Biology subjects

Dinh, T.

Publications and source records attributed to Dinh, T..

17 recordsLinked to original sources

bioRxiv2026

HIV-1 Integrase C-terminal Domain Mutations Regulate ALLINI Sensitivity

HIV-1 integrase (IN) performs two essential functions in virus replication: catalyzing the integration of reverse-transcribed viral cDNA into host DNA and promoting proper virion maturation. The latter is mediated by IN binding to the genomic RNA (gRNA) through basic residues in its C-terminal domain (CTD), thereby ensuring correct repackaging of the gRNA inside the remodeled capsid lattice. Allosteric integrase inhibitors (ALLINIs) bind within a V-shaped pocket at the interface of the catalytic core domain (CCD) of IN dimers, promote the engagement of the CTDs from adjacent dimers, and thereby occlude gRNA binding. However, virological evidence supporting a role for the IN-CTD in ALLINI mechanism of action is lacking, as mutations in CTD residues implicated in ALLINI-induced multimerization typically yield non-infectious viruses. Here we demonstrate that IN-CTD mutants bearing compensatory substitutions that restore infectivity exhibit markedly altered sensitivity to ALLINIs. Specifically, we show that R269A/K273A substitutions within the IN-CTD confer ~100-fold enhanced sensitivity to ALLINIs. This correlates with a loss of IN-gRNA binding at low ALLINI concentrations, while exerting minimal effects on IN multimerization in vitro and in virions. In contrast, we found that the IN I268T substitution which arose as a suppressor mutation of I268A confers partial resistance to ALLINI-induced inhibition of gRNA binding. Collectively, these findings provide the first direct genetic evidence that CTD residues, despite being distal to the primary drug binding site at the CCD dimer, modulate sensitivity to ALLINIs and highlights IN-gRNA binding as a central target of ALLINIs.

microbiology↗

bioRxiv2026

Distinct sensorimotor encoding in tuft dendrites and somata associated with action, correction, and learning

Frontal cortex plays critical roles in action control and motor skill learning. Within the layer 1 apical tuft dendrites of layer 5 (L5) neurons in frontal cortex, precise input patterns and back-propagating action potentials can trigger powerful regenerative events that may be essential for flexible computation and learning. However, it remains unclear whether tuft activity in frontal cortical L5 circuits encodes sensorimotor information that differs from the information conveyed by their outputs to downstream targets. Using longitudinal two-photon calcium imaging, we investigated sensorimotor encoding in the apical tuft dendrites and somata of L5 extratelencephalic (ET) neurons in the frontal cortex of mice during learning of a discrete change to a cued dexterous action. During learning, movement errors either triggered corrective action or did not, allowing us to dissociate error signals from signals selective for corrective action. Somatic activity tracked both instructional cues and action, whereas tuft activity predominantly tracked instructional cues. Movement errors during learning revealed additional distinct tuft activity that was selectively associated with corrective actions. Furthermore, learning induced divergent changes in the response gain and net selectivity of tuft dendrites compared to somata. Our measurements uncover systematic differences between the tuft dendrites and somata in sensorimotor selectivity, sensitivity to corrective action, and functional plasticity, providing a foundation for investigating the contributions of dendritic computation to motor skill learning.

neuroscience↗

bioRxiv2026

PanACRpred: Predicting Accessible Chromatin Regions in Pangenomes using Motif Chaining

In this work, we investigate using motif subsequence features to predict whether a genomic region is accessible to regulatory proteins, i.e. an accessible chromatin region (ACR), enabling transcription of associated genes. We focus on plants, whose agricultural and ecological importance make them interesting and important organisms to study, and whose complex genomes provide important stress tests for our algorithm. We show that motif sequence similarity as found by co-linear chaining can be used in combination with machine learning models to effectively predict ACRs in genome assemblies.

bioinformatics↗

bioRxiv2026

A Statistical Review of Virus Reduction in Coagulation, Flocculation, and Sedimentation Treatment Processes

