Vif is a RNA chaperone that could temporally regulate RNA dimerization and the early steps of HIV-1 reverse transcription

HIV-1 Vif (viral infectivity factor) is associated with the assembly complexes and packaged at low level into the viral particles, and is essential for viral replication in non-permissive cells. Viral particles produced in the absence of Vif exhibit structural defects and are defective in the early steps of reverse transcription. Here, we show that Vif is able to anneal primer tRNALys3 to the viral RNA, to decrease pausing of reverse transcriptase during (–) strand strong-stop DNA synthesis, and to promote the first strand transfer. Vif also stimulates formation of loose HIV-1 genomic RNA dimers. These results indicate that Vif is a bona fide RNA chaperone. We next studied the effects of Vif in the presence of HIV-1 NCp, which is a well-established RNA chaperone. Vif inhibits NCp-mediated formation of tight RNA dimers and hybridization of tRNALys3, while it has little effects on NCp-mediated strand transfer and it collaborates with nucleocapsid (NC) to increase RT processivity. Thus, Vif might negatively regulate NC-assisted maturation of the RNA dimer and early steps of reverse transcription in the assembly complexes, but these inhibitory effects would be relieved after viral budding, thanks to the limited packaging of Vif in the virions

A supramolecular assembly formed by influenza A virus genomic RNA segments

The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common ‘transition zone’ located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5′ region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration

Dimerization of the human immunodeficiency virus type 1 genomic RNA,functions in selective packaging and splicing

La nature diploïde du génome viral est une caractéristique spécifique et conservée à l'ensemble des rétrovirus, mettant en évidence le rôle crucial de la dimérisation dans le cycle réplicatif. Par ailleurs, les domaines de dimérisation et d'encapsidation, situés à l'extrémité 5 UTR de l'ARNg, se superposent et des mutations à l'origine de défauts de dimérisation réduisent l'efficacité d'encapsidation de l'ARNg, suggérant que la dimérisation serait impliquée dans le processus de sélection du génome viral. Or, le site d'initiation de la dimérisation (DIS) est présent sur tous les ARN viraux (génomique et épissés) et pourrait ainsi favoriser l'encapsidation des ARN épissés sous forme d'hétérodimères avec l'ARNg. En analysant la capacité de dimérisation des ARN viraux, nous avons montré qu in vitro les ARN épissés sont capables d'homodimériser et d'hétérodimériser avec l'ARNg par l'intermédiaire du DIS. Cependant, à l'inverse de l'ARNg, bien que le DIS des ARN épissés soit fonctionnel in vitro, il ne favorise pas leur encapsidation et ceux-ci sont largement exclus des particules virales, suggérant l'existence d'un système de discrimination des ARN conduisant à la sélection spécifique de l'ARNg. La localisation du DIS à proximité du site majeur d'épissage nous a alors conduit à analyser le lien possible entre la dimérisation et l'épissage des ARN viraux, qui pourrait moduler le processus de sélection de l'ARNg. Nous avons montré que le DIS et/ou la dimérisation régulent négativement l'épissage et positivement l'encapsidation. Ainsi les mécanismes de dimérisation et d'épissage pourraient entrer en compétition et favoriser l'encapsidation sélective de l'ARNg.The conservation of the genome dimeric nature among retroviruses underlies the importance of RNA dimerization for virus replication. Noticeably, dimerization and packaging domains, located at the 5 end of the HIV-1 gRNA, are superposed and mutations affecting dimerization reduce gRNA packaging efficiency, suggesting that dimerization could be involved in the process of viral genome selection. However, the dimerization initiation site (DIS) is present in all viral RNA species (spliced and genomic) and could thus favored spliced RNA packaging in the form of heterodimers with the gRNA. By analyzing viral RNA dimerization in vitro, we have shown that spliced RNAs are able to form homodimers and to heterodimerize with the gRNA through the DIS. But, conversely to the gRNA, although the spliced RNA DIS is fully functional in vitro, it does not promote their packaging and they are largely excluded from viral particles. Thus, the virus must have developed specific mechanisms to preferentially select the gRNA. Moreover, the proximity between the DIS and the SD site suggests that RNA dimerization could affect the fate of HIV-1 RNAs by modulating splicing and packaging. We have shown that the DIS and/or RNA dimerization itself could limit splicing efficiency, thus favoring the production of gRNA prone to be packaged. Altogether, our results show that the DIS or RNA dimerization could influence the fate of HIV-1 gRNA by regulating viral RNA splicing and its selection to allow optimal production of infectious virions

