Biology subjects

Khalid, M. M.

Publications and source records attributed to Khalid, M. M..

17 recordsLinked to original sources

bioRxiv2026

Epistasis between SARS-CoV-2 M and N Proteins Balances Particle Assembly and Immune Evasion

Since its emergence in the human population, SARS-CoV-2 has continuously evolved to evade immune responses and robustly establish global circulation. In this process, the structural viral membrane (M) protein has accumulated amino acid changes whose impact on viral particle assembly and innate immune evasion remains incompletely understood. Here, we designed a SARS-CoV-2 replicon system lacking M that assesses the influence of transiently transfected M protein variants on viral particle production independently of viral RNA replication. We found that M protein variants have reduced particle assembly while innate immune antagonism functions are strengthened. Notably, the assembly defect is rescued by co-evolving N protein variants, highlighting how SARS-CoV-2 evolution coordinates between two of its structural proteins to optimize viral infection. Our work underscores the complex evolutionary trajectories of SARS-CoV-2 variants across different viral proteins and informs future therapeutic strategies targeting viral assembly and limiting infection. Author summarySince the onset of the COVID-19, SARS-CoV-2 has been changing continuously in ways that help it spread efficiently and evade human defenses. The viral membrane (M) protein is a key structural component required to form new viral particles. Nevertheless, it has accumulated changes over time with overlooked functions. In this study, we developed an experimental system to examine the impact of these changes on virus assembly and host immune system. We found that recent M variants are better than older variants at suppressing immune responses but less efficient at forming viral particles. This defect seems to be compensated for by coordinated changes in another structural protein, nucleocapsid (N). These findings reveal that SARS-CoV-2 evolution involves trade-offs between different viral characteristics, with changes in one protein balancing changes in another. Understanding these trade-offs provides new insight into how the virus adapts to humans and may help guide therapeutic strategies to limit infection.

microbiology↗

bioRxiv2026

Ketone ester supplementation in aged mice reduces activation of B cell subsets

BackgroundAging in the immune system results in increased susceptibility to infections, exacerbated autoimmunity, and reduced responsiveness to vaccines. However, there are no current established interventions for immune aging. Ketogenic diets and fasting have been researched as interventions against other aspects of aging and age-related diseases, and they work in part by increasing circulating levels of ketone bodies, which have anti-inflammatory properties and can boost T cell function. Exogenous ketones, such as ketone esters, are currently being studied as a more accessible approach to obtain the benefits of ketone bodies through direct supplementation. Here, we investigated whether ketone ester supplementation improves immune function during aging. Aged (19-month-old) C57BL/6JN mice were given a diet supplemented with the ketone ester or a control diet for 15 weeks. ResultsWe found that the ketone ester diet decreased activation of B cells, especially age-associated B cells, in the spleen. The diet also reduced the proportion of age-associated B cells defined by CD11b and CD11c expression, though not of the broader CD21-CD23- population. Despite this decrease in activation, there was no impairment in antibody production, as mice on the ketone ester diet mounted a normal response to nitrophenyl-ovalbumin immunization. The ketone ester diet also reduced glucose dependence and translation in subsets of age-associated B cells. Treating splenocytes from aged mice with {beta}-hydroxybutyrate was sufficient to suppress B cell translation and reduced mTORC1 signaling, indicating that ketone bodies act directly on B cells. {beta}-hydroxybutyrate also decreased expression of T-bet, the signature transcription factor driving the autoimmune functions of age-associated B cells. ConclusionsOur study elucidates the effect of ketone esters on B cells in the context of aging and unveils a new immunoregulatory role of ketone bodies on B cells.

immunology↗

bioRxiv2025

Multi-omics Analysis of Human Blood Cells Reveals Unique Features of Age-associated Type2 CD8 Memory T cells

Aging impacts immune function, but the mechanisms driving age-related changes in immune cell subsets remain unclear. To explore age-dependent changes in immune cell populations, we analyzed human peripheral blood mononuclear cells (PBMCs) from a cohort of healthy donors aged 20-82 years using a 36-color spectral flow cytometry panel focused on T cells. We identified a unique population of memory CD8 T cells, which lack CXCR3 and produce a Th2-like cytokine response, accumulate with age. We discovered an age-dependent bias in naive CD8 T cells toward Th2 cytokine production, accompanied by transcriptional and epigenetic changes supporting this phenotype. Moreover, health outcome association analysis linked the accumulation of these unique CXCR3- central memory CD8 T cells to asthma, chronic liver conditions, and type 2 diabetes. Together, our results support the model that an age-dependent drift in epigenetic regulation towards a Th2-like phenotype drives a pathogenic Th2-like immune population.

