Biology subjects

Wong, H. T.

Publications and source records attributed to Wong, H. T..

7 recordsLinked to original sources

bioRxiv2026

Cross-source proteogenomic profiling of human microglia reveals a conserved core membrane protein and HERV signature

Microglia are essential immune cells of the brain parenchyma which are responsible for maintaining homeostasis and responding to brain injury. When activated, they induce immune signaling and become phagocytic. In the context of neurodegenerative diseases, they exhibit both pro- and anti-inflammatory properties. Human endogenous retroviruses (HERVs) are the remnants of ancient retroviral germline infections that integrated into the human genome, and their expression has been directly linked to microglial-mediated neurodegenerative diseases. Despite this, HERV expression is not consistently included in analyses characterizing microglia. Investigating the function of microglia across different diseases is critical for elucidating mechanisms of disease progression and for the development of effective therapeutic interventions. Approaches to studying microglia include isolation from post-mortem brain tissues, differentiation from peripheral blood monocytes or differentiation of induced pluripotent stem cells (iPSC). Ex vivo brain derived microglia (BdMG) are considered most physiologically relevant, but constraints including the unpredictable timing of availability, labor-intensive protocols, and low yields reduce their practicality. Existing alternatives are iPSC-derived microglia (iMG) and monocyte-derived microglia (MDMi) models that circumvent sample accessibility and scalability issues. Here, we explored the transcriptome and proteome of these three microglia sources and identified a common microglia surface marker expression profile, establishing that MDMi and iMG exhibit similar phenotypes relative to BdMG. We also identified a common HERV expression profile across BdMG, iMG and MDMi, indicating deeper similarities across different sources. Altogether, our findings indicate that given the phenotypic similarities of the 2 models, MDMi represent a practical and reliable model for investigating microglial function with iMG models serving for more molecular based approaches, such as CRISPRa/i.

neuroscience↗

bioRxiv2026

Three immunoregulatory signatures define non-productive HIV infection in CD4+ T memory stem cells

The persistent HIV reservoir constitutes the main obstacle to curing HIV/AIDS disease. Our understanding of how non-productive HIV infections are established in primary human CD4+ T cells during the first round of infection remains, however, incomplete. In this study, we leveraged the HIV reporter virus pMorpheus-V5 to delineate cellular expression patterns that are upregulated in non-productively infected primary CD4+ T memory stem cells (TSCM). We found that CD4+ TSCM harboring non-productive proviruses displayed a distinct transcriptomic signature comprising 118 upregulated genes. This non-productive expression profile was distinct from that of productively infected cells as well as from negative-exposed and mock-infected cells. Among the cellular genes most upregulated in CD4+ T cells harboring non-productive proviruses were CCR4-binding migratory chemokines (CCL22, CCL17), tryptophan catabolic enzymes (IDO1, KYNU), and genes encoding cytoskeletal rearrangement proteins (BASP1, TNFAIP2). Intracellular flow cytometry-based analyses confirmed that non-productively infected CD4+ TSCM cells were enriched for CCL22 and IDO1 co-expression compared to the other CD4+ memory subsets, underscoring a clear CD4+ T cell subset specificity for the upregulation of these two immune gene sets associated with non-productive infections. These findings suggest that primary human CD4+ TSCM harboring non-productive proviruses display a distinct immunoregulatory phenotype which may facilitate immune evasion and contribute to the persistence of the HIV reservoir.

microbiology↗

bioRxiv2025

HIV-1 Vpr drives epigenetic remodeling to enhance virus transcription and latency reactivation

Despite decades of research, the primary proviral function of the HIV-1 Vpr accessory protein remains enigmatic. Vpr is essential for pathogenesis in vivo and for virus replication in myeloid cells, but the underlying cause-and-effect mechanism(s) driving these phenomena are poorly understood. Canonically, Vpr hijacks a cellular ubiquitin ligase complex to target several dozen host proteins for proteasomal degradation. Many of these substrates were recently revealed to be involved in DNA damage repair (DDR), which rationalizes the longstanding observation that Vpr induces constitutive activation of DDR signaling. Here, we use a combination of functional, biochemical, and genetic approaches establish a clear mechanistic link between Vpr-induced DDR signaling and remodeling of the epigenetic landscape to enhance HIV-1 promoter activity during acute infection and virus reactivation from latency. Functional, genetic, and bimolecular fluorescence complementation experiments reveal that Vpr utilizes degradation-dependent and -independent mechanisms to induce epigenetic remodeling and that Vpr segregates into two discrete pools with dedicated activities--A multimeric pool in the nucleus that is associated with chromatin and a monomeric pool associated with DCAF1 in the cytoplasm. Vpr function in remodeling the nuclear environment is present in common HIV-1 subtypes worldwide and provides a mechanistic rationale for its essentiality in virus replication. Author summaryWhile HIV-1 Vpr plays an essential role in virus replication, the molecular mechanisms underlying its essentiality remain enigmatic. Vprs best characterized function is the ability to induce the depletion of several dozen host proteins by hijacking a cellular E3-ubiquitin ligase complex. Here, we establish that Vpr promotes global epigenetic remodeling to enhance HIV-1 promoter activity during acute infection and virus reactivation from latency. We demonstrate that epigenetic remodeling activity is linked to Vprs ability to induce constitutive DNA damage repair signaling, and that it occurs through both degradation-dependent and -independent mechanisms. Moreover, this Vpr function is present in common HIV-1 subtypes circulating globally. This study provides novel mechanistic insights into how HIV-1 exploits host DNA repair pathways and sheds light on Vprs proviral function.

