidated? By examining the RNA structure, one can find that the translation initiation codon of preC gene is located 33 nt upstream of ε structure, while core gene initiation codon is located at the 3’ region of ε structure. As the result, Nassal et al. (1990) proposed that, when polymerase binds to ε structure on pg RNA, the 40S ribosomal subunit is blocked for proceeding scanning. Thus, rather then translation, pg RNA is switched to encapsidation process. By contrast, in preC RNA, fully functioned 80S ribosome is able to resolve ε secondary structure, even when polymerase and core protein have already bound to ε structure. To test the translational inactivation of the encapsidation signal model proposed by Nassal et al., in this study, we compare the translation efficiency of preC RNA and pg RNA in resolving the ε structure using lacZ as a reporter. In addition, to mimic the situation of the formation of preassembly encapsidation complex, studies were performed in the presence of P protein or both P and C proteins. Furthermore, to improve the efficiency of preassembly encapsidation complex formation, P protein is provided in cis through IRES sequence. The results indicated that a small difference between the translatability of the HBV encapsidation sequence of preC RNA and pg RNA can be observed when cis-providing P or both P and C proteins. Furthermore, when encapsidation was examined in the shorten version of the test RNAs, selectivity of encapsidation could be observed. We also examined if the cis-assembly property of P and C proteins played a crucial role in encapsidation selectivity in the study of Nassal et al. and found no correlation between the level of cis-expressing P and C proteins and the accessibility for encapsidation. To conclude, in this study, we observed the correlation of inaccessibility for translation and accessibility for encapsidation, though the difference of translatability between preC RNA and pg RNA was small. It is possible that a strong and general repression on protein expression exerted by P protein masks the specific effect being searched for. Furthermore, the designs of prototype and mutant constructs were not proper. The most upstream AUG (C0 AUG) in prototype RNA was changed that might affect the translatability of pg RNA and preC RNA. Particularly, the ε mutation didn’t completely abolish its interaction with P protein. It is worth noting that, in the study, about 20% of pg RNAs were encapsidated, indicating only a small fraction of pg RNA could interact specifically with P protein. Apparently, these factors all contributed to the small translation effect observed.