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The production of chemicals, biologics, and therapeutics on an industrial scale increasingly uses simple microorganisms and complicated mammalian systems. Natural strains often generate limited and economically unviable quantities of the desired bioproduct and present physiological, experimental, and operational challenges when used at industrial scales. Therefore, systematically improving these biological systems is essential to smooth a bioprocess and maximize operation economics. This results-oriented approach towards enhancing industrially important producers’ genetic and physical characteristics is called strain improvement. The workflow has traditionally employed classical mutagenesis approaches, followed by screening and selecting enhanced mutants. With modern genetic, recombinant, and system biology tools, targeted modifications to the desired genes or whole pathway adjustments have become possible. Additionally, the ability to precisely and parallelly engineer genomes at single residue levels have increased the overall throughput of the strain improvement process. Here, the authors survey various classical to modern approaches to strain improvement and their collective implications for bioprocess engineering.

Viral proteins typically exist in the context of complex virions or in the even more complex host cells in which they replicate. Hence, meaningful insights into virus protein structure often need to account for this context. Various flavors of in situ cryo-electron microscopy (cryo-EM), such as cryo-electron tomography, are key methods for the contextual study of virus protein structure in pleomorphic virions and host cells. Here, we review recent in situ cryo-EM work on three selected phenomena in RNA virus replication: the maturation and nuclear entry of HIV-1, the membrane-bound replication organelles of positive-sense RNA viruses, and the membrane-less viral factories of negative-sense RNA viruses. We highlight cases where the imaged phenomena are targets of novel antiviral drugs (such as the recently approved antiretroviral Lenacapavir), drug candidates, and antiviral strategies. Finally, we discuss recent technical advances that extend the reach of in situ cryo-EM in virology.

The spread of SARS-CoV-2 led to the emergence of several variants of concern (VOCs). The spike glycoprotein, responsible for engaging the viral receptor, exhibits the highest density of mutations, suggesting an ongoing evolution to optimize viral entry. This study characterizes the bond formed by virion mimics carrying the SARS-CoV-2 spike protein and the plasma membrane of host cells in the early stages of virus entry. Contrary to the traditional analysis of isolated ligand-receptor pairs, we utilized well-defined biomimetic models and biochemical and biophysical techniques to characterize the multivalent interaction of VOCs with the complex cell membrane. We observed an overall increase in the binding affinity for newer VOCs. By progressively reducing the system complexity, we identify heparan sulfate (HS) as a main driver of this variation, with a 10-fold increase in affinity for Omicron BA.1 over that of the original strain. These results demonstrate the essential role of coreceptors, particularly HS, in the modulation of SARS-CoV-2 infection and highlight the importance of multiscale biophysical and biochemical assays that account for membrane complexity to fully characterize and understand the role of molecular components and their synergy in viral attachment and entry.

Background: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes.

Results: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes.

Conclusion: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C’s association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divides the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

During host cell invasion, microsporidian spores translocate: their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the Vairimorpha necatrix polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and uncovers new aspects of ribosome regulation and transport.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.