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Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety level containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines., (© 2024. The Author(s).)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can infect various human tissues and cell types, principally via interaction with its cognate receptor angiotensin-converting enzyme-2 (ACE2). However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After twenty passages of a spike-expressing recombinant vesicular stomatitis virus (VSV), cell-type-specific phenotypic changes were observed and sequencing allowed the identification of sixteen adaptive spike mutations. We used VSV pseudotyping to measure the entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, and entry pathway usage of all the mutants, alone or in combination. The fusogenicity of the mutant spikes was assessed with a cell-cell fusion assay. Finally, mutant recombinant VSVs were used to measure the fitness advantage associated with selected mutations. We found that the effects of these mutations varied across cell types, both in terms of viral entry and replicative fitness. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has significantly impacted global health, stressing the necessity of basic understanding of the host response to this viral infection. In this study, we investigated how SARS-CoV-2 remodels the landscape of small non-coding RNAs (sncRNA) from a large collection of nasopharyngeal swab samples taken at various time points from patients with distinct symptom severity. High-throughput RNA sequencing analysis revealed a global alteration of the sncRNA landscape, with abundance peaks related to species of 21-23 and 32-33 nucleotides. Host-derived sncRNAs, including microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), and small nucleolar RNA-derived small RNAs (sdRNAs) exhibited significant differential expression in infected patients compared to controls. Importantly, miRNA expression was predominantly down-regulated in response to SARS-CoV-2 infection, especially in patients with severe symptoms. Furthermore, we identified specific tsRNAs derived from Glu- and Gly-tRNAs as major altered elements upon infection, with 5' tRNA halves being the most abundant species and suggesting their potential as biomarkers for viral presence and disease severity prediction. Additionally, down-regulation of C/D-box sdRNAs and altered expression of tinyRNAs (tyRNAs) were observed in infected patients. These findings provide valuable insights into the host sncRNA response to SARS-CoV-2 infection and may contribute to the development of further diagnostic and therapeutic strategies in the clinic., (© 2024. The Author(s).)

Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.

Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus , Tobacco etch virus , and Potato virus X , in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.

Potassium (K + ) is a key monovalent cation necessary for multiple aspects of cell growth and survival. In plants, this cation also plays a key role in the control of stomatal movement. KAT1 and its homolog KAT2 are the main inward rectifying channels present in guard cells, mediating K + influx into these cells, resulting in stomatal opening. To gain further insight into the regulation of these channels, we performed a split-ubiquitin protein-protein interaction screen searching for KAT1 interactors in Arabidopsis ( Arabidopsis thaliana ). We characterized one of these candidates, BCL2-ASSOCIATED ATHANOGENE4 (BAG4), in detail using biochemical and genetic approaches to confirm this interaction and its effect on KAT1 activity. We show that BAG4 improves KAT1-mediated K + transport in two heterologous systems and provide evidence that in plants, BAG4 interacts with KAT1 and favors the arrival of KAT1 at the plasma membrane. Importantly, lines lacking or overexpressing the BAG4 gene show altered KAT1 plasma membrane accumulation and alterations in stomatal movement. Our data allowed us to identify a KAT1 regulator and define a potential target for the plant BAG family. The identification of physiologically relevant regulators of K + channels will aid in the design of approaches that may impact drought tolerance and pathogen susceptibility., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase.

The maintenance of ionic homeostasis is essential for cell viability, thus the activity of plasma membrane ion transporters must be tightly controlled. Previous studies in Saccharomyces cerevisiae revealed that the proper trafficking of several nutrient permeases requires the E3 ubiquitin ligase Rsp5 and, in many cases, the presence of specific adaptor proteins needed for Rsp5 substrate recognition. Among these adaptor proteins are nine members of the arrestin-related trafficking adaptor (ART) family. We studied the possible role of the ART family in the regulation of monovalent cation transporters. We show here that the salt sensitivity phenotype of the rim8/art9 mutant is due to severe defects in Ena1 protein accumulation, which is not attributable to transcriptional defects. Many components of the Rim pathway are required for correct Ena1 accumulation, but not for the accumulation of other nutrient permeases. Moreover, we observe that strains lacking components of the endosomal sorting complexes required for transport (ESCRT) pathway previously described to play a role in Rim complex formation present similar defects in Ena1 accumulation. Our results show that, in response to salt stress, a functional Rim complex via specific ESCRT interactions is required for the proper accumulation of the Ena1 protein, but not induction of the ENA1 gene., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The relative concentrations of ions and solutes inside cells are actively maintained by several classes of transport proteins, in many cases against their concentration gradient. These transport processes, which consume a large portion of cellular energy, must be constantly regulated. Many structurally distinct families of channels, carriers, and pumps have been characterized in considerable detail during the past decades and defects in the function of some of these proteins have been linked to a growing list of human diseases. The dynamic regulation of the transport proteins present at the cell surface is vital for both normal cellular function and for the successful adaptation to changing environments. The composition of proteins present at the cell surface is controlled on both the transcriptional and post-translational level. Post-translational regulation involves highly conserved mechanisms of phosphorylation- and ubiquitylation-dependent signal transduction routes used to modify the cohort of receptors and transport proteins present under any given circumstances. In this review, we will summarize what is currently known about one facet of this regulatory process: the endocytic regulation of alkali metal transport proteins. The physiological relevance, major contributors, parallels and missing pieces of the puzzle in mammals, yeast and plants will be discussed.