immunology↗

bioRxiv2025

Drug-induced phospholipidosis as an artifact in antiviral drug repurposing

Drug repurposing in principle can speed antiviral drug discovery. Among the molecules most frequently advanced in such repurposing efforts are a group of structurally diverse cationic amphiphilic drugs (CADs). While CADs have shown micromolar to mid-nanomolar antiviral activity in cell-based assays, they can induce phospholipidosis, confounding in COVID-19 repurposing. A barrier to the identification of phospholipidosis inducers has been the involved nature of the microscopy assays used to characterize them. To ease the identification of these artifacts, we describe a rapid microplate-based assay to detect phospholipidosis. Leveraging this assay, we quantified the prevalence of phospholipidosis-inducers across several cell-based antiviral repurposing screens. We selected 40 drugs reported to have micromolar antiviral activities and found that 26 of them (65%) induced phospholipidosis within the same concentration range as their reported antiviral activities. Intriguingly, we identified four non-CADs that also induce phospholipidosis, revealing a new group of drugs that can lead to this toxic event and highlighting the importance of facile experimental assays to detect it. Understanding how phospholipidosis can confound antiviral drug discovery, and its rapid detection, will help prevent what is an apparently general artifact, active across viruses, from distracting investigators from potentially more useful candidates.

cell biology↗

bioRxiv2024

HEMATO-CHEMICAL VARIATIONS- A DIAGNOSTIC TOOL FOR BABESIA, THEILERIA AND ANAPLASMA INFECTIONS IN CAPTIVE HOG DEER (AXIS PORCINUS)

Hog deer are an essential component of Pakistans biodiversity. Their conservation helps maintain integrity of local ecosystems, contributing to overall stability and balance of regions wildlife populations. In Pakistan, they are classified as an endangered species according to IUCN. Theileria, babesia and anaplasma are among most prevalent blood parasites in ruminants which require diagnosis at early stages of the infection so that treatment protocols can be followed and more severe pathological alterations can be prevented. The changes in hemato-chemical parameters caused by Theileria, babesia and anaplasma were recorded individually and then compared with healthy group of hog deer respectively. Alterations in hemato-chemical parameters produced by these parasite-infected hog deer were assessed as potential diagnostic tools for distinguishing between individual blood parasites. Total leukocyte count, monocytes and eosinophils were observed to be higher than normal in babesia infected hog deer while level was lower than normal hog deer in anaplasma and theileria infected hog deer. The main objective of this study is to promote early diagnosis of parasitic infections in hog deer to facilitate prompt treatment, pathological prevention and allocate resources for further research on hemato-chemical parameters as diagnostic tools, enhancing understanding and management of blood parasite infections in hog deer.

pathology↗

bioRxiv2024

Regulation of virion production by the ORF8 signal peptide across SARS-CoV-2 variants

The open reading frame 8 (ORF8), an accessory protein of SARS-CoV-2, is prone to deletions and mutations across different viral variants, which was first described in several Singapore variants. The reason why viral evolution favors loss or inactivation of ORF8 is not fully understood, although the effects of ORF8 on inflammation, immune evasion, and disease severity have been described. Here we show -using clinical ORF8-deficient viral isolates, virus-like particles (VLPs) and viral replicons- that ORF8 expression dampens viral particle production. ORF8 physically interacts with the viral Spike protein and induces Golgi fragmentation, overall contributing to less virus particle production. Using systematic ORF8 deletions, we mapped the particle-reducing function to its N-terminal signal peptide. Interestingly, this part of ORF8 is severely truncated in the recent XBB.1.5 variant, and when restored, suppresses viral particle production in the context of the entire viral genome. Collectively, our data supports the model that evolutionary pressure exists to delete ORF8 sequence and expression across SARS-CoV-2 variants to fully enable viral particle production. ImportanceSARS-CoV-2 variants continue to emerge worldwide with advantages in replication and immune evasion. Many variants have acquired distinct mutations in independent lineages to abolish ORF8 expression. To understand the molecular mechanisms behind this evolutionary trend, we utilized reverse genetics, molecular virology, and confocal microscopy to show that ORF8 has antiviral functions by dampening viral particle production and inducing Golgi stress during infection. Our data demonstrate that SARS-CoV-2 is continuing its adaptation to optimize viral particle production and other unknown aspects of viral infection.

microbiology↗

bioRxiv2023

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38{+/-}10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N- protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

biophysics↗

bioRxiv2023

Colloidal aggregation confounds cell-based Covid-19 antiviral screens

Colloidal aggregation is one of the largest contributors to false-positives in early drug discovery and chemical biology. Much work has focused on its impact on pure-protein screens; here we consider aggregations role in cell-based infectivity assays in Covid-19 drug repurposing. We began by investigating the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal-particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate antiviral efficacy of the drugs in cells we used spike pseudotyped lentivirus and pre-saturation of the colloids with BSA. The antiviral potency of the aggregators was diminished by at least 10-fold and often entirely eliminated in the presence of BSA, suggesting antiviral activity can be attributed to the non-specific nature of the colloids. In confocal microscopy, the aggregates induced fluorescent puncta of labeled spike protein, consistent with sequestration of the protein on the colloidal particles. Addition of either non-ionic detergent or of BSA disrupted these puncta. These observations suggest that colloidal aggregation is common among cell-based anti-viral drug repurposing, and perhaps cell-based assays more broadly, and offers rapid counter-screens to detect and eliminate these artifacts, allowing the community invest resources in compounds with true potential as a Covid-19 therapeutic.

biochemistry↗

bioRxiv2023

Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (30kb). Here, we designed a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

microbiology↗

bioRxiv2022

SARS-CoV-2 ORF8 limits expression levels of Spike antigen.