Coagulation, flocculation, and sedimentation (CFS) is widely applied as a combined unit process in the treatment of drinking water, wastewater, and recycled water; however, virus reduction through CFS has not been sufficiently characterized to assign pathogen log reduction value (LRV) credits. This study collected data through a systematic review that yielded over 1000 LRVs from 43 manuscripts covering 46 viruses to characterize virus reduction through CFS. The results demonstrate that CFS is effective at reducing viruses, with 68% of virus LRVs greater than 1. A mixed-effects model was used to identify potential mechanisms of virus reduction with ferric and aluminum coagulants, as well as factors associated with variability in performance. Key insights from the model show that virus reduction is: (1) improved at lower pH, similar to natural organic matter (NOM) reduction, (2) lower in secondary effluent than surface water for drinking water treatment, (3) virus-dependent, and (4) dependent on virus enumeration methods, with lower LRVs observed for molecular techniques. These findings demonstrate the potential for CFS to provide consistent and explainable virus reduction, potentially establishing a foundation for regulatory crediting in potable reuse applications. Future crediting frameworks will need to account for the factors impacting performance to accurately quantify and assign credit for virus reduction.

microbiology↗

bioRxiv2025

CRISPR/Cas9 screenings reveal the role of STX1A and CDK1 in Cathepsin G entering and killing colorectal cancer cells

Neutrophils are the major populations of white blood cells and have been reported to facilitate cancer metastasis. Meanwhile, emerging evidence has recently suggested the anti-cancer role of neutrophils. Our previous study revealed that CB-839 and 5-FU-treated colorectal cancer (CRC) tumors recruited neutrophils and induced neutrophil extracellular traps (NETs). Cathepsin G (CTSG), which is released during NET formation, enters CRC cells through the receptor for advanced glycation end products (RAGE) and cleaves 14-3-3{varepsilon} to promote apoptosis. However, the detailed mechanism underlying CTSGs anti-tumor function remains less studied. In this study, we report that CTSG enters CRC cells through RAGE-mediated endocytosis. Knocking out RAGE or inhibiting endocytosis blocks CTSG from entering CRC cells and attenuates CTSG-induced apoptosis. Furthermore, the clathrin coat assembly complex and SNARE proteins were enriched in an arrayed CRISPR/Cas9 screening targeting human membrane trafficking genes. Knocking out SNARE protein STX1A prevents the spread of CTSG in CRC cells and the induction of cleaved PARP. A pooled genome-wide CRISPR/Cas9 screening further identifies the role of CDK1 in the NET-induced killing of CRC cells. Inhibiting CDK1 protected CRC cells from killing by CTSG. Our study reveals novel mechanisms by which CTSG enters and kills CRC cells.

cancer biology↗

bioRxiv2025

Efineptakin alfa (NT-I7) improves overall survival and induces tertiary lymphoid structures in murine lung tumors

Tertiary lymphoid structures (TLSs) are emerging as good predictive biomarkers of response to cancer immunotherapy. However, therapeutic strategies to induce these structures are currently limited. We evaluated the therapeutic benefit of efineptakin alfa (NT-I7), a long-acting form of IL-7, and its ability to induce TLSs in a murine lung tumor model. NT-I7 improved overall survival in tumor-bearing mice. It also increased the abundance of T, B, dendritic cells, and stem-like CD8 T cells and promoted the formation of immune aggregates in the tumor microenvironment (TME). Stem-like CD8 T cells were preferentially located in the immune aggregates. Spatial transcriptomic analyses of the TME further demonstrated that the immune aggregates induced by NT-I7 included TLSs with enrichment of Cd274 (PD-L1) transcripts and genes involved in antigen processing and presentation. Upregulation of Cd274 in the TLSs may provide opportunities for synergy between NT-I7 and PD-1-targeted immunotherapy. STATEMENT OF SIGNIFICANCEThis study demonstrates the ability of efineptakin alfa (NT-I7) to potentially augment the clinical efficacy of cancer immunotherapy by inducing tertiary lymphoid structures in the tumor microenvironment.

immunology↗

bioRxiv2025

Common Gene Networks Orchestrate Organelle Architecture and Inter-Organelle Metabolic Flows for Mucin Production in High Endothelial and Goblet Cells

