The hepatitis delta virus (HDV) is the smallest human pathogenic RNA virus and relies heavily on host proteins for its replication. The objective of my research was to observe the effect of HDV replication on host gene expression, using a HEK-293-based cell system engineered to mimic HDV replication. A high-throughput sequencing was performed and allowed to establish a total of 3,561 genes differentially expressed by HDV RNA. Among those genes, 3,278 were upregulated by HDV RNA and 283 downregulated. A Gene Ontology (GO) enrichment analysis was performed on those dysregulated genes and revealed that upregulated genes were predominantly part of these four pathways: RNA processing, G-protein coupled receptor signaling pathway, protein transport, and organelle organization. On the other hand, downregulated genes were part of the nucleosome assembly pathway. The expression of several genes was confirmed by RT-qPCR. Moreover, protein complexes whose expression at the gene level was affected were identified. A total of 30 complexes were found to be significantly affected by HDV replication. Among them, we found many chromatin and histone related complexes. Lastly, a flow cytometry analysis revealed an increase in cell cycle arrest in G0/G1 and a reduction in the percentage of cell in S phase. Moreover, there was a difference in cell size for arrested cells in G0/G1 in HDV replicating cells. Overall, my results support the hypothesis that HDV replication induces cell cycle dysregulation.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

<p>HDV antigenomic RNA is processed in two distinct pathways; it can be cleaved at the polyA site and polyadenylated to become mRNA for the delta antigens, or the RNA can be cleaved by the antigenomic ribozyme to become full-length antigenomic RNA that is used for synthesis of genomic HDV RNA. The polyA site is located just 33 nucleotides upstream of the ribozyme cleavage site. If processing occurs primarily at the upstream polyA site, there may not be enough full-length antigenomic RNA to support replication. On the other hand, ribozyme cleavage downstream of the polyA site could inhibit polyadenylation by interfering with polyadenylation complex assembly. Thus, it appears that HDV may need a mechanism to control RNA processing so that both products can be generated in the proper amounts during the infection cycle. </p><p>A model has been proposed in which the choice between ribozyme cleavage and polyadenylation is determined by alternative RNA secondary structures formed by the polyA sequence (Wadkins and Been 2002). One of the hypothetical structures, AltP2, is a pairing between part of the upstream polyA sequence and the 3' end of the ribozyme sequence. For this model, the same upstream sequence that forms AltP2 could also form a stem loop, P(-1), within the leader, by pairing with sequences located farther upstream. A processing choice is possible because AltP2 is predicted to inhibit ribozyme cleavage and favor polyadenylation resulting in mRNA production, whereas P(-1) would inhibit polyadenylation and favor ribozyme cleavage resulting in full-length replication product. </p><p>The P(-1) vs. AltP2 model was tested using an antigenomic HDV ribozyme construct with the 60-nucleotide sequence upstream of the ribozyme cleavage site. This leader sequence contains the proposed polyA sequence elements. In vitro analysis of this construct revealed that the kinetic profile of ribozyme self-cleavage was altered in two ways. Relative to the ribozyme without upstream sequences, the fraction of precursor RNA that cleaved decreased to about 50%, but the active ribozyme fraction cleaved faster. Native gel electrophoresis revealed that the active and inactive precursor RNAs adopted persistent alternative structures, and structure mapping with Ribonuclease T1 and RNase H provided evidence for structures resembling P(-1) and AltP2.</p><p>Sequence changes in the 5' leader designed to alter the relative stability of P(-1) and AltP2 increased or decreased the extent of ribozyme cleavage in a predictable way, but disrupting AltP2 did not completely restore ribozyme activity. The analysis of deletion and base change variants supported a second alternative pairing, AltP4, formed by the pyrimidine-rich sequence immediately 5' of the ribozyme cleavage site and a purine-rich sequence from the 5' side of P4. A similar approach was used to test if the effect of disrupting both AltP2 and AltP4 might be additive, and the results suggested that ribozyme precursors with 5' leader sequences could fold into multiple inactive conformations, which can include, but may not be limited to, AltP2, AltP4, or a combination of both.</p><p>Luciferase expression constructs with HDV polyA and ribozyme sequences were used to investigate the effects of RNA structure and ribozyme cleavage on polyadenylation in cells. One hypothesis was that P(-1) could inhibit polyadenylation by making the polyA sequence elements less accessible to polyA factors, but sequence changes designed to alter the stability of the stem loop had no effect on polyadenylation. The model also predicts that the ribozyme sequence downstream of the polyA site could affect polyadenylation, possibly in two different ways. Ribozyme cleavage could interfere with polyadenylation by uncoupling transcription from processing, however, the ribozyme sequence might also influence polyadenylation in a manner independent of the ribozyme cleavage activity. As such, the AltP2 structure could potentially have a positive effect on polyadenylation either by inhibiting ribozyme cleavage or by making the polyA signal sequences more accessible to the polyA factors. To distinguish between the effects of ribozyme cleavage and alternative RNA structures, luciferase expression levels from constructs with an HDV polyA sequence followed by the active wild-type ribozyme or the inactive C76u version of the ribozyme were compared. For the wild-type HDV polyA sequence, the active ribozyme reduced expression, whereas the inactive ribozyme control had no effect on expression. However, for the modified leader sequences, which were efficiently polyadenylated in the absence of ribozyme, there were changes in expression that appeared to be independent of ribozyme cleavage. Based on these findings, two alternative models are proposed. One model predicts that protein factors might affect antigenomic RNA processing, and the other model suggests that additional alternative structures, such as AltP4, might influence the choice between ribozyme cleavage and polyadenylation.</p> / Dissertation

