Identification and characterization of multiple TRIM proteins that inhibit hepatitis B virus transcription

Abstract

Tripartite motif (TRIM) proteins constitute a family of over 100 members that share conserved tripartite motifs and exhibit diverse biological functions. Several TRIM proteins have been shown to restrict viral infections and regulate host cellular innate immune responses. In order to identify TRIM proteins that modulate the infection of hepatitis B virus (HBV), we tested 38 human TRIMs for their effects on HBV gene expression, capsid assembly and DNA synthesis in human hepatoma cells (HepG2). The study revealed that ectopic expression of 8 TRIM proteins in HepG2 cells potently reduced the amounts of secreted HBV surface and e antigens as well as intracellular capsid and capsid DNA. Mechanistic analyses further demonstrated that the 8 TRIMs not only reduced the expression of HBV mRNAs, but also inhibited HBV enhancer I and enhancer II activities. Studies focused on TRIM41 revealed that a HBV DNA segment spanning nucleotide 1638 to nucleotide 1763 was essential for TRIM41-mediated inhibition of HBV enhancer II activity and the inhibitory effect depended on the E3 ubiquitin ligase activity of TRIM41 as well as the integrity of TRIM41 C-terminal domain. Moreover, knockdown of endogenous TRIM41 in a HepG2-derived stable cell line significantly increased the level of HBV preC/C RNA, leading to an increase in viral core protein, capsid and capsid DNA. Our studies have thus identified eight TRIM proteins that are able to inhibit HBV transcription and provided strong evidences suggesting the endogenous role of TRIM41 in regulating HBV transcription in human hepatoma cells.

Figure 1. Identification of TRIMs that regulate HBV viral protein expression, capsid assembly and DNA replication.

HepG2 cells in 12 well plates were co-transfected with 0.4 µg of pHBV1.3 and 0.4 µg of plasmid expressing an indicated TRIM protein or a control vector. 72 hours after transfection, the levels of the secreted HBeAg and HBsAg were determined by ELISA. The amounts of intracellular capsid and capsid DNA were determined by a viral particle gel assay. The levels of AFP in cultured media were determined by ELISA and served as a cellular function and viability control. The levels of HBeAg, HBsAg, capsid, capsid DNA and AFP from the samples co-transfected with pHBV1.3 and an indicated TRIM-expressing plasmid were expressed as a ratio over that obtained from samples co-transfected with pHBV1.3 and vector plasmids. The mean and standard deviations (n = 3) were presented. * and ** indicate P<0.05 and 0.01, respectively.

Figure 2. Eight TRIM proteins can efficiently reduce the amount of HBV mRNA in HepG2 cells.

HepG2 cells in 12 well plates were co-transfected with 0.4 µg of pHBV1.3 and 0.4 µg of vector plasmid or plasmid expressing one of eight TRIM proteins. Two days post transfection, cells were harvested and total cellular RNAs were extracted. The levels of preC/C RNA and total viral mRNA were quantified by a quantitative RT-PCR assay. The mean and standard deviations (n = 3) were presented. * and ** indicate P<0.05 and 0.01, respectively.

Figure 3. Effects of the eight TRIM proteins on HBV Enhancer I and Enhancer II activity in HepG2 cells.

0.2 µg of pGL4.10-HBV Enh II or pGL4.10-HBV Enh I was co-transfected into HepG2 cells in 24 well plates with 0.2 µg of a vector plasmid or a plasmid expressing an indicated TRIM protein, with 0.1 µg of pCMV-renilla luciferase as an internal control. After two days post transfection, cells were harvested and the luciferase activity was analyzed with a dual-luciferase kit. The mean and standard deviations (n = 4) were presented. * and ** indicate P<0.05 and 0.01, respectively.

Figure 4. TRIM41 inhibits HBV replication by reducing the HBV mRNA levels.

