Cis-mediated interactions of the SARS-CoV-2 frameshift RNA alter its conformations and affect function.

The RNA genome of SARS-CoV-2 contains a frameshift stimulatory element (FSE) that allows access to an alternative reading frame through -1 programmed ribosomal frameshifting (PRF). -1PRF in the 1a/1b gene is essential for efficient viral replication and transcription of the viral genome. -1PRF efficiency relies on the presence of conserved RNA elements within the FSE. One of these elements is a three-stemmed pseudoknot, although alternative folds of the frameshift site might have functional roles as well. Here, by complementing ensemble and single-molecule structural analysis of SARS-CoV-2 frameshift RNA variants with functional data, we reveal a conformational interplay of the 5' and 3' immediate regions with the FSE and show that the extended FSE exists in multiple conformations. Furthermore, limiting the base pairing of the FSE with neighboring nucleotides can favor or impair the formation of the alternative folds, including the pseudoknot. Our results demonstrate that co-existing RNA structures can function together to fine-tune SARS-CoV-2 gene expression, which will aid efforts to design specific inhibitors of viral frameshifting.

The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting.

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

From the Inside Out: an Epibiotic Bdellovibrio Predator with an Expanded Genomic Complement.

Bdellovibrio and like organisms are abundant environmental parasitoids of prokaryotes that show diverse predation strategies. The vast majority of studied Bdellovibrio bacteria and like organisms deploy intraperiplasmic replication inside the prey cell, while few isolates with smaller genomes consume their prey from the outside in an epibiotic manner. The novel parasitoid "Candidatus Bdellovibrio qaytius" was isolated from a eutrophic freshwater pond in British Columbia, where it was a continual part of the microbial community. "Ca Bdellovibrio qaytius" was found to preferentially prey on the betaproteobacterium Paraburkholderia fungorum without entering the periplasm. Despite its epibiotic replication strategy, "Ca Bdellovibrio" encodes a large genomic complement more similar to that of complex periplasmic predators. Functional genomic annotation further revealed several biosynthesis pathways not previously found in epibiotic predators, indicating that "Ca Bdellovibrio" represents an intermediate phenotype and at the same time narrowing down the genomic complement specific to epibiotic predators. In phylogenetic analysis, "Ca Bdellovibrio qaytius" occupies a widely distributed, but poorly characterized, basal cluster within the genus Bdellovibrio This suggests that epibiotic predation might be a common predation type in nature and that epibiotic predation could be the ancestral predation type in the genus.IMPORTANCEBdellovibrio and like organisms are bacteria that prey on other bacteria and are widespread in the environment. Most of the known Bdellovibrio species enter the space between the inner and outer prey membrane, where they consume their prey cells. However, one Bdellovibrio species has been described that consumes its prey from the outside. Here, we describe "Ca Bdellovibrio qaytius," a novel member of the genus Bdellovibrio that also remains outside the prey cell throughout its replication cycle. Unexpectedly, the genome of "Ca Bdellovibrio" is much more similar to the genomes of intracellular predators than to the species with a similar life cycle. Since "Ca Bdellovibrio" is also a basal representative of this genus, we hypothesize that extracellular predation could be the ancestral predation strategy.

Chromulinavorax destructans, a pathogen of microzooplankton that provides a window into the enigmatic candidate phylum Dependentiae.

Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes.