The Dihydroquinolizinone Compound RG7834 Inhibits the Polyadenylase Function of PAPD5 and PAPD7 and Accelerates the Degradation of Matured Hepatitis B Virus Surface Protein mRNA

Abstract

Hepatitis B virus (HBV) mRNA metabolism is dependent upon host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 are cellular, noncanonical, poly(A) polymerases (PAPs) whose main function is to oligoadenylate the 3' end of noncoding RNA (ncRNA) for exosome degradation. HBV seems to exploit these two ncRNA quality-control factors for viral mRNA stabilization, rather than degradation. RG7834 is a small-molecule compound that binds PAPD5/7 and inhibits HBV gene production in both tissue culture and animal study. We reported that RG7834 was able to destabilize multiple HBV mRNA species, ranging from the 3.5-kb pregenomic/precore mRNAs to the 2.4/2.1-kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7-kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization was initiated by a shortening of the poly(A) tail, followed by an accelerated degradation process in both the nucleus and cytoplasm. In cells expressing HBV mRNA, both PAPD5/7 were found to be physically associated with the viral RNA, and the polyadenylating activities of PAPD5/7 were susceptible to RG7834 repression in a biochemical assay. Moreover, in PAPD5/7 double-knockout cells, viral transcripts with a regular length of the poly(A) sequence could be initially synthesized but became shortened in hours, suggesting that participation of PAPD5/7 in RNA 3' end processing, either during adenosine oligomerization or afterward, is crucial for RNA stabilization.

Keywords: HBV surface protein; HBs mRNA; PAPD5; PAPD7; RG7834; ZCCHC14; hepatitis B virus; polyadenylation.

FIG 1

PAPD5/7 inhibitor RG7834 accelerated the turnover of multiple species of HBV mRNA. (A) HepG2-tTA25 cells were infected with adenoviruses that expressed HBV pgRNA, LHBs, M/SHBs, or HBx in a doxycycline (dox)-controllable manner (vector maps were shown in Fig. S1). Three days after adenovirus infection, dox was added back to the culture media at time 0 to terminate viral gene transcription. RG7834 treatment, at 1 μM, was initiated at the same time when dox was added. Total cellular RNA was extracted at indicated time points for Northern blot. (B) Similar to the experimental design in A, the change of 2.1-kb SHBs mRNA under RG7834 treatment was monitored at additional time points for half-life calculation. RNA signal was measured using ImageJ software. Average and standard deviation values shown in the plot were based on 3 independent experiments. (C) HepG2-tTA25 cells were infected with adenovirus expressing a CMV-IE-driven HBx gene. Twenty-four hours after adenovirus infection, cells were treated with 1 μM RG7834 for 3 days and HBx mRNA was detected in Northern blot. All the experiments were conducted at least three times, and representative images were presented. Dox(+), doxycycline was always present in culture medium.

FIG 2

RG7834 induced a shortening of the HBV mRNA 3′ tail. (A) HBV transcription in HepAD38 cells was terminated by the addition of dox at time 0. Treatment of the cells with 1 μM RG7834 was simultaneously initiated at time 0, as well. Cellular RNA was extracted at indicated time points and analyzed with Northern blot. (B) RNA derived from the 6-hour time point in A was ligated to an RNA adaptor and was used for RT-PCR to measure the 3′ tail length using an HBV-specific primer and a primer located in the adaptor (Pf1 and Pr1). RNA size between the poly(A) signal and 5′ transcription start site was monitored with PCR primers Pf2 and Pr2. PCR products were resolved in agarose gel, and Lambda/HindIII fragments were used as DNA ladders. *, PCR was carried out on RNA samples without reverse transcription.

FIG 3

SHBs mRNA was associated with PAPD5/7. (A) HEK293 cells were transfected with pCMV-FlagD5 or pCMV-FlagD7 plus SHBs-expressing vector pCMV-S. Cells transfected with pCMV-S alone were used as a specificity control for anti-Flag antibody. Two days after transfection, the cell lysate was subjected to immune precipitation with an equal amount of Flag antibody or nonspecific mouse IgG. A fraction of the cell lysate and immune precipitates were denatured in Laemmli buffer and resolved in an SDS-PAGE gel, followed by detection with anti-Flag antibody. (B) RNA bound to precipitates was extracted with phenol-chloroform after proteinase K digestion, followed by DNase I clearance. Before RNA extraction, in vitro-transcribed tetracycline transactivator (tTA) RNA was spiked into the RNP precipitates for normalizing RNA extraction variation. Purified RNA was RT-qPCR quantified using gene-specific primers against HBV, tTA, and β-actin. RNA detection in the precipitated RNP complex was carried out in triplicates for statistical analysis. qPCR measurements of SHBs mRNA were consecutively normalized with readings from tTA and β-actin detection. Mock, cells transfected with empty vector; *, Student’s t test, P < 0.01.

FIG 4

RG7834 inhibited the polyadenylase activities of PAPD5/7. (A) HEK293 cells were transfected with plasmid pCMV-FlagD5. Two days posttransfection, the tagged PAPD5 polypeptides were precipitated with anti-Flag antibody. The precipitated beads, mixed with 1 mM ATP, UTP, CTP, GTP, or NTP (mixture of 1 mM of each nucleotide), were incubated with P³²-labeled RNA oligonucleotides at 37°C for 20 minutes. The enzymatic reaction was stopped by the addition of 2× RNA loading buffer followed by Urea-TBE gel electrophoresis. Radioactive signals were detected with phosphorimager. (B) Serially diluted RG7834 was applied to the RNA tailing reaction mixture containing 1 mM ATP as the nucleotide substrate. After 20 minutes of incubation at 37°C, the end product was resolved in a Urea-TBE gel for phosphorimager quantification. (C and D) Similar to A and B, flag-tagged PAPD7 was incubated with 1 mM of individual nucleotides or a mixture of 4 nucleotides at concentration of 1 mM each at 37°C for RNA elongation. Serially diluted RG7834 was also applied in the in vitro tailing assay using ATP as the substrate. Note, the incubation time for the PAPD7-catalyzed reaction lasted for 3 h. Arrowheads indicated elongated RNA oligonucleotides containing guanosines. Experiments were conducted at least three times, and representative images were presented.