HepG2 cells in 12 well plates were co-transfected with 0.4 µg of pHBV1.3 and the indicated amounts of plasmid expressing TRIM41, supplemented with vector plasmid to make a total of 0.8 µg plasmid. (A) Cells were harvested at two days post transfection. The levels of HBV preC/C RNA and total mRNA were quantified by a real-time RT-PCR assay. (B) The amount of intracellular core protein was determined by a Western blot assay. The level of β-actin was used as a loading control. (C and D) The amounts of intracellular capsid (C) and capsid DNA (D) were determined by a viral particle gel assay. (E) HBV RNAs were determined by Northern blot hybridization. The positions of HBV preC/C RNA (3.5 kb), surface RNAs (sRNA; 2.4 kb and 2.1 kb) and HBx RNA (0.7 kb) are indicated. Ribosomal RNAs (28S and 18S) served as loading controls. (F) The cytoplasmic HBV capsid DNA was detected by Southern blot hybridization. The position of viral single strand DNA (ssDNA) is indicated. The mean and standard deviations were presented (n = 3). * and ** indicate P<0.05 and 0.01, respectively.

Figure 5. Mapping the cis element that mediates TRIMs inhibition of HBV Enh II activity.

(A) Schematic representation to map the cis element mediating TRIMs inhibition of HBV Enh II activity. Three overlapping DNA fragments covering the entire HBV Enh II region were placed in the up-stream of a firefly luciferase coding sequence to yield three reporter plasmids, pGL4.10-HBV Enh II-1, pGL4.10-HBV Enh II-2 and pGL4.10-HBV Enh II-3, respectively. (B to D) 0.2 µg of the indicated reporter plasmid was co-transfected into HepG2 cells in 24 well plates with 0.2 µg of a vector plasmid or a plasmid expressing the indicated TRIM proteins, with 0.1 µg of pCMV-renilla luciferase as an internal control. Two days post transfection, cells were harvested and the luciferase activity was analyzed with a dual-luciferase kit. The mean and standard deviations (n = 4) were presented. * and ** indicate P<0.05 and 0.01, respectively.

Figure 6. RING finger and PRYSPRY domains of TRIM41 are essential for the inhibition of HBV Enh II activity.

0.4 µg of plasmids expressing wild type or mutant TRIM41 were transfected into HepG2 in 24 well plates. The cells were harvested at two days post transfection. Expression of TRIM41 was determined by Western blot assays (A) or immunofluorescence (B). (C) 0.2 µg of pGL4.10-HBV Enh II was co-transfected into HepG2 cells in 24 well plates with 0.2 µg of a vector plasmid or plasmid expressing the wild type or indicated mutant TRIM41, with 0.1 µg of pCMV-renilla luciferase as an internal control. Two days post transfection, cells were harvested and the luciferase activity was analyzed with a dual-luciferase kit. The mean and standard deviations (n = 4) were presented. * and ** indicate P<0.05 and 0.01, respectively.

Figure 7. Endogenous TRIM41 inhibits HBV transcription in HepG2 cells.

(A) HepG2 cells in 12 well plates were transfected with 20 nM of scramble siRNA or one of three siRNA specifically targeting TRIM41. Six hours later, the cells were co-transfected with 0.4 µg of pHBV1.3 and 0.4 µg of a plasmid expressing TRIM41. The cells were re-transfected with 20 nM of siRNA 24 hours later and harvested at 48 hours post pHBV1.3 and TRIM41-expressing plasmid transfection. Core protein was tested by Western blot with β-actin as a loading control. Capsid and capsid DNA were analyzed by a particle gel assay. (B) 15 nM of TRIM41 siRNAs or scramble siRNA were transfected into HepG2.2.15. Two days post transfection, cells were harvested. The total cellular RNAs were extracted and reverse transcribed. TRIM41 siRNA knockdown efficiency was tested by quantification of TRIM41 mRNA level with a quantitative RT-PCR assay (upper panel). The level of TRIM41 protein in the transfected cultures were determined by immunoprecipitation and Western blot assays with a polyclonal antibody against human TRIM41 (lower panel). (C) HBV preC/C RNA and total viral mRNA were quantified by a quantitative RT-PCR assay and normalized with β-actin mRNA as an internal control. (D) Core protein, capsid and capsid DNA levels were tested by Western blot or Southern blot assays. The same amounts of cell lysates were loaded after BCA quantification. The level of β-actin was used as a loading control. The mean and standard deviations (n = 4) were presented. * and ** indicate P<0.05 and 0.01, respectively.