Covalent attachment of active enzymes to upconversion phosphors allows ratiometric detection of substrates

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface?accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide?thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state

An eIF3d-dependent switch regulates HCMV replication by remodeling the infected cell translation landscape to mimic chronic ER stress

Regulated loading of eIF3-bound 40S ribosomes on capped mRNA is generally dependent upon the translation initiation factor eIF4E; however, mRNA translation often proceeds during physiological stress, such as virus infection, when eIF4E availability and activity are limiting. It remains poorly understood how translation of virus and host mRNAs are regulated during infection stress. While initially sensitive to mTOR inhibition, which limits eIF4E-dependent translation, we show that protein synthesis in human cytomegalovirus (HCMV)-infected cells unexpectedly becomes progressively reliant upon eIF3d. Targeting eIF3d selectively inhibits HCMV replication, reduces polyribosome abundance, and interferes with expression of essential virus genes and a host gene expression signature indicative of chronic ER stress that fosters HCMV reproduction. This reveals a strategy whereby cellular eIF3d-dependent protein production is hijacked to exploit virus-induced ER stress. Moreover, it establishes how switching between eIF4E and eIF3d-responsive cap-dependent translation can differentially tune virus and host gene expression in infected cells

Expanding the scope of biomolecule monitoring with ratiometric signaling from rare-earth upconverting phosphors

Upconversion (UC) is a powerful mulitphoton mechanism that converts low‐energy photons into higher energy emission. One of the most investigated UC systems is upconverting phosphors (UCPs). Here, a new, one‐pot synthetic procedure was used to prepare water dispersible, visibly emissive, rare‐earth doped UCPs that were capped with the functional groups oleic acid (OA), 6‐aminohexanoic acid (AHA), and 6‐maleimidohexanoic acid (MHA). These synthesized UCPs were characterized by UC luminescence, dynamic light scattering (DLS), transmission electron microscopy (TEM), and powder X‐ray diffraction (pXRD). This study expands upon our previous proof‐of‐principle work in demonstrating the use of UCPs (both synthesized and commercial) to detect on the level and function of biological analytes, from enzymes to key disease biomarkers (PETNR, glucose oxidase, vitamin B12, and cytochrome c). By tailoring the absorption profile of the biomolecule cofactors to the UCP emission, a wide‐range of analytes can be utilized. We also demonstrate the ability of our system to reversibly monitor the addition of enzyme substrates via repeat oxidation and reduction of pentaerythritol tetranitrate reductase (PETNR)

Targeting the m(6)A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication

N-6-methyladenosine (m(6)A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m(6)A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m(6)A-modified. How the cellular m(6)A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human beta-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m(6)A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m(6)A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m(6)A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m(6)A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m(6)A-modified and host m(6)A pathway components control beta-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m(6)A pathway to restrict coronavirus reproduction