High endothelial cells (HECs) are specialized vascular gatekeepers that control lymphocyte entry into lymph nodes, a process essential for immune surveillance and adaptive responses. However, how HECs coordinate their high biosynthetic demands with the secretory apparatus to support immune function remains unclear. Using multi-omic approaches, we identify IRE1-XBP1-centered transcriptional networks that, together with CREB3L2-associated gene programs, coordinate inter-organelle metabolic and secretory pathways required for glycosylation and assembly of peripheral node addressin (PNAd), a sulfated sialomucin critical for lymphocyte homing. Genetic or pharmacological perturbation of these pathways disrupts HEC morphology and lymphocyte recruitment during homeostasis and impairs HEC induction during inflammation. Parallel transcriptional programs operate in mucin-producing intestinal goblet cells, suggesting common regulatory pathways linking metabolism and sulfated mucin specialization with the associated expansion of secretory organelles across immune and barrier tissues. Thus, our findings identify transcriptional programs that coordinately scale metabolic pathways and secretory organelles to support the biosynthetic infrastructure underlying the morphology and immune trafficking functions of HEVs. Key pointsXBP1-centered transcriptional networks coordinate metabolic and secretory programs for PNAd biosynthesis. IRE1 or S1P inhibition flattens HEVs, prevents ectopic HEV formation and, reduces lymphocyte recruitment Endothelial XBP1 deletion disrupts HEV morphology and lymphocyte trafficking. Goblet cells share secretory transcriptional programs and regulatory logic with HEV.

immunology↗

bioRxiv2025

How Nasal Cavity Structure Influences Empty Nose Syndrome Severity: A CFD-Based Analysis

BackgroundEmpty Nose Syndrome (ENS) is a debilitating condition that can occur after partial or total turbinectomy, leading to impaired nasal airflow sensation, breathing difficulties, and sleep disturbances. While ENS is often diagnosed using the ENS6Q questionnaire, its precise causes remain unclear. Some patients with significant turbinate loss develop minor ENS symptoms, whereas others experience severe symptoms after minor mucosal cauterization. Understanding the structural and aerodynamic factors contributing to ENS is crucial for improving diagnosis and prevention. ObjectiveThis study aims to identify correlations between the ENS6Q score and key anatomical and aerodynamic parameters obtained from computational fluid dynamics (CFD) simulations in ENS patients. MethodsWe reconstructed patient-specific nasal cavity models from computed tomography (CT) scans and performed CFD simulations. The analysis focused on five key parameters: the remaining turbinate volume, total mucosal surface area, nasal resistance, average cross-sectional area, and airflow imbalance between the two nasal cavities. These parameters were then compared to ENS6Q scores. ResultsPreliminary findings suggest that a lower remaining turbinate volume, reduced mucosal surface area are associated with higher ENS6Q scores. Additionally, significant airflow asymmetry between the two nasal cavities appears to correlate with more severe symptoms. Furthermore, our data indicate that individuals with larger nasal cavities and greater preoperative mucosal surface area tend to be more resilient to turbinectomy. For an equivalent amount of turbinate resection, patients with initially smaller nasal cavities thus having less mucosal surface experience more severe ENS symptoms. ConclusionBy quantifying the anatomical and aerodynamic characteristics of ENS patients, this study provides new insights into the structural factors contributing to ENS severity. These findings may help refine diagnostic criteria and guide surgical approaches to minimize ENS risk.

biophysics↗

bioRxiv2025

In Vivo Multiplexed Modeling Reveals Diverse Roles of the TBX2 Subfamily and Egr1 in Ras-Driven Lung Adenocarcinoma

The TBX2 subfamily of T-box transcription factors (including Tbx2, Tbx3, Tbx4, Tbx5) plays an essential role in lung development. Downregulation of these genes in human Lung adenocarcinoma (LUAD) suggests that these genes may be tumor suppressive, however because downregulation appears to occur primarily via epigenetic change, it remains unclear if these changes causally drive tumor progression or are merely the consequence of upstream events. Herein, we developed the first multiplexed mouse model to study the impact of TBX2 subfamily loss, alongside associated signaling genes Egr1, Chd2, Tnfaip3a, and Atf3, in Ras-driven lung cancer. Using TuBa-seq, a high-throughput tumor-barcoding system, we quantified the growth effects of these knockouts during early and late tumorigenesis. Chd2 loss consistently suppressed tumor progression, while Tbx2 loss exhibited stage-dependent effects. Notably, Egr1 emerged as a potent tumor suppressor, with its knockout increasing tumor size ([~]5x) at 20 weeks, surpassing Rb1 loss. Transcriptomic analyses of Egr1-deficient tumors suggested immune dysregulation, including heightened inflammation and potential markers of T cell exhaustion in the tumor microenvironment. These findings indicate that Egr1 may play a role in suppressing tumor growth through modulating immune dynamics, offering new insights into the interplay between tumor progression and immune regulation in LUAD.

