Biology subjects

Swanstrom, R.

Publications and source records attributed to Swanstrom, R..

15 recordsLinked to original sources

bioRxiv2026

Cell-type-specific parallel pathways in the canonical cortical microcircuit

Information processing in the cortex depends on the integration of bottom-up and top-down signals through recurrent microcircuits spanning layers. Although the canonical microcircuit provides a framework for this integration, how these interactions are implemented at synapse resolution remains unclear. Here, we use large-volume electron microscopy reconstructions of mouse primary visual cortex to map the intralaminar and interlaminar connectivity of intratelencephalic (IT) neurons in layers 2/3 and 5. We find that layer 2/3 IT neurons formed a depth-dependent gradient of recurrent connectivity, with superficial (L2) and deeper (L3) neurons potentially forming two channels associated with top-down and bottom-up processing, respectively. These channels are preserved across layers via cell-type-specific pathways involving distinct L5 IT types, rather than collapsing into a single integrative pool. Moreover, each channel is regulated by a largely separate cohort of inhibitory interneurons, stabilizing recurrent excitation while limiting crosstalk. Together, these results reveal parallel, cell-type-specific processing streams embedded within the canonical circuit.

neuroscience↗

bioRxiv2026

A quantitative census of millions of postsynaptic structures in a large electron microscopy volume of mouse visual cortex

Neurons display remarkable sub-cellular specificity in their synaptic targeting, which varies by cell type--for example, excitatory neurons prefer to target the spines of other excitatory cells. Modern electron microscopy connectomes enable the study of this sub-cellular specificity and its context in a circuit at unprecedented scale and resolution. However, this scale has also made it challenging to create accurate and efficient methods for classifying and segmenting fine cell components (including spines) across entire volumes. Here, we present a cost-efficient computational pipeline for classifying postsynaptic targets and segmenting structures such as spines. Our method relies only on having a mesh representation of a neuron and avoids processing image or segmentation data directly. Instead, we leverage tools from geometry processing to create features capturing the local geometry of a neurons surface. We couple this core technique with computational and storage optimizations, enabling reliable deployment over hundreds of thousands of neurons for a few hundred dollars in cloud compute cost. We then show that a simple classifier trained on the MICrONS mouse visual cortex dataset can use these mesh-based features to accurately classify synapses as targeting somas, dendritic shafts, or spines (weighted F1 score 0.961). Using this pipeline, we create a map of the postsynaptic structures at over 207.3 million synapses in MICrONS. We present an overview of this census of postsynaptic targeting, finding expected patterns (e.g., excitatory neurons preferentially targeting excitatory spines) as well as unexpected exceptions (e.g., Layer 5 near-projecting and Layer 6 corticothalamic cells often connecting to excitatory neuron shafts). We also demonstrate that these tools can be used to detect spines receiving multiple synaptic inputs, revealing surprising variability in their frequency across cells even within a cell type. We make our postsynaptic target predictions available for study, as well as the code for the computational pipeline and cloud deployment. Beyond MICrONS, we find that the model generalizes well to the H01 connectome without retraining (weighted F1 score 0.949), indicating that these tools will be useful in future connectomics reconstructions. More generally, our work demonstrates that representations derived from neuronal meshes can be a scalable and generalizable primitive for describing morphologies.

neuroscience↗

bioRxiv2025

HIV-1 Rebound Virus Consists of a Small Number of Lineages That Entered the Reservoir Close to ART Initiation

