Viral hijacking of the TENT4–ZCCHC14 complex protects viral RNAs via mixed tailing

Kim, Dong-Wan; Lee, Young-Suk; Jung, Soo‐Jin; Yeo, Jinah; Seo, Jenny J.; Lee, Young-Yoon; Lim, Ju Young; Chang, Hyeshik; Song, Jaewon; Yang, Jian; Kim, Jong Seo; Jung, Gyuweon; Ahn, Kyu Jeung; Kim, V. Narry

doi:10.1038/s41594-020-0427-3

Cited by 51 publications

(103 citation statements)

References 66 publications

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“…However, deletion of the RRE, WPRE or both elements together did not substantially affect cytoplasmic genomic RNA abundance, indicating that they are not required for nuclear export of this transcript. The hepatitis B virus post-transcriptional regulatory element and WPRE have recently been shown to promote gene expression by recruiting the TENT4-ZCCHC14 complex 16 . This leads to a 'mixed tail' at the 3' end of mRNAs that protects them from degradation and may be the mechanism by which the WPRE promotes gene expression from lentiviral vector genomes.…”

Section: Discussionmentioning

confidence: 99%

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HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

Sertkaya

Ficarelli

Sweeney

et al. 2021

Preprint

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Many lentiviral vector genomes used for gene therapy are derived from HIV-1 and contain sequences required for genome expression, encapsidation and reverse transcription. In addition, they contain substantial regions of gag and env. Extraneous viral sequence in the genome reduces the maximum size of the transgene expression cassette and potentially increases biosafety concerns. Importantly, it remains unclear whether all of the HIV-1 sequence is required for transduction efficiency. To determine the functional relevance of the viral sequences in the vector genome, we performed a deletion analysis. Most of gag could be deleted without compromising vector titre. Within env, only the RRE is necessary, allowing over 50% of this region to be deleted. Oxford Nanopore sequencing showed that deleting 507 nt of env decreased the number of pre-mRNA splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining the deletion in gag with moving the RRE downstream of the 3’ R to prevent its reverse transcription showed that substantial amounts of HIV-1 sequence can be removed from the vector genome without compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve biosafety and increase the transgene capacity.

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Section: Discussionmentioning

confidence: 99%

“…The function of this element remains unclear, but it may improve vector titre and/or promote transgene expression by promoting RNA nuclear export or stability [10][11][12][13][14][15][16] .…”

Section: Introductionmentioning

confidence: 99%

HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

Sertkaya

Ficarelli

Sweeney

et al. 2021

Preprint

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“…It was recently reported that PAPD5/7 extended HBV mRNA poly(A) tails with intermittent guanosine (G), and the incorporation of G could shield them from rapid de-adenylation by cellular deadenylases (32). Since AB-452 inhibited PAPD5/7 enzymatic activities and shortened poly(A) tail lengths, we therefore hypothesized that the G content within the HBV poly(A) tails would be affected by AB-452 treatment.…”

Section: Ab-452 Promotes Hbv Rna Degradation Through Inhibiting Papd5/7 and Blocking Guanosine Incorporation Within Hbv Poly(a) Tailsmentioning

confidence: 98%

“…In particular, the CAGGC pentaloop sequence/structure of stem-loop alpha (SLα) within the PREα sub-element has been predicted to bind sterile-alpha-motif domain containing proteins (24). Recently, ZCCHC14 (a sterile-alpha-motif containing protein), together with PAPD5 and PAPD7 (the non-canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7), were identified as the cellular binding proteins that interacted with the HBV SLα sequence (32).…”

Section: Introductionmentioning

confidence: 99%

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Host poly(A) polymerases PAPD5 and PAPD7 provide two layers of protection that ensure the integrity and stability of hepatitis B virus RNA

Liu

Lee²,

Guo³

et al. 2021

Preprint

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Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize HBV RNA via the interaction with the viral post-transcriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, HBsAg production and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies, therefore it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (EC50 ranged from 1.4 to 6.8 nM) and in vivo by 0.93 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity towards the inhibitory effect of AB-452. Although neither single knock-out (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO could not result in any measurable phenotypic changes, but displays a similar antiviral effect as AB-452 treatment when PAPD5 is depleted simultaneously. PAPD5/7 double KO confers complete resistance to AB-452 treatment. Our results thus indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5 targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication.

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Functions and mechanisms of RNA tailing by metazoan terminal nucleotidyltransferases

Liudkovska

Dziembowski

2020

WIREs RNA

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Termini often determine the fate of RNA molecules. In recent years, 3′ ends of almost all classes of RNA species have been shown to acquire nontemplated nucleotides that are added by terminal nucleotidyltransferases (TENTs). The best‐described role of 3′ tailing is the bulk polyadenylation of messenger RNAs in the cell nucleus that is catalyzed by canonical poly(A) polymerases (PAPs). However, many other enzymes that add adenosines, uridines, or even more complex combinations of nucleotides have recently been described. This review focuses on metazoan TENTs, which are either noncanonical PAPs or terminal uridylyltransferases with varying processivity. These enzymes regulate RNA stability and RNA functions and are crucial in early development, gamete production, and somatic tissues. TENTs regulate gene expression at the posttranscriptional level, participate in the maturation of many transcripts, and protect cells against viral invasion and the transposition of repetitive sequences. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein‐RNA Recognition RNA Processing > 3′ End Processing RNA Turnover and Surveillance > Regulation of RNA Stability

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Viral hijacking of the TENT4–ZCCHC14 complex protects viral RNAs via mixed tailing

Cited by 51 publications

References 66 publications

HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

Host poly(A) polymerases PAPD5 and PAPD7 provide two layers of protection that ensure the integrity and stability of hepatitis B virus RNA

Functions and mechanisms of RNA tailing by metazoan terminal nucleotidyltransferases

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