Combined proteomic–RNAi screen for host factors involved in human hepatitis delta virus replication

Abstract

Human hepatitis delta virus (HDV) is the only animal virus known to replicate its RNA genome using a host polymerase because its only virally encoded proteins, the small and large hepatitis delta antigens (HDAg-S and HDAg-L), lack polymerase activity. Although this makes HDV an ideal model system to study RNA-directed transcription in mammalian cells, little is known about the host factors involved in its replication. To comprehensively identify such host factors, we created a stable cell line carrying a functional FLAG-HDAg-S. Anti-Flag immunopurification and mass spectrometry identified >100 proteins associated with FLAG-HDAg-S, many of which had predicted roles in RNA metabolism. The biological relevance of this screen was strongly supported by the identification of nine out of the 12 subunits of the RNA polymerase II complex thought to mediate HDV replication. To further investigate the significance of these factors for HDV replication, we selected 65 proteins to look for factors that would also affect the accumulation of HDV RNA following siRNA knockdown. Fifteen and three factors were found to regulate HDV RNA accumulation negatively and positively, respectively, upon RNAi knockdown. Our results provide a valuable resource for future research to advance our mechanistic understanding of HDV replication and RNA-directed transcription in mammalian cells.

Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme

“…Interestingly, they include: (i) nine of the 12 subunits of Pol II, highlighting again the key role of this polymerase in HDV replication and (ii) the putative RNA helicase MOV 10 reported to interact with an Argonaute complex and, like its Arabidopsis homologue SDE3, involved in RNA amplification. Moreover, RNAi-knockdown of a fraction of these polypeptides, including MOV 10 and Argonaute 4 (an specific effector of RNA silencing), affected negatively HDV replication, 53,86 thus validating the reliability and potential of this approach, and establishing a link between HDV replication and RNA silencing. Viroid replication and even pathogenesis, might also be connected to RNA silencing.…”

“…Given the intrinsic experimental limitations of some of these approaches, and the lack of confirmatory work on the physiological relevance of a good number of the interactions detected, a combined proteomic-RNAi screen for host proteins interacting with S-∂Ag has been performed. 86 To this end, a stable cell line expressing a Flag-tagged S-∂Ag, which supports in trans replication of an HDV variant with an early nonsense mutation in S-∂Ag, was created and subsequently used to immunoprecipitate and identify by MS more than 100 polypeptides. Interestingly, they include: (i) nine of the 12 subunits of Pol II, highlighting again the key role of this polymerase in HDV replication and (ii) the putative RNA helicase MOV 10 reported to interact with an Argonaute complex and, like its Arabidopsis homologue SDE3, involved in RNA amplification.…”

Human tRNA-derived small RNAs in the global regulation of RNA silencing

“…6C), cand45-targ2, cand45-+2.45, cand45-+4.45, and cand45-2.45 overexpression, the cloning vector was digested with BbsI, de-phosphorylated, and the following phosphorylated and annealed oligos inserted: In the cotransfection studies in Figure 4C, 2 mg of cand45 and 2 mg of FLAG-protein expression vectors were cotransfected into 6-cm dishes of 293 cells. The Brf1 overexpression vector was created by cloning the Brf1 ORF from pTRE2-Brf1 into pcDNA3 just downstream from an N-terminal FLAG epitope (Cao et al 2009), while in the negative control vector the Brf1 ORF had been replaced with the truncated ORF of a replicationdeficient early nonsense mutant version of HDAg (Haussecker et al 2008).…”

HSPC117 Is the Essential Subunit of a Human tRNA Splicing Ligase Complex

“…We have identified HSPC117 as an essential component of the prevalent human tRNA splicing pathway. Recently, HSPC117 or the RNA>p ligase pathway have also been implicated in RNA processing during viral replication (29,30). The high degree of conservation of HSPC117/RtcB proteins is suggestive of shared roles for this protein family in organisms as distantly related as humans and E. coli.…”