Transcription-Based Amplified Colorimetric Thrombin Sensor Using Non-Crosslinking Aggregation of DNA-Modified Gold Nanoparticles

Gold nanoparticles (AuNPs) have been employed as colorimetric biosensors due to the color difference between their dispersed (red) and aggregated (blue) states. Although signal amplification reactions triggered by structural changes of the ligands on AuNPs have been widely used to improve measurement sensitivity, the use of ligands is limited. In this study, we designed a AuNP-based signal-amplifying sandwich biosensor, which does not require a conformational change in the ligands. Thrombin was used as a model target, which is recognized by two different probes. In the presence of the target, an extension reaction occurs as a result of hybridization of the two probes. Then RNA synthesis is started by RNA polymerase activation due to RNA promoter duplex formation. The amplified RNA drives aggregation or dispersion of the AuNPs, and a difference of the color if the AuNP solution is observed. As this detection system does not require a conformational change in the ligand, it can be generically applied to a wide range ligands.

The Role of the Stem-Loop A RNA Promoter in Flavivirus Replication

An essential challenge in the lifecycle of RNA viruses is identifying and replicating the viral genome amongst all the RNAs present in the host cell cytoplasm. Yet, how the viral polymerase selectively recognizes and copies the viral RNA genome is poorly understood. In flaviviruses, the 5′-end of the viral RNA genome contains a 70 nucleotide-long stem-loop, called stem-loop A (SLA), which functions as a promoter for genome replication. During replication, flaviviral polymerase NS5 specifically recognizes SLA to both initiate viral RNA synthesis and to methylate the 5′ guanine cap of the nascent RNA. While the sequences of this region vary between different flaviviruses, the three-way junction arrangement of secondary structures is conserved in SLA, suggesting that viruses recognize a common structural feature to replicate the viral genome rather than a particular sequence. To better understand the molecular basis of genome recognition by flaviviruses, we recently determined the crystal structures of flavivirus SLAs from dengue virus (DENV) and Zika virus (ZIKV). In this review, I will provide an overview of (1) flaviviral genome replication; (2) structures of viral SLA promoters and NS5 polymerases; and (3) and describe our current model of how NS5 polymerases specifically recognize the SLA at the 5′ terminus of the viral genome to initiate RNA synthesis at the 3′ terminus.

Processive RNA polymerization and promoter recognition in an RNA World

Early life is thought to have required the self-replication of RNA by RNA replicases. However, how such replicases evolved and subsequently enabled gene expression remains largely unexplored. We engineered and selected a holopolymerase ribozyme that uses a sigma factor–like specificity primer to first recognize an RNA promoter sequence and then, in a second step, rearrange to a processive elongation form. Using its own sequence, the polymerase can also program itself to polymerize from certain RNA promoters and not others. This selective promoter–based polymerization could allow an RNA replicase ribozyme to define “self” from “nonself,” an important development for the avoidance of replicative parasites. Moreover, the clamp-like mechanism of this polymerase could eventually enable strand invasion, a critical requirement for replication in the early evolution of life.

Development of a Reverse Genetics System for Toscana Virus (Lineage A)

Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, order Bunyavirales, found in the countries surrounding the Mediterranean. TOSV is an important cause of seasonal acute meningitis and encephalitis within its range. Here, we determined the full sequence of the TOSV strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007) and used this in combination with other sequence information to construct functional cDNA plasmids encoding the viral L, M, and S antigenomic sequences under the control of the T7 RNA promoter to recover recombinant viruses. Importantly, resequencing identified two single nucleotide changes to a TOSV reference genome, which, when corrected, restored functionality to the polymerase L and made it possible to recover infectious recombinant TOSV (rTOSV) from cDNA, as well as establish a minigenome system. Using reverse genetics, we produced an NSs-deletant rTOSV and also obtained viruses expressing reporter genes instead of NSs. The availability of such a system assists investigating questions that require genetic manipulation of the viral genome, such as investigations into replication and tropism, and beyond these fundamental aspects, also the development of novel vaccine design strategies.

mSphere of Influence: Resolution of the Structure of an Influenza Virus Polymerase Is a Game Changer

ABSTRACT Mathilde Richard works in the field of virology, more specifically on the evolution and pathogenesis of influenza viruses. In this mSphere of Influence article, she reflects on how the two articles “Structure of Influenza A Polymerase Bound to the Viral RNA Promoter” by A. Pflug, D. Guilligay, S. Reich, and S. Cusack (Nature 516:355–360, 2014, https://doi.org/10.1038/nature14008) and “Structural Insight into Cap-Snatching and RNA Synthesis by Influenza Polymerase” by S. Reich, D. Guilligay, A. Pflug, H. Malet, I. Berger, et al. (Nature 516:361–366, 2014, https://doi.org/10.1038/nature14009) made an impact on her by providing new grounds to study the influenza virus polymerase and its role in virus biology and evolution.