cancer biology↗

bioRxiv2025

Phenotypic pleiotropy of missense variants in human B cell-confinement receptor P2RY8

Missense variants can have pleiotropic effects on protein function and predicting these effects can be difficult. We performed near-saturation deep mutational scanning of P2RY8, a G-protein-coupled receptor that promotes germinal center B cell confinement. We assayed the effect of each variant on surface expression, migration, and proliferation. We delineated variants that affected both expression and function, affected function independently of expression, and discrepantly affected migration and proliferation. We also used cryo-electron microscopy to determine the structure of activated, ligand-bound P2RY8, providing structural insights into the effects of variants on ligand binding and signal transmission. We applied the deep mutational scanning results to both improve computational variant effect predictions and to characterize the phenotype of germline variants and lymphoma-associated variants. Together, our results demonstrate the power of integrating deep mutational scanning, structure determination, and in silico prediction to advance the understanding of a receptor important in human health.

genomics↗

bioRxiv2025

Targeting Osteosarcoma heterogeneity to improve therapeutic response

Intra tumor heterogeneity complicates cancer therapy by providing tumors with the ability to alter their phenotypes and become more therapy resistant. Here, we tested the hypothesis that identifying and modulating expression of key state-specific transcription factors could be used as a strategy for driving cells to a more therapy-sensitive state. Recent single-cell studies have explored the inter and intra tumoral heterogeneity of osteosarcoma and identified gene pathways enriched in specific cell states. For example, metastatic tumors are characterized by an expression of genes in the TNF-, PI3K, TGF{beta} and mTOR pathways. We identified similar profiles in osteosarcoma patient-derived xenograft-derived cell lines and potential transcription factor drivers of these states. We then used perturb-seq to downregulate expression of key transcription factors and evaluated the effect of these modulations on single cell RNA profiles and drug responses. Knockdown of NFE2L3 or NR0B1 increased the proportion of cells sensitive to targeted therapy. This approach, which could potentially be applied to other cancers, could be used as a strategy to increase the response to targeted therapies by increasing the proportion of cells in a drug-sensitive state. HighlightsO_LIDistinct transcriptomic states were identified in osteosarcoma cell lines using single-cell RNA sequencing. C_LIO_LILineage tracing identified states with differential sensitivity to therapy. C_LIO_LIUsing perturb-seq, we identified transcription factors that drive cells towards a more sensitive state. C_LIO_LIThe transcription factor NFE2L3 was identified as targets capable of reprogramming cells to a sensitive state. C_LI

genomics↗

bioRxiv2024

Oligomeric HIV-1 Integrase Structures Reveal Functional Plasticity for Intasome Assembly and RNA Binding

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.

biochemistry↗

bioRxiv2024

The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents which potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into Phase 2a clinical trials. Previous cell culture based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. While both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150- fold resistance to the inhibitor compared to the WT virus. By rationally modifying PIR we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was [~]14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use. IMPORTANCEAntiretroviral therapies save the lives of millions of people living with HIV (PLWH). However, evolution of multi-drug-resistant viral phenotypes is a major clinical problem, and there are limited or no treatment options for heavily treatment-experienced PLWH. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a novel class of antiretroviral compounds which work by a unique mechanism of binding to the non-catalytic site on the viral protein and inducing aberrant integrase multimerization. Accordingly, ALLINIs potently inhibit both wild type HIV-1 and all drug-resistant viral phenotypes that have so far emerged against currently used therapies. Pirmitegravir, a highly potent and safe investigational ALLINI, is currently advancing through clinical trials. Here we have elucidated structural and mechanistic bases behind the emergence of HIV-1 integrase mutations in infected cell that confer resistance to pirmitegravir. In turn, our findings allowed us to rationally develop an improved ALLINI with substantially enhanced potency against the pirmitegravir resistant virus.

microbiology↗

bioRxiv2022

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes.

