Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage.

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage.

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants.

The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered "landing pad" site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting and insufficient for more complex cell-based assays. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable alternative avenues for using mammalian synthetic biology to characterize transgenic protein variants. By designing plasmids flanked by a second pair of auxiliary recombination sites, we demonstrate that we can avoid the genomic integration of undesirable bacterial DNA elements using the same starting cells engineered for whole-plasmid integration. We also created "double landing pad" cells simultaneously harboring two orthogonal Bxb1 recombinase sites at separate genomic loci, allowing complex cell-based genetic assays. Integration of a genetically encoded calcium indicator allowed for the real-time monitoring of intracellular calcium signaling dynamics, including kinetic perturbations that occur upon overexpression of the wild-type or variant version of the calcium signaling relay protein STIM1. A panel of missense mutants of the HIV-1 accessory protein Vif was paired with various paralogs within the human Apobec3 innate immune protein family to identify combinations capable or incapable of interacting within cells. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, enabling new functional readouts for large-scale genetic assays for protein function.

Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains.

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV) and SARS-CoV-2 enter cells via engagement of angiotensin converting enzyme-2 (ACE2), while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clades 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from Rhinolophus alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection.

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.

Transcranial Direct Current Stimulation Targeting Primary Motor Versus Dorsolateral Prefrontal Cortices: Proof-of-Concept Study Investigating Functional Connectivity of Thalamocortical Networks Specific to Sensory-Affective Information Processing.

The pain matrix is comprised of an extensive network of brain structures involved in sensory and/or affective information processing. The thalamus is a key structure constituting the pain matrix. The thalamus serves as a relay center receiving information from multiple ascending pathways and relating information to and from multiple cortical areas. However, it is unknown how thalamocortical networks specific to sensory-affective information processing are functionally integrated. Here, in a proof-of-concept study in healthy humans, we aimed to understand this connectivity using transcranial direct current stimulation (tDCS) targeting primary motor (M1) or dorsolateral prefrontal cortices (DLPFC). We compared changes in functional connectivity (FC) with DLPFC tDCS to changes in FC with M1 tDCS. FC changes were also compared to further investigate its relation with individual's baseline experience of pain. We hypothesized that resting-state FC would change based on tDCS location and would represent known thalamocortical networks. Ten right-handed individuals received a single application of anodal tDCS (1 mA, 20 min) to right M1 and DLPFC in a single-blind, sham-controlled crossover study. FC changes were studied between ventroposterolateral (VPL), the sensory nucleus of thalamus, and cortical areas involved in sensory information processing and between medial dorsal (MD), the affective nucleus, and cortical areas involved in affective information processing. Individual's perception of pain at baseline was assessed using cutaneous heat pain stimuli. We found that anodal M1 tDCS and anodal DLPFC tDCS both increased FC between VPL and sensorimotor cortices, although FC effects were greater with M1 tDCS. Similarly, anodal M1 tDCS and anodal DLPFC tDCS both increased FC between MD and motor cortices, but only DLPFC tDCS modulated FC between MD and affective cortices, like DLPFC. Our findings suggest that M1 stimulation primarily modulates FC of sensory networks, whereas DLPFC stimulation modulates FC of both sensory and affective networks. Our findings when replicated in a larger group of individuals could provide useful evidence that may inform future studies on pain to differentiate between effects of M1 and DLPFC stimulation. Notably, our finding that individuals with high baseline pain thresholds experience greater FC changes with DLPFC tDCS implies the role of DLPFC in pain modulation, particularly pain tolerance.

The effect of motor overflow on bimanual asymmetric force coordination.