microbiology↗

bioRxiv2023

A Sweeter Future: Using protein language models for exploring sweeter brazzein homologs

Reducing sugar intake lowers the risk of obesity and associated metabolic disorders. Currently, this is achieved using artificial non-nutritive sweeteners, where their safety is widely debated and their contributions in various diseases is controversial. Emerging research suggests that these sweeteners may even increase the risk of cancer and cardiovascular problems, and some people experience gastrointestinal issues as a result of using them. A safer alternative to artificial sweeteners could be sweet-tasting proteins, such as brazzein, which do not appear to have any adverse health effects. In this study, protein language models were explored as a new method for protein design of brazzein. This innovative approach resulted in the identification of unexpected mutations, which opened up new possibilities for engineering thermostable and potentially sweeter versions of brazzein. To facilitate the characterization of the brazzein mutants, a simplified procedure was developed for expressing and analyzing related proteins. This process involved an efficient purification method using Lactococcus lactis (L. lactis), a generally recognized as safe (GRAS) bacterium, as well as taste receptor assays to evaluate sweetness. The study successfully demonstrated the potential of computational design in producing a more heat-resistant and potentially more palatable brazzein variant, V23.

synthetic biology↗

bioRxiv2022

Construction of a constitutively active type III secretion system for heterologous protein secretion

Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells, purified, and resolubilized. The Salmonella pathogenicity island 1 type III secretion system (T3SS) is a protein secretion complex in Salmonella that has been engineered to secrete heterologous proteins and addresses the problems associated with bacterial protein production. However, the current best practices method of T3SS pathway activation for secretion is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer to the culture media and adds significant cost to the production media. Plasmid-based expression is also subject to instability in large-scale fermentation. Here, we show that we can constitutively activate the T3SS by modulating the upstream transcriptional regulator, hilD, either through knocking out hilE, a repressor of HilD, or by adding transcriptional fusions to hilD. Finally, we combine the two most promising genomic modifications to build a constitutively active T3SS capable of secreting a range of heterologous proteins at titers comparable to those reported with synthetic induction of hilA. These improvements further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. ImportanceProteins are used in our everyday lives as therapeutics (insulin), industrial enzymes (laundry detergent), and bio-based materials (spider silk). Current industrial protein production in bacteria is costly because it requires purification of the target protein from the other proteins inside the cell. We solve this problem by engineering the Salmonella pathogenicity island 1 type III secretion system (T3SS) to export the target protein into the cell growth media. This makes the protein purification process more efficient and cheaper. However, this system currently requires an expensive inducer reagent to activate it which significantly increases the cost of the production media. We show here the creation of a constitutively active T3SS, meaning the T3SS pathway is always on. In doing so, we successfully created a Salmonella strain that eliminates the need for the inducer reagent and exports proteins at levels comparable to the inducer-activated system, ultimately reducing the cost of T3SS protein production.

synthetic biology↗

bioRxiv2021

Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism

Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds the nuclear pore components Nup98-Rae1 to inhibit nuclear translocation of activated STAT1/IRF3 transcription factors. However, a direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants can still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6/Nup98 interaction domain robustly inhibited nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral-membrane protein, opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication.

cell biology↗

bioRxiv2020

Variation in the Gut Microbiota of Common Marmosets: Differences with Colony of Origin and Integration

Characterization of the gut microbiome may aid understanding and management of natural and experimental disease states in research animals, thereby promoting reproducibility. In this study, the rectal bacterial communities of three separate common marmoset (Callithrix jacchus) breeding colonies were defined using 16S rRNA sequencing of rectal swab samples. Study animals originated from two German colonies and a United States colony (JHU). The two German cohorts, previously fed the same diet, were imported into the JHU facility; they were then isolated, transitioned onto JHU diet, and then moved into rooms housing JHU animals. To dissect the contributions of diet and integration in shaping the rectal bacterial community, samples were collected from German origin marmosets upon JHU arrival (baseline), following diet transition (100 d), and following cohousing (390 d). Baseline and 390 d samples were collected from stably maintained JHU marmosets. Bacterial community composition was distinct between all three cohorts at baseline, suggesting that factors other than primary diet confer significant differences between captive populations. Beta-diversity of the animals from the two German colonies converged by 100 d but remained distinct from JHU sample beta-diversity throughout the 390-d study, indicating that diet had greater influence on bacterial community composition than did housing animals within the same room. Our results demonstrate substantial differences in gut bacteria between different captive marmoset colonies, with persistence of these differences following husbandry standardization and housing integration. Goals of rigor and reproducibility in research underscore the need to consider microbial differences between marmosets of diverse origin. ImportanceCharacterizing gut microbial populations is expected to promote health and enhance research reproducibility in animal studies. As use of common marmosets as animal models of human diseases expands, evaluating the marmoset gut bacterial community will be critical for interpreting research findings, especially as marmosets are prone to gastrointestinal inflammation. In this study, using 16S rRNA sequencing of rectal swab samples, we compared bacterial community among three captive colonies of marmosets at baseline and following importation of cohorts from two of the colonies into the third colony. Diet history had sustained influence on bacterial community composition, while housing the animals within the same room over a period of eight months did not appear to be a major factor. These persistent differences in marmoset gut bacterial community highlight the need for careful consideration of animal origin as a variable in marmoset research studies.

microbiology↗