FIG 5

The polyadenylase function of PAPD5/7 was regulated by HSLα structure. (A) Adenovirus expressing SHBs mRNA with wild-type HSLα (AdHSLα-WT) or mutated HSLα (AdHSLα-MUT) were used for HepG2 cell infection. Mutations in the loop and adjacent stem region of HSLα were marked in gray circles. (B) HepG2 cells were transfected with 20 nM siRNAs against PAPD5/7 or ZCCHC14, followed by AdHSLα-WT and AdHSLα-MUT infection. Twelve hours after adenovirus infection, treatment with DMSO or 1 μM RG7834 was initiated, and cells were harvested 3 days later for Northern detection using a P³²-labeled HBV riboprobe. A representative blot of three duplicates was shown. (C) RNA samples derived from B were subjected to RT-qPCR quantification using HBV-specific primers and normalized with GAPDH. *, Student’s t test, P < 0.01.

FIG 6

RG7834-induced SHBs mRNA tail trimming occurred in both the nucleus and cytoplasm. (A) HepG2-tTA25 cells were infected with adenovirus expressing doxycycline (dox)-controlled SHBs (AdTRE-SHBs, tet-off manner). Three days after adenovirus infection, dox was added to the culture medium to stop viral transcription. Cells were next harvested and fractionated at the indicated time points for Northern analysis. Lanes labeled with “Dox(+)” meant dox was always present during cell culture. (B) SHBs mRNA signals from A were quantified with the ImageJ program and proportioned to nuclear and cytoplasmic compositions based on total RNA yield from each compartment. (C) HepG2-tTA25 cells infected with AdTRE-SHBs were treated with dox for 0, 6, or 6 hours followed by an additional 4-hour of 1 μM RG7834. Cells were then fractionated and analyzed with Northern blotting. Small nucleolar RNA63 (snoRA63) was used as a nuclear fraction marker. Numbers listed beneath the blot were values of SHBs mRNA measured with ImageJ software. Representative blots from 3 duplicated experiments were shown. HPD, hours post-dox addition.

FIG 7

PAPD5 and PAPD7 were preferentially localized in the nucleus and cytoplasm, respectively. (A) HepG2 cells were transfected with plasmids pCMV-FlagD5 and pCMV-FlagD7, respectively. Transfected cells were reseeded onto the coverslip for an additional 2 days, followed by immune fluorescent staining with anti-Flag antibody. (B) HBV replicating HepG2-2.2.15 cells were employed for PAPD5/7 immune fluorescent staining as described in A. A representative image from five duplicated experiments was shown.

FIG 8

PAPD5/7 were not required for the de novo synthesis of viral mRNA poly(A) tail. (A) Plasmid map of adenovector Ad3G-ilovS. Fluorescent protein iLov (111 amino acid [aa]; accession no. QCO69681) was fused with SHBs, and the expression cassette was placed under tetracycline regulatory promoter (tet-on manner). Sequence numbering of the HBV genome was based on gene annotation of variant U95551. Underlined hexamer TATAAA depicted a polyadenylation signal in the HBV genome. Cassette harboring a 3rd generation tetracycline transactivator (tTA-3G) was placed downstream of the ilovS gene. (B) Huh7.5.1 and derived PAPD5/7 and ZCCHC14 knockout cell lines were infected with Ad3G-ilovS. Two days postadenoinfection, doxycycline (dox) was added into the culture media to induce ilovS expression. RNA was harvested at indicated time points and detected with a P³²-labeled HBV probe. Numbers beneath the loading gel were ImageJ quantifications of ilovS mRNA. The signal at time 0 of Huh7.5.1 cells was set as the background. (C) Selected RNA samples at time points 1 and 8 hours from B were loaded onto a Northern gel with adjusted amounts. The numbers beneath the loading gel represented proportions of RNA amount that were used in B. A representative blot of three duplicates was shown. (D) Proposed model for PAPD5/7-mediated HBV RNA stabilization. HBV mRNA is transcribed by host RNA polymerase II (RNAPII). After the first 20 to 30 nts are synthesized, the 5′ end of the transcripts is 7-methyl-guanosine capped and protected by the cap binding protein complex (black circle). The transcription of a hexanucleotide UAUAAA polyadenylation signal (PAS; green rectangle) at the 3′ UTR recruits canonical polyadenylating polymerase alpha (PAPα). The RNA sequence 20 nts downstream of the PAS will be endolytically cleaved (read arrowhead) followed by PAPα-mediated polyadenylation. During the extending of poly(A) tail, noncanonical PAPs PAPD5/7 are recruited by ZCCHC14 to incorporate guanosine (red G) into the poly(A) tail for RNA stabilization. However, it is also possible that PAPD5/7 can be retained on the viral mRNA that has a finished tail to protect it from being shortened by exoribonucleases (Exo). RG7834 blocks guanosine incorporation mediated by PAPD5/7 and consequently destabilizes viral RNA.