Motor overflow, typically described in the context of unimanual movements, refers to the natural tendency for a 'resting' limb to move during movement of the opposite limb and is thought to be influenced by inter-hemispheric interactions and intra-cortical networks within the 'resting' hemisphere. It is currently unknown, however, how motor overflow contributes to asymmetric force coordination task accuracy, referred to as bimanual interference, as there is need to generate unequal forces and corticospinal output for each limb. Here, we assessed motor overflow via motor evoked potentials (MEPs) and the regulation of motor overflow via inter-hemispheric inhibition (IHI) and short-intra-cortical inhibition (SICI) using transcranial magnetic stimulation in the presence of unimanual and bimanual isometric force production. All outcomes were measured in the left first dorsal interosseous (test hand) muscle, which maintained 30% maximal voluntary contraction (MVC), while the right hand (conditioning hand) was maintained at rest, 10, 30, or 70% of its MVC. We have found that as higher forces are generated with the conditioning hand, MEP amplitudes at the active test hand decreased and inter-hemispheric inhibition increased, suggesting reduced motor overflow in the presence of bimanual asymmetric forces. Furthermore, we found that subjects with less motor overflow (i.e., reduced MEP amplitudes in the test hemisphere) demonstrated poorer accuracy in maintaining 30% MVC across all conditions. These findings suggest that motor overflow may serve as an adaptive substrate to support bimanual asymmetric force coordination.

Influence of Corticospinal Tracts from Higher Order Motor Cortices on Recruitment Curve Properties in Stroke.

BACKGROUND: Recruitment curves (RCs) acquired using transcranial magnetic stimulation are commonly used in stroke to study physiologic functioning of corticospinal tracts (CST) from M1. However, it is unclear whether CSTs from higher motor cortices contribute as well. OBJECTIVE: To explore whether integrity of CST from higher motor areas, besides M1, relates to CST functioning captured using RCs. METHODS: RCs were acquired for a paretic hand muscle in patients with chronic stroke. Metrics describing gain and overall output of CST were collected. CST integrity was defined by diffusion tensor imaging. For CST emerging from M1 and higher motor areas, integrity (fractional anisotropy) was evaluated in the region of the posterior limb of the internal capsule, the length of CST and in the region of the stroke lesion. RESULTS: We found that output and gain of RC was related to integrity along the length of CST emerging from higher motor cortices but not the M1. CONCLUSIONS: Our results suggest that RC parameters in chronic stroke infer function primarily of CST descending from the higher motor areas but not M1. RCs may thus serve as a simple, in-expensive means to assess re-mapping of alternate areas that is generally studied with resource-intensive neuroimaging in stroke.

Reproducibility of transcranial magnetic stimulation metrics in the study of proximal upper limb muscles.

OBJECTIVE: Reproducibility of transcranial magnetic stimulation (TMS) metrics is essential in accurately tracking recovery and disease. However, majority of evidence pertains to reproducibility of metrics for distal upper limb muscles. We investigate for the first time, reliability of corticospinal physiology for a large proximal muscle - the biceps brachii and relate how varying statistical analyses can influence interpretations. METHODS: 14 young right-handed healthy participants completed two sessions assessing resting motor threshold (RMT), motor evoked potentials (MEPs), motor map and intra-cortical inhibition (ICI) from the left biceps brachii. Analyses included paired t-tests, Pearson's, intra-class (ICC) and concordance correlation coefficients (CCC) and Bland-Altman plots. RESULTS: Unlike paired t-tests, ICC, CCC and Pearson's were >0.6 indicating good reliability for RMTs, MEP intensities and locations of map; however values were <0.3 for MEP responses and ICI. CONCLUSIONS: Corticospinal physiology, defining excitability and output in terms of intensity of the TMS device, and spatial loci are the most reliable metrics for the biceps. MEPs and variables based on MEPs are less reliable since biceps receives fewer cortico-motor-neuronal projections. Statistical tests of agreement and associations are more powerful reliability indices than inferential tests. SIGNIFICANCE: Reliable metrics of proximal muscles when translated to a larger number of participants would serve to sensitively track and prognosticate function in neurological disorders such as stroke where proximal recovery precedes distal.