Dimerization of the human immunodeficiency virus type 1 genomic RNA,functions in selective packaging and splicing

La nature diploïde du génome viral est une caractéristique spécifique et conservée à l'ensemble des rétrovirus, mettant en évidence le rôle crucial de la dimérisation dans le cycle réplicatif. Par ailleurs, les domaines de dimérisation et d'encapsidation,The conservation of the genome dimeric nature among retroviruses underlies the importance of RNA dimerization for virus replication. Noticeably, dimerization and packaging domains, located at the 5 end of the HIV-1 gRNA, are superposed and mutations aff

A Mass Optimisation Study of the Lunar Zebro Chassis

A prototype lunar rover is in development by students of the TU Delft since 2017. It is a nanorover based on the terrestrial ZeBRo design, now named the Lunar Zebro. The Lunar Zebro is a prototype design as a proof of concept for nanorover capabilities. With a chassis of 200 by 140 by 60 millimeters, fitting on a sheet of A5 paper, the Lunar Zebro is intended to be the smallest and lightest autonomous rover on the Moon to date. The objective is to traverse a distance of 200 meters during a lunar day, surviving the harsh environment and strong solar radiation. Due to the limited time budget there is little refinement in the structural design. The resulting functional but heavy design leaves many opportunities for optimization. While there are many examples of successful planetary rover missions, little is published concerning the design of the structures. This report contains further analysis of the design of satellite structures. The various structure types and design requirements highlight the importance of thermal transport and resistance to mechanical launch loads. Compared to the deployed planetary rovers, the Lunar Zebro is unique in many ways. The small size facilitates production of the current monolithic chassis which is ideal in its thermal conduction and environmental sealing properties. However with a constant plate thickness and no reinforcing substructure, the structure is not an efficient loadbearing design. Due to the many requirements and unique mission profile of the Lunar Zebro, there is no clear method by which to optimise the structure.To better understand the current structure and reduce the mass, a case study is performed with Finite Element Methods. After validating a modelling approach for thin plate reinforcement, a simplified chassis structure is generated. Maintaining the essential configuration of the chassis and connected components, the response to the static launch load of 10G is analysed. Several methods for rib placement design are tested while reducing the plate thickness. Buckling behaviour and CNC production limitations are accounted for in this approach. To minimally affect the other design requirements, the stiffness of the structure is maintained. While the placement of ribs is sensitive to the vicinity of connected components, equally stiff designs can be obtained with reinforcement grids. Reducing the plate thickness by 66.6%, a mass reduction in the order of 50% can be achieved without sacrificing stiffness. However, local adjustments are required to prevent plastic deformation in high stress areas. From there the analysis and design of reinforcement grids is investigated further. Grids are often seen in aerospace applications due to the convenient geometries for CNC production, light weight and predicable orthotropic or isotropic behaviour. A smeared stiffness approach is investigated that relates the rib and plate interaction to composite plate theory. Applying this analysis method provides beneficial insight in the parameters and related stiffness behaviour of a grid reinforced plate. By modelling three common grid sections on a hypothetical plate design scenario with varying boundary conditions, the important criteria for the selection and design of a reinforcement grid are provided.Mechanical Engineerin