HIV-1 persists as a latent reservoir during suppressive antiretroviral therapy (ART). Viral rebound occurs upon ART interruption, posing a challenge to cure efforts. Characterizing viral populations fuelling rebound is imperative to curing HIV-1. We used longitudinal samples collected pretherapy from women in the CAPRISA 002 cohort to create an evolutionary time- line to determine the pretherapy timepoint when the rebound virus originally entered the long- lived reservoir. Participants (N=10) were untreated for an average of 5 years then on ART for an average of 2 years before viral rebound (defined as >1000 RNA copies/ml). env sequences were used to characterize the longitudinal pre-ART evolving viral RNA population, the proviral DNA reservoir during ART, and viral RNA in the plasma during rebound. For each participant, between 1 and 3 major viral lineages were identified in the plasma during rebound. A total of 20 rebound virus lineages were examined for the 10 participants, and 19 were found to have entered the reservoir around the time of therapy initiation. The one lineage estimated to enter the reservoir more than a year before therapy was observed in a participant who was untreated for more than 8 years, yet retained moderate CD4 T cell counts. Analysis of the viral DNA reservoir, from which the rebound viruses emanated, revealed that while 95% of rebounding lineages dated to the year before ART initiation, only 61% of unique proviruses dated to that time period. Strikingly, for three participants with DNA reservoirs dominated by viruses from earlier in untreated infection, only 33% of unique proviruses dated to the year before ART initiation, yet 83% of rebounding lineages dated to that time. Our results show that rebound virus almost exclusively comes from the portion of the latent reservoir that formed around the time of therapy initiation, even when the reservoir is composed of diverse sequences from across the pre-ART time period. Author SummaryHIV-1 is maintained in a long-lived reservoir during suppressive therapy. Virus rebounds if therapy is discontinued. We found that in most cases rebound virus comes from a pool of viral sequences that entered the long-lived reservoir around the time of therapy initiation. While the viral DNA reservoir is on average also skewed toward sequences replicating around the time of therapy initiation, the rebound virus almost exclusively comes from this portion of the latent reservoir, even when the reservoir contained proviruses from much earlier in untreated infection. Thus, we hypothesize that there are features of the viruses forming the latent reservoir around the time of therapy initiation, or features of the host at that time, that select these viruses as initiators of rebound during therapy discontinuation.

microbiology↗

bioRxiv2024

HIV-1 single transcription start site mutants display complementary replication functions that are restored by reversion

HIV-1 transcription initiates at two positions, generating RNAs with either cap1G or cap3G 5' ends. The replication fates of these RNAs differ, with viral particles encapsidating almost exclusively cap1G RNAs and cap3G RNAs retained in cells where they are enriched on polysomes and among spliced viral RNAs. Here, we studied replication properties of virus promoter mutants that produced only one RNA 5' isoform or the other: separately, in combination, and during spreading infection. Results showed that either single start RNA could serve as both mRNA and genomic RNA when present as the only form in cells, although cap3G RNA was more efficiently translated and spliced while cap1G RNA was packaged into nascent virions slightly better than RNAs from the parental virus. When co-expressed from separate vectors, cap1G RNA was preferentially packaged into virions. During spreading infection cap1G-only virus displayed only minor defects but cap3G-only virus showed severe replication delays in both the highly permissive MT-4 cell line and in primary human CD4+ T cells. Passage of cap3G-only virus yielded revertants that replicated as well as the twinned (cap1G+ cap3G) transcription start site parent. These revertants displayed restored packaging and splicing levels and had regained multiple transcription start site use. ImportanceHIV-1 generates two RNAs during its replication that differ by only two nucleotides in length. Despite this very minor difference, the RNAs perform different and complementary replication functions. When mutants that expressed only one RNA were forced to revert, they regained functions associated with the second RNA.

microbiology↗

bioRxiv2024

The role of Nef in the long-term persistence of the replication-competent HIV reservoir in South African women