Survival from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of SARS-CoV-2 mechanisms counteracting antibody-mediated immunity is needed. Here, we report that ORF8 is a SARS-CoV-2 factor that controls cellular Spike antigen levels. ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. With limited Spike availability, ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Our studies propose an ORF8-dependent SARS-CoV-2 strategy that allows immune evasion of infected cells for extended viral production.

microbiology↗

bioRxiv2022

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-{kappa}B inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more I{kappa}B expression in infected than bystander cells, but found concurrent nuclear translocation of NF-{kappa}B that I{kappa}B usually prevents. Overexpressing a nondegradable I{kappa}B mutant reduced NF-{kappa}B translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-{kappa}B feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

microbiology↗

bioRxiv2021

Hepatitis C Virus Infects and Perturbs Liver Stem Cells

Hepatitis C virus (HCV) is the leading cause of death from liver disease. How HCV infection causes lasting liver damage and increases cancer risk beyond viral clearance remains unclear. We identify bipotent liver stem cells as novel targets for HCV infection, and their erroneous differentiation as the potential cause of impaired liver regeneration and cancer development. We show 3D organoids generated from liver stem cells from actively HCV-infected individuals carry replicating virus and maintain low-grade infection over months. Organoids can be infected with a primary HCV isolate. Virus-inclusive single-cell RNA-sequencing uncovered extensive transcriptional reprogramming in HCV+ cells supporting hepatocytic differentiation, cancer stem cell development and viral replication while stem cell proliferation and interferon signaling are disrupted. Our data adds a pathogenesis factor - infection of liver stem cells - to the biology of HCV infection that explains persistent liver damage and enhanced cancer risk through an altered stem cell state.

microbiology↗

bioRxiv2021

Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles

Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M). One-Sentence SummaryR203M substitution within SARS-CoV-2 N, found in delta variant, improves RNA packaging into virus-like particles by 10-fold.

microbiology↗

bioRxiv2021

Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.

synthetic biology↗

bioRxiv2020

Zika Virus Infection Prevents Host mRNA Nuclear Export by Disrupting UPF1 Function

Zika virus (ZIKV) is a mosquito-borne RNA virus that can infect fetuses in utero causing characteristic neurodevelopmental disorders including microcephaly. We previously showed that ZIKV infection downregulates expression of up-frameshift protein 1 (UPF1), a helicase/ATPase and central regulator of the nonsense-mediated mRNA decay pathway. Here, we identify a novel function of nuclear UPF1 in mRNA export. Using crosslinking immunoprecipitation of UPF1 followed by sequencing of associated transcripts as well as fluorescence in situ hybridization experiments, we find widespread mRNA accumulation in the nucleus of human neural progenitor cells (NPCs) upon ZIKV infection or UPF1 knockdown. Knockdown of FREM2, a top UPF1 target transcript encoding an extra-cellular matrix protein critical in fetal development, decreased expression of pluripotency markers and increased expressed neuronal differentiation in NPCs, consistent with the model that trapping FREM2 mRNA in the nucleus perturbs proper NPC function. Collectively, our data uncover a new posttranscriptional mechanism by which ZIKV "shuts off" host mRNA export via UPF1. As we find UPF1 linked to many neurodevelopment pathways, we propose that the lack of host mRNA export contributes to the neurodevelopmental defects associated with ZIKV infection.

microbiology↗

bioRxiv2019

Human liver organoids; a patient-derived primary model for HBV Infection and Related Hepatocellular Carcinoma

The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV infected organoids produced cccDNA, expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor NTCP by lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for disease surveillance and development of HCC in HBV infected patients.

cancer biology↗

bioRxiv2018

The cellular NMD pathway restricts Zika virus infection and is targeted by the viral capsid protein

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly1, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice2-4. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1)5. Endogenous UPF1 interacted with the viral capsid protein in co-immunoprecipitation experiments and capsid expression post-transcriptionally downregulated UPF1, a process that we confirmed occurs during de novo ZIKV infection. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures. We therefore propose that ZIKV, via the capsid protein, has evolved a strategy to dampen antiviral activities of NMD6,7, which subsequently contributes to neuropathology in vivo.

microbiology↗