The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay.

The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.

The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: RNA packaging into HIV-1 Virions. Includes bibliographical references (152-174). Also available online through Digital Dissertations.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24270号 / 生博第484号 / 新制||生||64(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 原田 浩, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism used by many viral and cellular mRNAs. PRF occurs at a heptanucleotide slippery sequence and is stimulated by a downstream RNA structure, most often in the form of a pseudoknot. The utilization of −1/ +1 PRF to produce proteins encoded by the −1/+1 reading frame is wide-spread in RNA viruses, but relatively rare in cellular mRNAs. In human, only three such cases of −1 PRF events have been reported, all involving retroviral-like genes and protein products. To evaluate the extent of PRF utilization in the human transcriptome, we have developed a computational scheme for identifying putative pseudoknot-dependent −1 PRF events and applied the method to a collection of 45,000 human mRNAs in the NCBI RefSeq database. Using core program PKscan, we have performed a large-scale search for putative pseudoknot dependent PRF. In addition to the three reported cases, our study identified more than two dozen putative −1 PRF cases. The genes involved in these cases are genuine cellular genes without a viral origin. Moreover, in more than half of these cases, the frameshift site locates far upstream from the stop codon of the 0 reading frame, which is nonviral-like.Using dual luciferase assays in HEK293FT cells, we confirmed that the −1 PRF signals in the mRNAs of CDK5R2 and SEMA6C are functional in inducing efficient frameshifting. We also present evidence to show that the mRNA of human inorganic pyrophosphatase 1 (PPA1) harbors functional cis-acting signals for +1 PRF. The consequence of frameshifting is the production of a longer PPA1 protein in which the C-terminal 25 residues of normal PPA1 are replaced by 68 residues translated from the +1 reading frame. To the best of our knowledge, the human PPA1 mRNA is the only other mammalian cellular mRNA known to date to utilize the +1 PRF mechanism, besides the antizyme mRNAs. Results from the studies on the involvement of PPA1 in tumorigenesis suggest that PPA1 is a potential prognostic biomarker for certain cancers, and strategies for PPA1 down-regulation may have therapeutic potential for the treatment of cancers. This study also recognized a new pseudoknot structure in SARS-CoV that has been validated as a cis-acting regulation factor in viral frameshifting event, referred to 'intertwined double pseudoknots’. They are present at the −1 PRF site in SARS-CoV-1/2 and many other coronaviruses. An even larger scale analysis on transcriptome-wide study in all available human mRNAs identified many of them have the potential to form the same structure, which may involve in regulation of translation initiation, or mRNA stability. Finally, preliminary design of CRISPR-inspired strategy to induce -1 ribosomal frameshifting by trans-acting factors was verified to be functional. Our findings have significant implications in expanding the repertoire of the PRF phenomenon in both human and viral mRNAs and the protein-coding capacity of the human transcriptome.

Le virus de la grippe A est un virus à ARN négatif appartenant à la famille des Orthomyxoviriadea dont la réplication se produit dans le noyau des cellules infectées. L'organisation du génome est segmentée en huit segments d'ARNv de polarité négative, codant pour un minimum de 16 protéines virales différentes. Ces ARN viraux (ARNv) sont en complexe avec de nombreuses copies de nucléoprotéines et liés par leurs extrémités 5' et 3' au complexe hétérotrimérique de l'ARN-polymérase ARN-dépendante composé des sous unités PA, PB1 et PB2. Cet assemblage macromoléculaire (ARNv / polymérase / NP) nommée Ribonucléoprotéine (RNP) constitue une entité génomique indépendante. Dans le contexte de la RNP, l'ARN-polymérase assure à la fois la transcription et la réplication du génome ARNv. En assurant ces deux fonctions, l'ARN-polymérase joue un rôle majeur dans la réplication virale et constitue une cible antivirale privilégiée. Les travaux de recherche présentés dans cette thèse se concentrent sur les éléments structuraux participants à l'assemblage de l'ARN polymérase et son interaction avec les avec les ARNv. Pour atteindre ces objectifs, notre laboratoire, en collaboration avec d'autres groupes, a mis en place un système d'expression en polyprotéines permettant d'exprimer la polymérase. Plus encore, cette méthode a aussi permis de reconstituer des complexes entre l'ARN-polymérase et des partenaires cellulaires, notamment RanBP5 qui appartient à la famille des importines-β. / Influenza A virus is a negative-strand RNA virus belonging to the Orthomyxoviriadea family whose replication occurs in the nucleus of infected cells. The genome organisation of influenza virus is segmented in eight vRNA segments of negative polarity coding for at least 16 different viral proteins. Each vRNA is bound to multiple copies of nucleoprotein (NP) and to the heterotrimeric RNA-dependent RNA-polymerase complex (PA, PB1 and PB2) through its 5' and 3' extremities. This macromolecular assembly (vRNA/polymerase/NP) forms the ribonucleoprotein (RNP) particle, which acts as a separate genomic entity within the virion. The RNP complex is at the core of viral replication and in the context of RNPs, the polymerase performs both transcription and replication of the vRNA genome. As such, the polymerase constitutes a major antiviral drug target. The research work presented within this thesis focuses on the underlying determinants of the RNA polymerase assembly process and its interaction with its vRNA genome. To fulfill these goals, our lab, in collaboration with other groups, has set up a novel polyprotein expression system to express the polymerase but also to reconstitute polymerase and cellular partner complexes, notably RanBP5, which belongs to the importin-β family.

The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.

The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.

Cellular T-cell intracellular antigen-1 related protein (TIAR) binds to the 3' terminal stem-loop of the West Nile virus minus-strand RNA [WNV 3'(-) SL RNA]. TIAR binding sites were previously mapped on loop 1 (L1) and loop 2 (L2) of the 3' (-) SL RNA and mutations of these sites in a WNV infectious clone inhibited virus replication. In the present study, data from in vitro binding assays suggested that multiple TIAR proteins bind to each WNV 3′ (-) SL RNA in a positively cooperative manner. The tertiary structure of WNV 3′ (-) SL RNA was predicted and it was suggested that L2 forms an exposed loop while L1 forms an embedded loop. We propose that TIAR binds first to L2 and that this interaction facilitates the binding of a second TIAR molecule to L1. Data from in vitro assays also showed that TIAR binds specifically to the WNV 3' (-) SL RNA but not to the complementary WNV 5' (+) SL RNA and that the C-terminal prion domain of TIAR contributes to RNA binding specificity. Immunoprecipitation experiments indicated that TIAR interacts with the WNV 3' (-) SL RNA in cells. Colocalization of TIAR and viral dsRNA in the perinuclear region of WNV-infected cells was visualized using a proximity ligation assay. In WNV-infected, TIAR-overexpressing cells, increased extracellular virus yields, intracellular viral protein and RNA levels, and an increased ratio of viral plus-strand RNA to minus-strand RNA were observed. These data suggest that TIAR enhances WNV plus-strand RNA synthesis from the minus-strand template. WNV infections induce small TIAR foci formation in primate cells but not rodent cells. The TIAR foci are located in the perinuclear region and differ in size and location from arsenite-induced stress granules (SGs). However, the small TIAR foci contain many SG components, such as G3BP, PABP, and eIF3A, but not HuR. Arsenite-induced SG formation is still inhibited by WNV infection in these cells. eIF2a phosphorylation was observed in some infected cells that contained WNV-induced TIAR foci but viral NS3 protein accumulation was not inhibited. The data suggest that WNV-induced TIAR foci in primate cells are not canonical SGs.