Hepatitis B virus (HBV), which causes liver cirrhosis and hepatocellular carcinoma, has a small, compact genome. The only transcript required for genome replication is the 3.5-kb pregenomic (pg) RNA. It serves as the dicistronic mRNA for the translation of both core and P proteins, and also as the genome precursor to be encapsidated and converted by the P protein to double stranded DNA. Since a capsid is assembled from 240 copies of core protein but packages probably just one molecule of P protein, efficient genome replication requires a proper ratio of core / P protein translation. P protein translation involves ribosomal leaky scanning of upstream AUG codons including those of the core gene, but the control mechanisms remain ill defined. Genotype G harbors a unique 36-nt insertion at the 5'end of its core gene, which markedly enhances core protein translation whether in genotype G or when artificially introduced to other genotypes. This increase in core protein translation is rather associated with impaired genome replication in non-G genotypes, most likely due to a corresponding reduction in P protein translation. Paradoxically, deleting the 36nt from genotype G also impaired genome replication. We propose that the unique structural features of genotype G provide a window of opportunity to elucidate the control mechanisms regulating the translation of core vs. P protein.
Aim 1 of this R21 grant application will verify the hypothesis that the insertion creates a hairpin structure downstream of core gene AUG to augment translation initiation.
Aim 2 will validate a small open reading frame upstream of the core gene (the uORF) as a positive regulator of P protein translation.
Aim 3 will investigate why genotype G harbors two nonsense mutations in the precore region. Besides pg RNA, HBV produces another 3.5-kb RNA with about 30-nt extension at the 5'end. This precore (pc) RNA is devoted exclusively to the translation of precore/core protein, the precursor to hepatitis B e antigen (HBeAg). The late stage of chronic HBV infection often selects for the G1896A nonsense mutation in the precore region to abolish HBeAg expression. Curiously, genotype G harbors an extra C1817T nonsense mutation at codon 2. We propose that C1817T enables translational re-initiation at the uORF instead of the core gene, thus redirecting the pc RNA towards P protein translation and rescuing genotype G replication despite the 36-nt insertion. Our studies will clarify translational control of core and P protein expression, and help understand how different HBV genetic variants avoid deviating from an optimal core/P protein ratio required for efficient genome replication.

Public Health Relevance

Hepatitis B virus genotype G has unusual structural features and clinical manifestations. We will employ genotype G to investigate translational control of core and P proteins, the two viral components essential for genome replication. Our study will help understand why genotype G infection is associated with great risk for liver fibrosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI107618-01
Application #
8571336
Study Section
Virology - B Study Section (VIRB)
Program Officer
Koshy, Rajen
Project Start
2013-08-06
Project End
2015-07-31
Budget Start
2013-08-06
Budget End
2014-07-31
Support Year
1
Fiscal Year
2013
Total Cost
$224,190
Indirect Cost
$83,190
Name
Rhode Island Hospital
Department
Type
DUNS #
075710996
City
Providence
State
RI
Country
United States
Zip Code
02903
Lee, Jiwon; Zong, Li; Krotow, Alexander et al. (2018) N-Linked Glycosylation Is Not Essential for Sodium Taurocholate Cotransporting Polypeptide To Mediate Hepatitis B Virus Infection In Vitro. J Virol 92:
Zong, Li; Qin, Yanli; Jia, Haodi et al. (2017) Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region. Virology 505:155-161
Qin, Yanli; Wang, Yong-Xiang; Zhang, Jiming et al. (2017) Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization. Methods Mol Biol 1540:219-226
Zhang, Fei; Tang, Xiaoli; Garcia, Tamako et al. (2017) Characterization of contrasting features between hepatitis B virus genotype A and genotype D in small envelope protein expression and surface antigen secretion. Virology 503:52-61
Tong, Shuping; Revill, Peter (2016) Overview of hepatitis B viral replication and genetic variability. J Hepatol 64:S4-S16
Zong, Li; Qin, Yanli; Jia, Haodi et al. (2016) Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers. J Virol Methods 233:46-50
Li, Jisu; Zong, Li; Sureau, Camille et al. (2016) Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor. J Virol 90:8302-13
Shapiro, Jason M; Chung, Waihong; Ogawa, Kosuke et al. (2016) Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase. PLoS One 11:e0165227
Li, Jisu; Tong, Shuping (2015) From DCPD to NTCP: the long journey towards identifying a functional hepatitis B virus receptor. Clin Mol Hepatol 21:193-9
Tong, Shuping; Li, Jisu (2014) Identification of NTCP as an HBV receptor: the beginning of the end or the end of the beginning? Gastroenterology 146:902-5