PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization

Abstract

RG7834 is a potent, orally bioavailable small‐molecule inhibitor of hepatitis B virus (HBV) gene expression that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound‐based adaptation version of the yeast three‐hybrid screen to identify the cognate cellular protein targets, the non‐canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide‐mediated knockdown studies that phenocopied the result seen with RG7834‐treated HBV‐infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down, suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. In addition, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusion: We identify PAPD5 and PAPD7 as cellular host factors required for HBV RNA stabilization and as therapeutic targets for the HBV cure.

A genome-wide CRISPR screen identifies ZCCHC14 as a host factor required for hepatitis B surface antigen production

“…One of the key factors involved in mRNA stability regulation by the TRAMP complex is the length of the polyA tail (Vanacova et al, 2005). Consistent with previous reports (Mueller et al, 2019), we saw a decrease in RNA abundance and moderate increases in electrophoretic mobility of all HBV transcripts (including pre-genomic RNA) isolated from cells treated with RG7834, TENT4B siRNA, or TENT4A/B siRNAs; knockdown of either TENT4A or ZCCHC7 had no observable impact on HBV RNA mobility ( Figure 3E). To evaluate the tail lengths of HBV transcripts, we performed an RNA tail length assay ( Figure 3F) and using HBV-specific forward and reverse primers positioned upstream of the RNA tailing site verified target-specific amplification.…”

“…One of the strongest confirmed pro-HBsAg factors identified from the genome-wide CRISPR screen was ZCCHC14. Interestingly, ZCCHC14 also came up as an efficacy target of RG7834, along with a previously identified target of the compound, TENT4B (Mueller et al, 2019). Our results suggest a novel complex consisting of ZCCHC14 and TENT4A/B, which functions to stabilize HBV mRNA by promoting 3'-end tailing and thereby likely preventing degradation of the tail by exonucleases ( Figure 4J).…”

“…To investigate mechanisms of reducing HBsAg, we performed a genome-wide CRISPR screen resulting in the identification of several novel host factors required for HBsAg expression. Using this approach, we also interrogated the mechanism of a small molecule inhibitor of HBsAg expression (RG7834), which was recently shown to destabilize HBV transcripts through interactions with the non-canonical poly(A) RNA polymerases TENT4B (PAPD5) and TENT4A (PAPD7) (Han et al, 2018;Mueller et al, 2019;Mueller et al, 2018). Here we provide strong evidence that a newly implicated host factor, ZCCHC14, together with TENT4B and TENT4A, stabilizes HBsAg expression through HBV RNA tailing (nontemplated addition to the 3' terminus of the RNA).…”

“…For the HBV Rluc cell line, the Rluc reporter was inserted in HBV genotype B genome under internal S1/S2 promoters disrupting P polymerase. For the 4xSLα mutant, four mutations were introduced into the SLα region (ntd 1292-1321) of the HBV Rluc plasmid as previously described in.RG7834 and RO0321 were synthesized at Novartis based on the reported structures(Han et al, 2018;Mueller et al, 2019;Mueller et al, 2018). The EC50 value for RG7834 on intracellular HBsAg in Cas9 HepG2-HBV cell line was determined by a 16-point dose-response ranging from 0-10 µM.…”

Efficacy of an Inhibitor of Hepatitis B Virus Expression in Combination With Entecavir and Interferon‐α in Woodchucks Chronically Infected With Woodchuck Hepatitis Virus

“…( 15 ) RG7834 was shown to reduce viral messenger RNA and to accelerate RNA degradation by targeting the host proteins noncanonical poly(A) RNA polymerase‐associated domain‐containing protein 5 and 7 (PAPD5 and PAPD7). ( 16 ) Both PAPD5 and PAPD7 are essential host components that are required for HBV RNA stabilization.…”