Genome-Scale CRISPR Screening Reveals Host Factors Required for Ribosome Formation and Viral Replication.

Viruses are known to co-opt host machinery for translation initiation, but less is known about which host factors are required for the formation of ribosomes used to synthesize viral proteins. Using a loss-of-function CRISPR screen, we show that synthesis of a flavivirus-encoded fluorescent reporter depends on multiple host factors, including several 60S ribosome biogenesis proteins. Viral phenotyping revealed that two of these factors, SBDS, a known ribosome biogenesis factor, and the relatively uncharacterized protein SPATA5, were broadly required for replication of flaviviruses, coronaviruses, alphaviruses, paramyxoviruses, an enterovirus, and a poxvirus. Mechanistic studies revealed that loss of SPATA5 caused defects in rRNA processing and ribosome assembly, suggesting that this human protein may be a functional ortholog of yeast Drg1. These studies implicate specific ribosome biogenesis proteins as viral host dependency factors that are required for synthesis of virally encoded protein and accordingly, optimal viral replication. IMPORTANCE Viruses are well known for their ability to co-opt host ribosomes to synthesize viral proteins. The specific factors involved in translation of viral RNAs are not fully described. In this study, we implemented a unique genome-scale CRISPR screen to identify previously uncharacterized host factors that are important for the synthesis of virally encoded protein. We found that multiple genes involved in 60S ribosome biogenesis were required for viral RNA translation. Loss of these factors severely impaired viral replication. Mechanistic studies on the AAA ATPase SPATA5 indicate that this host factor is required for a late step in ribosome formation. These findings reveal insight into the identity and function of specific ribosome biogenesis proteins that are critical for viral infections.

Ribosomal control in RNA virus-infected cells.

Viruses are strictly intracellular parasites requiring host cellular functions to complete their reproduction cycle involving virus infection of host cell, viral genome replication, viral protein translation, and virion release. Ribosomes are protein synthesis factories in cells, and viruses need to manipulate ribosomes to complete their protein synthesis. Viruses use translation initiation factors through their own RNA structures or cap structures, thereby inducing ribosomes to synthesize viral proteins. Viruses also affect ribosome production and the assembly of mature ribosomes, and regulate the recognition of mRNA by ribosomes, thereby promoting viral protein synthesis and inhibiting the synthesis of host antiviral immune proteins. Here, we review the remarkable mechanisms used by RNA viruses to regulate ribosomes, in particular, the mechanisms by which RNA viruses induce the formation of specific heterogeneous ribosomes required for viral protein translation. This review provides valuable insights into the control of viral infection and diseases from the perspective of viral protein synthesis.

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution, new validation tools, and implications for the future.

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) "Resolution Revolution" made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.

Macromolecular Structure Assembly as a Novel Antibiotic Target.

This review discusses the inhibition of macromolecular structure formation as a novel and under-investigated drug target. The disruption of cell wall structures by penicillin-binding protein interactions is one potential target. Inhibition of DNA polymerase III assembly by novel drugs is a second target that should be investigated. RNA polymerase protein structural interactions are a third potential target. Finally, disruption of ribosomal subunit biogenesis represents a fourth important target that can be further investigated. Methods to examine these possibilities are discussed.

Characterization of Aurintricarboxylic Acid (ATA) Interactions with Plasma Transporter Protein and SARS-CoV-2 Viral Targets: Correlation of Functional Activity and Binding Energetics.

In an effort to identify functional-energetic correlations leading to the development of efficient anti-SARS-CoV-2 therapeutic agents, we have designed synthetic analogs of aurintricarboxylic acid (ATA), a heterogeneous polymeric mixture of structurally related linear homologs known to exhibit a host of biological properties, including antiviral activity. These derivatives are evaluated for their ability to interact with a plasma transporter protein (human serum albumin), eukaryotic (yeast) ribosomes, and a SARS-CoV-2 target, the RNA-dependent RNA polymerase (RdRp). The resultant data are critical for characterizing drug distribution, bioavailability, and effective inhibition of host and viral targets. Promising lead compounds are selected on the basis of their binding energetics which have been characterized and correlated with functional activities as assessed by inhibition of RNA replication and protein synthesis. Our results reveal that the activity of heterogeneous ATA is mimicked by linear compounds of defined molecular weight, with a dichlorohexamer salicylic-acid derivative exhibiting the highest potency. These findings are instrumental for optimizing the design of structurally defined ATA analogs that fulfill the requirements of an antiviral drug with respect to bioavailability, homogeneity, and potency, thereby expanding the arsenal of therapeutic regimens that are currently available to address the urgent need for effective SARS-CoV-2 treatment strategies.

Proteins of SARS CoV-2, which causes COVID-19, and the Interacting proteins. (Proteins of SARS CoV-2, which causes COVID-19, and the Interacting proteins.)

This journal issue on the topic of COVID proteins features four articles: (1) A review on the biochemical activities of the nonstructural proteins (NSPs) and the spike protein and a description of a recent ribosomal profiling study that revealed 23 new SARS CoV-2 proteins (2) An in silico study of the mutational stability of SARS CoV-2 proteins (3) A docking study of milk peptides binding to various SARS CoV-2 proteins (4) A docking study of possible anti-SARS CoV-2 main protease inhibitors.

Promising Assays for Examining a Putative Role of Ribosomal Heterogeneity in COVID-19 Susceptibility and Severity.

The heterogeneity of ribosomes, characterized by structural variations, arises from differences in types, numbers, and/or post-translational modifications of participating ribosomal proteins (RPs), ribosomal RNAs (rRNAs) sequence variants plus post-transcriptional modifications, and additional molecules essential for forming a translational machinery. The ribosomal heterogeneity within an individual organism or a single cell leads to preferential translations of selected messenger RNA (mRNA) transcripts over others, especially in response to environmental cues. The role of ribosomal heterogeneity in SARS-CoV-2 coronavirus infection, propagation, related symptoms, or vaccine responses is not known, and a technique to examine these has not yet been developed. Tools to detect ribosomal heterogeneity or to profile translating mRNAs independently cannot identify unique or specialized ribosome(s) along with corresponding mRNA substrate(s). Concurrent characterizations of RPs and/or rRNAs with mRNA substrate from a single ribosome would be critical to decipher the putative role of ribosomal heterogeneity in the COVID-19 disease, caused by the SARS-CoV-2, which hijacks the host ribosome to preferentially translate its RNA genome. Such a protocol should be able to provide a high-throughput screening of clinical samples in a large population that would reach a statistical power for determining the impact of a specialized ribosome to specific characteristics of the disease. These characteristics may include host susceptibility, viral infectivity and transmissibility, severity of symptoms, antiviral treatment responses, and vaccine immunogenicity including its side effect and efficacy. In this study, several state-of-the-art techniques, in particular, chemical probing of ribosomal components or rRNA structures, proximity ligation to generate rRNA-mRNA chimeras for sequencing, nanopore gating of individual ribosomes, nanopore RNA sequencing and/or structural analyses, single-ribosome mass spectrometry, and microfluidic droplets for separating ribosomes or indexing rRNAs/mRNAs, are discussed. The key elements for further improvement and proper integration of the above techniques to potentially arrive at a high-throughput protocol for examining individual ribosomes and their mRNA substrates in a clinical setting are also presented.