Biology subjects

Torbett, B. E.

Publications and source records attributed to Torbett, B. E..

9 recordsLinked to original sources

bioRxiv2026

Optimizing Lentiviral Vector-Based Delivery of SCN1A transgenes to Mammalian Cells

The SCN1A gene encodes NaV1.1, a voltage-gated sodium channel protein that is necessary for neuronal excitability and whose loss-of-function mutations cause Dravet syndrome, a treatment-resistant childhood onset epilepsy. Gene replacement strategies for this syndrome are challenged by the large size of SCN1A and difficulty achieving stable cellular expression. Lentiviral vectors (LVVs) offer sufficient packaging capacity and genomic integration for defective SCN1A gene replacement. Here, we evaluated LVV-mediated delivery of different engineered SCN1A transgene sequences in human cells. LVV-transduced cells expressed full-length NaV1.1 protein that trafficked to the membrane and produced functional sodium currents. However, SCN1A transgene expression declined over time despite stable vector copy number, indicating post-integration regulatory limitations. Expression efficiency varied by SCN1A transgene sequence, with a codon-optimized variant showing higher expression despite lower LVV copy number. Treatment with sodium butyrate, a histone deacetylase inhibitor, significantly enhanced SCN1A transgene expression and partially rescued expression decay in a sequence-dependent manner. Incorporation of a ubiquitous chromatin opening element (UCOE) upstream of the promoter to maintain expression resulted in a trend of increased expression and increased responsiveness to butyrate. These findings demonstrate that sequence-specific and epigenetic factors may influence expression of large transgenes following lentiviral delivery, highlighting key challenges and design considerations for therapeutic SCN1A transgene expression.

synthetic biology↗

bioRxiv2026

Lenacapavir prevents production of infectious HIV-1 by abrogating immature virus assembly.

The HIV-1 capsid effector Lenacapavir (LEN) acts by disrupting the early (reverse transcription and nuclear entry) and late (assembly and maturation) stages of the viral lifecycle. The early stage requires an intact Capsid consisting of a lattice of capsid protein (CA) hexamers and pentamers. Phenylalanine-Glycine (FG) containing nuclear pore proteins interact with Capsid hexamers at their FG pockets, facilitating nuclear entry. Disruption of Capsid lattice stability, or competitive binding to the FG pocket, by LEN blocks infection. Here, we provide insight into the effects of LEN on the late stage. Using a combination of cryo-EM structure determination, in vitro assembly, and in situ viral assays, we determined that treatment of producer cells with LEN abrogates the production of infectious virus via multiple mechanisms. Previous studies have shown that HIV-1 produced from LEN treated cells have improperly formed Capsids. However, how LEN interacts with the CA domain of the Gag polyprotein during assembly, prior to maturation, is unclear. Using a viral protease (PR) defective HIV-1 clone, which traps the Gag lattice in an immature state, we found that LEN dramatically remodulates the CA domain. The resulting CA layer was mature-like despite the absence of PR cleavage. We dubbed this unnatural state as "premature" lattice. Proper immature and mature lattice formation requires inositol hexakisphosphate (IP6), but our cryo-EM work revealed a lack of IP6 in the premature lattice. These findings provide insight into the mechanisms by which LEN prevents infectious HIV-1 production.

molecular biology↗

bioRxiv2024

MrHAMER2: high-accuracy long-read RNA sequencing to decode isoform-specific variation in viral transcripts during latency

Alternative splicing (AS) greatly expands the repertoire of proteins encoded by the human genome. Viruses have been shown to hijack AS cellular pathways to sustain replication or lead to latency. In HIV-1 infection, the virus integrates into the host genome, becoming a transcriptional unit that directly engages in AS to regulate its gene expression. Sequencing advances have enabled insights into HIV-1 gene expression dynamics during productive replication. However, viral isoform dynamics during latency remain largely uncharacterized due to the low abundance of spliced viral transcripts in associated CD4+ T cell subsets, making their accurate detection and quantification challenging. MrHAMER2 is a high-accuracy long-read RNA sequencing method that leverages dual Unique Molecular Identifier (UMI) tagging of cDNA to accurately capture and quantify full-length isoforms with high dynamic range and 99.968% single-nucleotide accuracy. We used MrHAMER2 to decode the spliced HIV-1 transcriptome in a primary CD4+ T cell model of latency and showed substantial changes in viral isoforms bearing intron retentions accompanied by changes in their potential to generate translatable protein.

genomics↗

bioRxiv2024

APHIX: Analysis Pipeline for HIV-1 Isoform eXploration Using Long-read RNA Sequencing Data