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

The Hepatitis Delta Virus (HDV) is an RNA pathogen that uses the host DNA-dependent RNA polymerase II (RNAP II) to replicate. Previous studies identified the right terminal domain of genomic polarity (R199G) of HDV RNA as an RNAP II promoter, but the features required for HDV RNA to be used as an RNA promoter were unknown. In order to identify the structural features of an HDV RNA promoter, I analyzed 473,139 sequences representing 2,351 new R199G variants generated by high-throughput sequencing of a viral population replicating in 293 cells. To complement this analysis, I also analyzed the same region from HDV sequences isolated from various hosts. Base pair covariation analysis indicates a strong selection for the rod-like conformation. Several selected RNA motifs were identified, including a GC-rich stem, a CUC/GAG motif and a uridine at the initiation site of transcription. In addition, a polarization of purine/pyrimidine content was identified, which might represent a motif favourable for the binding of the host Polypyrimidine tract-binding protein-associated-splicing-factor (PSF), p54 and Paraspeckle Protein 1 (PSP1). Previously, it was shown that R199G binds both RNAP II and PSF, that PSF increased the HDV levels during in vitro transcription and that p54 binds R199G. In the present study, I showed that PSP1 also associates with HDV RNA and I investigated whether these proteins are required for HDV replication. My results show that knockdown of PSF, p54 and PSP1 resulted in a decrease of HDV accumulation. These proteins are highly concentrated in paraspeckles, which are nuclear structures involved in storage of transcripts generated by RNAP II. I found that upon viral replication in 293 cells, PSP1 appeared as bigger foci present outside of the nucleus, while PSF and p54 foci remained in the nucleus. NEAT1 is a long non-coding RNA essential for the formation of paraspeckles. Upon HDV replication, I found an increase of the intensity and size of NEAT1 foci that correlates with an increase of NEAT1 transcripts. Altogether, these data suggest that HDV replication results in an alteration of the paraspeckles structures, providing foundation for further investigation of the paraspeckles role in HDV cycle. Overall, the present study addresses the importance of the HDV RNA structure and of the host paraspeckle proteins for HDV replication.