The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Assembly of a nucleus-like structure during viral replication in bacteria.

We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201φ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.

A bacteriophage tubulin harnesses dynamic instability to center DNA in infected cells.

Dynamic instability, polarity, and spatiotemporal organization are hallmarks of the microtubule cytoskeleton that allow formation of complex structures such as the eukaryotic spindle. No similar structure has been identified in prokaryotes. The bacteriophage-encoded tubulin PhuZ is required to position DNA at mid-cell, without which infectivity is compromised. Here, we show that PhuZ filaments, like microtubules, stochastically switch from growing in a distinctly polar manner to catastrophic depolymerization (dynamic instability) both in vitro and in vivo. One end of each PhuZ filament is stably anchored near the cell pole to form a spindle-like array that orients the growing ends toward the phage nucleoid so as to position it near mid-cell. Our results demonstrate how a bacteriophage can harness the properties of a tubulin-like cytoskeleton for efficient propagation. This represents the first identification of a prokaryotic tubulin with the dynamic instability of microtubules and the ability to form a simplified bipolar spindle.

The structure and assembly mechanism of a novel three-stranded tubulin filament that centers phage DNA.

Tubulins are a universally conserved protein superfamily that carry out diverse biological roles by assembling filaments with very different architectures. The underlying basis of this structural diversity is poorly understood. Here, we determine a 7.1 Å cryo-electron microscopy reconstruction of the bacteriophage-encoded PhuZ filament and provide molecular-level insight into its cooperative assembly mechanism. The PhuZ family of tubulins is required to actively center the phage within infected host cells, facilitating efficient phage replication. Our reconstruction and derived model reveal the first example of a three-stranded tubulin filament. We show that the elongated C-terminal tail simultaneously stabilizes both longitudinal and lateral interactions, which in turn define filament architecture. Identified interaction surfaces are conserved within the PhuZ family, and their mutagenesis compromises polymerization in vitro and in vivo. Combining kinetic modeling of PhuZ filament assembly and structural data, we suggest a common filament structure and assembly mechanism for the PhuZ family of tubulins.

Complete genome sequence of Pyrobaculum oguniense.

Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative analyses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, reveals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illuminated some of the genome dynamics potentially shared with other species in this genus.