La dimérisation de l'ARN génomique du virus de l'immunodéficience humaine de type 1 (HIV-1) (rôle dans l'encapsidation sélective et l'épissage)

La nature diploïde du génome viral est une caractéristique spécifique et conservée à l ensemble des rétrovirus, mettant en évidence le rôle crucial de la dimérisation dans le cycle réplicatif. Par ailleurs, les domaines de dimérisation et d encapsidation, situés à l extrémité 5 UTR de l ARNg, se superposent et des mutations à l origine de défauts de dimérisation réduisent l efficacité d encapsidation de l ARNg, suggérant que la dimérisation serait impliquée dans le processus de sélection du génome viral. Or, le site d initiation de la dimérisation (DIS) est présent sur tous les ARN viraux (génomique et épissés) et pourrait ainsi favoriser l encapsidation des ARN épissés sous forme d hétérodimères avec l ARNg. En analysant la capacité de dimérisation des ARN viraux, nous avons montré qu in vitro les ARN épissés sont capables d homodimériser et d hétérodimériser avec l ARNg par l intermédiaire du DIS. Cependant, à l inverse de l ARNg, bien que le DIS des ARN épissés soit fonctionnel in vitro, il ne favorise pas leur encapsidation et ceux-ci sont largement exclus des particules virales, suggérant l existence d un système de discrimination des ARN conduisant à la sélection spécifique de l ARNg. La localisation du DIS à proximité du site majeur d épissage nous a alors conduit à analyser le lien possible entre la dimérisation et l épissage des ARN viraux, qui pourrait moduler le processus de sélection de l ARNg. Nous avons montré que le DIS et/ou la dimérisation régulent négativement l épissage et positivement l encapsidation. Ainsi les mécanismes de dimérisation et d épissage pourraient entrer en compétition et favoriser l encapsidation sélective de l ARNg.The conservation of the genome dimeric nature among retroviruses underlies the importance of RNA dimerization for virus replication. Noticeably, dimerization and packaging domains, located at the 5 end of the HIV-1 gRNA, are superposed and mutations affecting dimerization reduce gRNA packaging efficiency, suggesting that dimerization could be involved in the process of viral genome selection. However, the dimerization initiation site (DIS) is present in all viral RNA species (spliced and genomic) and could thus favored spliced RNA packaging in the form of heterodimers with the gRNA. By analyzing viral RNA dimerization in vitro, we have shown that spliced RNAs are able to form homodimers and to heterodimerize with the gRNA through the DIS. But, conversely to the gRNA, although the spliced RNA DIS is fully functional in vitro, it does not promote their packaging and they are largely excluded from viral particles. Thus, the virus must have developed specific mechanisms to preferentially select the gRNA. Moreover, the proximity between the DIS and the SD site suggests that RNA dimerization could affect the fate of HIV-1 RNAs by modulating splicing and packaging. We have shown that the DIS and/or RNA dimerization itself could limit splicing efficiency, thus favoring the production of gRNA prone to be packaged. Altogether, our results show that the DIS or RNA dimerization could influence the fate of HIV-1 gRNA by regulating viral RNA splicing and its selection to allow optimal production of infectious virions.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

De Zype[,] Beemster[,] de Purmer. Gemeeten ende geteekent door mr Lucas Jansen Sinck anno 1622[,] de Wormer[,] caerte van Waterland vertonende de gelegentheyt der meeren onlangs bedyckt als Buyckslooter Broecker en Belmer meer met de naest gelegen steden

Deze twee kaarten verschillen in zoverre van elkaar, dat die van de Zijpe (1597 drooggelegd) een gewone grootschalige topografische kaart is, terwijl die van de Beemster (1612 drooggelegd) een plan van indeling behelst. Als zodanig is het dus een vroege planologische kaart en vergelijkbaar met het verkavelingsplan voor het Haarlemmermeer uit circa 1855, die ook in deze collectie gedigitaliseerde thematische kaarten is opgenomen