HIV-1 Nef mediates immune evasion and viral pathogenesis in part through downregulation of cell surface cluster of differentiation 4 (CD4) and major histocompatibility complex class I (MHC-I) on infected cells. While Nef function of circulating viral populations found early in infection has been associated with reservoir size in early-treated cohorts, there is limited research on how its activity impacts reservoir size in people initiating treatment during chronic infection. In addition, there is little research on its role in persistence of viral variants during long-term antiretroviral therapy (ART). Phylogenetically distinct nef genes (n=82) with varying estimated times of reservoir entry were selected from viral outgrowth variants stimulated from the reservoir of South African women living with HIV who initiated ART during chronic infection (n=16). These nef genes were synthesized and used in a pseudovirus infection assay that measures CD4 and MHC-I downregulation via flow cytometry. Downregulation measures were compared to the size of the replication-competent viral reservoir (RC-VR), estimated by quantitative viral outgrowth assay (QVOA) at 5 years after treatment initiation, as well as proviral survival time. Maximum Nef-mediated MHC-I downregulation was significantly associated with RC-VR size (p=0.034), but this association was not observed for CD4 downregulation. Conversely, we did not find a consistent association between intraparticipant MHC-I or CD4 downregulation and the variant timing of entry into the reservoir. These data support a role for Nef-mediated MHC-I downregulation in determining RC-VR size, but more work is needed to determine Nefs role in the survival of individual viral variants over time. Author summaryUnderstanding the HIV reservoir, including viral determinants of reservoir formation and maintenance, is key for the rational design of cure interventions. In addition, for cure interventions to be equitable, it needs to be broadly applicable. While African women bear the greatest burden of HIV globally, most cure research has been performed in men living in the global North. Our study aims to elucidate attributes of the virus that contribute to reservoir dynamics in South African women on ART. We investigated the ability of the HIV accessory protein Nef to reduce the cell surface levels of two cellular proteins, CD4 and MHC-I, and compared this downregulation capacity with the size of the HIV reservoir and survival of cells infected with a given viral isolate. We found a positive association between an individuals measured reservoir size and MHC-I downregulation, but not CD4 downregulation. There was little evidence for a survival benefit for stronger Nef MHC-I reduction, but more research is needed on this subject. These data support earlier work and suggest that Nefs interaction with MHC-I may be a target to restrict the latent reservoir and inform alternate cure strategies.

pathology↗

bioRxiv2024

Changes in Cerebrospinal Fluid Proteins across the Spectrum of Untreated and Treated Chronic HIV-1 Infection

Using the Olink Explore 1536 platform, we measured 1,463 unique proteins in 303 cerebrospinal fluid (CSF) specimens from four clinical centers that included uninfected controls and 12 groups of people living with HIV-1 infection representing the spectrum of progressive untreated and treated chronic infection. We present three initial analyses of these measurements: an overview of the CSF protein features of the sample; correlations of the CSF proteins with CSF HIV-1 RNA and neurofilament light chain protein (NfL) concentrations; and comparison of the CSF proteins in HIV-associated dementia (HAD) and neurosymptomatic CSF escape (NSE). These reveal a complex but coherent picture of CSF protein changes that includes highest concentrations of many proteins during CNS injury in the HAD and NSE groups and variable protein changes across the course of neuroasymptomatic systemic HIV-1 progression, including two common patterns, designated as lymphoid and myeloid patterns, related to the principal involvement of their underlying inflammatory cell lineages. Antiretroviral therapy reduced CSF protein perturbations, though not always to control levels. The dataset of these CSF protein measurements, along with background clinical information, is posted online. Extended studies of this unique dataset will provide more detailed characterization of the dynamic impact of HIV-1 infection on the CSF proteome across the spectrum of HIV-1 infection, and further the mechanistic understanding of HIV-1-related CNS pathobiology.

microbiology↗

bioRxiv2023

N4-Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness

A diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N4-hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.

microbiology↗

bioRxiv2021

Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing

Previous work in several laboratories has demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of DNA and RNA viruses, yet the underlying mechanisms responsible for this positive effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how m6A readers regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is in large part mediated by the the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs while YTHDF2 binding correlates with increased HIV-1 transcript stability. Author SummaryThis manuscript reports that the expression of mRNAs encoded by the pathogenic human retrovirus HIV-1 is regulated by the methylation of a small number of specific adenosine residues. These in turn recruit a nuclear RNA binding protein, called YTHDC1, which modulates the alternative splicing of HIV-1 transcripts, as well as a cytoplasmic RNA binding protein, called YTHDF2, which stabilizes viral mRNAs. The regulation of HIV-1 gene expression by adenosine methylation is therefore critical for the effective and ordered expression of HIV-1 mRNAs and could represent a novel target for antiviral development.

molecular biology↗

bioRxiv2020

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we show that the ribonucleoside analog {beta}-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (b-D-N4-hydroxycytidine-5-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses.

microbiology↗

bioRxiv2020

Acetylation of cytidine residues boosts HIV-1 gene expression by increasing viral RNA stability