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large populations wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

microbiology↗

bioRxiv2022

A new cortical parcellation based on systematic review of primate anatomical tracing studies on corticostriatal projections

Corticostriatal projections form the input level of a circuitry that connects the cerebral cortex, basal ganglia, and thalamus. Three distinct, functional subcircuits exist according to the tripartite model: Sensorimotor cortices projecting mainly to the dorsolateral striatum; associative cortices projecting to the dorsomedial striatum and limbic cortices projecting to the ventral striatum. However, there is to date no atlas that allows researchers to label cortical projection areas belonging to each of these subcircuits separately. To address this research gap, the aim of this study was threefold: First, to systematically review anatomical tracing studies that focused on corticostriatal projections in non-human primates, and to classify their findings according to the tripartite model. Second, to develop an atlas of the human cerebral cortex based on this classification. Third, to test the hypothesis that labels in this atlas show structural connectivity with specific striatal subregions in humans using diffusion-based tractography in a sample of 24 healthy participants. In total, 98 studies met the inclusion criteria for our systematic review. Information about projections from the cortex to the striatum was systematically extracted by Brodmann area, and cortical areas were classified by their dominant efferent projections. Taking known homological and functional similarities and differences between non-human primate and human cortical regions into account, a new human corticostriatal projection (CSP) atlas was developed. Using human diffusion-based tractography analyses, we found that the limbic and sensorimotor atlas labels showed preferential structural connectivity with the ventral and dorsolateral striatum, respectively. However, the pattern of structural connectivity for the associative label showed the greatest degree of overlap with other labels. We provide this new atlas as a freely available tool for neuroimaging studies, where it allows for the first-time delineation of anatomically informed regions-of-interest to study functional subcircuits within the corticostriatal circuitry. This tool will enable specific investigations of subcircuits involved in the pathogenesis of neuropsychiatric illness such as schizophrenia and bipolar disorders. Highlights- Systematic review of anatomical projections from the cerebral cortex to the striatum in non-human primates. - Development of a novel cortical atlas for use in neuroimaging studies focusing on the corticostriatal brain circuitry. - Tractography in human diffusion-weighted imaging data to test if associative, limbic, and sensorimotor cortical atlas labels show preferential connectivity to regions within the striatum.

neuroscience↗

bioRxiv2022

An NKX-COUP-TFII genomic code for mucosal vascular addressins and organ morphogenesis

Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules, providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define a genomic address code for targeted expression of mucosal vascular addressins and implicate NCCE in fundamental processes in cell specification and development.

immunology↗

bioRxiv2021

Emergence of compensatory mutations reveal the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

ABSTRACTHIV-1 integrase (IN) has a non-catalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit IN-gRNA binding and result in non-infectious viruses marked by mislocalization of the gRNA within virions. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA. However, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R and D279R substitutions was sufficient to restore IN-RNA binding and infectivity for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding. Finally, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Taken together, our findings highlight the essential role of CTD in gRNA binding and ALLINI sensitivity, and reveal the importance of pliable electrostatic interactions between the IN- CTD and the gRNA. IMPORTANCEIn addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues within its C-terminal domain (CTD) and regulates proper virion maturation. Here we show that compensatory mutations in nearby acidic residues (i.e. D256N and D270N) restore the ability to bind gRNA for IN variants bearing substitutions in these positively charged CTD residues. Similarly, charge reversals through individual D-to-R and D-to-K substitutions at these positions enabled the respective IN mutants to bind gRNA and restore virion infectivity. Further, we show that specific residues within the IN-CTD required for RNA binding also influence sensitivity to allosteric integrase inhibitors, a class of novel IN- targeting compounds that target the non-catalytic function of IN. Taken together, our findings reveal the importance of electrostatic interactions in IN-gRNA binding and provide key evidence for a crucial role of the IN-CTD in allosteric integrase inhibitor mechanism of action.

microbiology↗