HIV-1 uses 4 major splice donors and 8 major splice acceptors as well as dozens of minor, cryptic, and uncharacterized splice sites to produce over one hundred distinct transcript isoforms from a single 9.2 kb genome. As a result, existing bioinformatic pipelines struggle to accurately analyze spliced HIV sequences due to the complex nature of HIV alternative splicing compared to human mRNA splicing. Previous approaches to identify HIV isoforms from long-read sequencing data used pipelines that are not publicly available, are convoluted to operate, or are locked into a specific HIV strain, which limits their wide adoption to other experimental designs or systems. To address this gap, we have developed a bioinformatic pipeline called APHIX that fully automates spliced isoform assignment, splice site usage quantification, and non-coding exon detection. APHIX takes a FASTQ/A of long-read transcripts and a HIV genome reference sequence and fully automates HIV isoform analysis. APHIX calculates splice site usage counts and percentages for each donor and acceptor site and their pairwise combinations, accurately assigns isoforms, and automatically identifies transcripts containing non-coding exons. APHIX is compatible with long-reads sequences generated from multiple platforms and library preps, including direct DNA and RNA sequencing. APHIX can also be adapted to multiple HIV-1 clades and strains by providing the appropriate reference sequence during bioinformatic processing. Overall, APHIX enables comprehensive processing of spliced sequences with reproducible results in a manner that is faster and easier to run compared to other methods.

bioinformatics↗

bioRxiv2024

IP6 and PF74 affect HIV-1 Capsid Stability through Modulation of Hexamer-Hexamer Tilt Angle Preference

The HIV-1 capsid is an irregularly shaped complex of about 1200 protein chains containing the viral genome and several viral proteins. Together, these components are the key to unlocking passage into the nucleus, allowing for permanent integration of the viral genome into the host cell genome. Recent interest into the role of the capsid in viral replication has been driven by the approval of the first-in-class drug lenacapavir, which marks the first drug approved to target a non-enzymatic HIV-1 viral protein. In addition to lenacapavir, other small molecules such as the drug-like compound PF74, and the anionic sugar inositolhexakisphosphate (IP6), are known to impact capsid stability, and although this is widely accepted as a therapeutic effect, the mechanisms through which they do so remain unknown. In this study, we employed a systematic atomistic simulation approach to study the impact of molecules bound to hexamers at the central pore (IP6) and the FG-binding site (PF74) on capsid oligomer dynamics, compared to apo hexamers and pentamers. We found that neither small molecule had a sizeable impact on the free energy of binding of the interface between neighboring hexamers but that both had impacts on the free energy profiles of performing angular deformations to the pair of oligomers akin to the variations in curvature along the irregular surface of the capsid. The IP6 cofactor, on one hand, stabilizes a pair of neighboring hexamers in their flattest configurations, whereas without IP6, the hexamers prefer a high tilt angle between them. On the other hand, having PF74 bound introduces a strong preference for intermediate tilt angles. These results suggest that structural instability is a natural feature of the HIV-1 capsid which is modulated by molecules bound in either the central pore or the FG-binding site. Such modulators, despite sharing many of the same effects on non-bonded interactions at the various protein-protein interfaces, have decidedly different effects on the flexibility of the complex. This study provides a detailed model of the HIV-1 capsid and its interactions with small molecules, informing structure-based drug design, as well as experimental design and interpretation.

biophysics↗

bioRxiv2022

HSPA8 chaperone complex drives chaperone-mediated autophagy regulation in acute promyelocytic leukemia differentiation

Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by the hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapy are focusing on tumor debulking, all-trans retinoic acid (ATRA) induces differentiation in acute promeylocytic leukemia (APL) a particular subtype. Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML has remained elusive. To answer our questions we took advantages of human AML primary samples and databases. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally, but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cAMP. Here, we report that CMA related mRNA transcripts are higher expressed in immature hematopoietic cells as compared to neutrophils. In line, lysosomal degradation of mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed the ATRA resistant NB4-R1 cells to differentiate upon ATRA treatment, but reduced association of LAMP-2A and HSPA8 is necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Overall, our findings demonstrate that both normal and APL neutrophil differentiation require CMA downregulation and this pathway is predominantly dependent on HSPA8 assisted by other co-chaperones.

cancer biology↗

bioRxiv2022

Selective Ablation of 3' RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host cell and Viral Transcripts

Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read-lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3 RNA ends (termed CASPR) which decreases ribosomal RNA reads and enriches for poly-adenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

genomics↗

bioRxiv2021

Co-variation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa

Adaptation of viruses to their environments occurs through the acquisition of both novel Single-Nucleotide Variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

genetics↗

bioRxiv2021

MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

genomics↗