Covalent modifications added to individual nucleotides on mRNAs, called epitranscriptomic modifications, have recently emerged as key regulators of both cellular and viral mRNA function1,2 and RNA methylation has now been shown to enhance the replication of human immunodeficiency virus 1 (HIV-1) and several other viruses3-11. Recently, acetylation of the N4 position of cytidine (ac4C) was reported to boost cellular mRNA function by increasing mRNA translation and stability12. We therefore hypothesized that ac4C and N-acetyltransferase 10 (NAT10), the cellular enzyme that adds ac4C to RNAs, might also have been subverted by HIV-1 to increase viral gene expression. We now confirm that HIV-1 transcripts are indeed modified by addition of ac4C at multiple discreet sites and demonstrate that silent mutagenesis of a subset of these ac4C addition sites inhibits HIV-1 gene expression in cis. Moreover, reduced expression of NAT10, and the concomitant decrease in the level of ac4C on viral RNAs, inhibits HIV-1 replication by reducing HIV-1 RNA stability. Interestingly Remodelin, a previously reported inhibitor of NAT10 function13,14, also inhibits HIV-1 replication without affecting cell viability, thus raising the possibility that the addition of ac4C to viral mRNAs might emerge as a novel cellular target for antiviral drug development.

microbiology↗

bioRxiv2020

Deep Sequencing Reveals Compartmentalized HIV-1 in the Semen of Men with and without STI-associated Urethritis

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 env gene was done for paired blood and semen samples from ART-naive men living in Malawi with (n = 19) and without (n = 5) STI-associated urethritis; for a subset of samples full length env genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of pro-inflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population indicating a recent bottleneck followed by limited viral replication. We documented a case of superinfection where the new strain was restricted to the genital tract. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, where a superinfecting strain can persist, and where specific genotypes can be amplified perhaps initially by cellular proliferation but further by limited viral replication. ImportanceHIV-1 is a sexually transmitted infection that co-exists with other STIs. Here we examine the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.

microbiology↗

bioRxiv2019

Selection of HIV-1 for Resistance to Fourth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in the high drug concentrations that are achived in vivo. Little is known about the de novo resistance pathway for DRV. We have selected for resistance to high drug concentrations against ten PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small chemical changes in the inhibitor P1 side chain led to preferential use of one pathway over the other. Changes in the inhbitor P2 side chain determined differences in potency that were retained in the resistant viruses. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fourth generation PIs, and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.

microbiology↗

bioRxiv2019

The Replication-Competent HIV-1 Latent Reservoir is Primarily Established Near the Time of Therapy Initiation

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. Little is known about the dynamics of reservoir formation and this reservoir forms even when ART is initiated early after infection. The reservoir of individuals who initiate therapy in chronic infection is generally larger and genetically more diverse than that of individuals who initiate in acute infection, suggesting the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting CD4+ T cells from nine women on therapy to viral sequences circulating in blood collected longitudinally prior to therapy. We found that 78% of viruses from the latent reservoir were most genetically similar to viruses replicating just prior to therapy initiation. This proportion is far greater than expected if the reservoir forms continuously and is always long-lived. Thus, therapy alters the host environment in a way that allows the formation of a majority of the long-lived latent HIV-1 reservoir.\n\nOne Sentence SummaryMost of the long-lived, replication-competent HIV-1 reservoir is formed at the time of therapy initiation.

microbiology↗

bioRxiv2018

HIV-1 Protease Uses Bi-Specific S2/S2’ Subsites To Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PR) have a unique specificity that allows cleavage of sites with or without a P1 proline. A P1 proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 iterations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1-P2), with two complementary sets of rules. When P1 is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2 amino acid interacts with a hydrophobic region of the S2 subsite. When P1 is not proline, the orientations of the P2 and P2 side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite while the P2 amino acid usually engages hydrophilic amino acids in the S2 subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2 subsites to accommodate the steric effects imposed by a P1 proline on the orientation of P2 and P2 substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.

biochemistry↗

bioRxiv2018

Constrained mutational sampling of amino acids in HIV-1 protease evolution

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution are interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available dataset of 32,163 sequenced isolates from drug-naive individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple base mutations in HIV-1 protease is strongly influenced by mutational sampling.

evolutionary biology↗