Dr Nicole Robb

Abstract:

Influenza A, a negative-sense RNA virus, has a genome that consists of eight single-stranded RNA segments. Influenza co-infections can result in reassortant viruses that contain gene segments from multiple strains, causing pandemic outbreaks with severe consequences for human health. The outcome of reassortment is likely influenced by a selective sequence-specific genome packaging mechanism. To uncover the contributions of individual segment pairings to selective packaging, we set out to statistically analyse packaging defects and inter-segment distances in individual A/Puerto Rico/8/34 (H1N1) (PR8) virus particles. To enable such analysis, we developed a multiplexed DNA-PAINT approach capable of assessing the segment stoichiometry of >10 000 individual virus particles in one experiment; our approach can also spatially resolve the individual segments inside complete virus particles with a localization precision of ∼10 nm. Our results show the influenza genome can be assembled through multiple pathways in a redundant and cooperative process guided by preferentially interacting segment pairs and aided by synergistic effects that enhance genome assembly, driving it to completion. Our structural evidence indicates that the interaction strength of segment pairs affects the spatial configuration of the gene segments, which appears to be preserved in mature virions. As our method quantified the interactions of whole influenza segments instead of identifying individual sequence-based interactions, our results can serve as a template to quantify the contributions of individual sequence motifs to selective packaging.

Abstract:

SUMMARYPleomorphism in influenza viruses, characterized by diverse morphological forms ranging from spherical virions to elongated filaments, has been suggested to present significant implications for pathogenesis. This review examines the role of pleomorphism on the influenza virus life cycle, encompassing viral attachment and entry, replication, assembly, and budding, as well as transmission dynamics. It explores the determinants' underlying morphological variability in virions and their impact on viral fitness and host interactions. Insights into how pleomorphic forms of the virus influence disease severity and the efficacy of antivirals are discussed. Understanding the implications of pleomorphism in influenza virus pathogenesis is crucial for the development of effective disease prevention, control, and treatment strategies.

Abstract:

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in significant global mortality, with over 7 million cases reported. Despite extensive research and high vaccination rates, highly mutated forms of the virus continue to circulate. It is therefore important to understand the viral lifecycle and the precise molecular mechanisms underlying SARS-CoV-2 replication. To address this, we developed a single-molecule Förster resonance energy transfer (smFRET) assay to directly visualize and analyse in vitro RNA synthesis by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). We purified the minimal replication complex, comprising nsp12, nsp7, and nsp8, and combined it with fluorescently labelled RNA substrates, enabling real-time monitoring of RNA primer elongation at the single-molecule level. This platform allowed us to investigate the mechanisms of action of key inhibitors of SARS-CoV-2 replication. In particular, our data provides evidence for remdesivir’s mechanism of action, which involves polymerase stalling and subsequent chain termination dependent on the concentration of competing nucleotide triphosphates. Our study demonstrates the power of smFRET to provide dynamic insights into SARS-CoV-2 replication, offering a valuable tool for antiviral screening and mechanistic studies of viral RdRp activity.

Abstract:

Plaque assays quantify the amount of active, replicating virus to study and detect infectious diseases by application of samples to monolayers of cultured cells. Due to the time taken in thawing, propagating, plating, counting, and then conducting the assay, the process can take over a week to gather data. Here, we introduce assay-ready cryopreserved Vero monolayers in multiwell plates, which can be used directly from the freezer with no cell culture to accelerate the process of plaque determination. Standard dimethyl sulfoxide cryopreservation resulted in just 25% recovery, but addition of polyampholytes (macromolecular cryoprotectants) increased post-thaw recovery and viability in 12- and 24-well plate formats. Variability between individual wells was reduced by chemically induced ice nucleation to prevent supercooling. Cryopreserved cells were used to determine influenza viral plaques in just 24 h, matching results from nonfrozen controls. This innovation may accelerate viral detection and quantification and facilitate automation by eliminating extensive cell culturing.