Biology subjects

Cohen, M. S.

Publications and source records attributed to Cohen, M. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Mono-ADP-ribosylation-driven immunosuppression and cross-resistance to therapy through cancer cell intrinsic and extrinsic mechanisms

Mono-ADP-ribosylation (MARylation) is emerging as an important regulator of anti-cancer immunity and immunosuppressive tumor microenvironment (TME). Our previous studies showed that PARP11, one of several enzymes that facilitate MARylation, regulates the activities of intratumoral cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs). Here, we demonstrate that stimuli such as adenosine, epinephrine, or glucagon-like peptide-1 (GLP1) induced PARP11 in cancer cells. Upregulation of PARP11 in cancer cells led to PARP11-mediated MARylation, ubiquitination, and accelerated degradation of MHC-I through the autophagy-lysosomal pathway. Induction of PARP11 protected cancer cells from killing by specific CTLs and stimulated tumor growth and progression. Genetic ablation of PARP11 attenuated MHC-I MARylation, ubiquitination, and interaction with autophagy receptors. Pharmacologic inhibition of PARP11 in pancreatic ductal adenocarcinoma (PDAC) cells restored their MHC-I levels, sensitized them to killing by CTLs, inhibited tumor growth, and impeded their initial resistance to chemotherapy and their acquired resistance to targeted therapy with RAS inhibitors. Moreover, inhibition of PARP11 prevented hyperprogressive disease in a mouse melanoma model treated with immune checkpoint inhibitors (ICBs), suggesting that PARP11 is a major therapeutically actionable driver of immunosuppression in tumors. SYNOPSISInduction of PARP11 in the tumor microenvironment mediates immunosuppression. This study reports that PARP11-driven MARylation and ubiquitination of MHC-I in cancer cells drives immune evasion, tumor growth and resistance to therapies.

cancer biology↗

bioRxiv2026

Beyond Neural Noise: Critical Dynamics Predict Slower Reaction Times in Adults With and Without ADHD

Historically, neural variability observed during task was interpreted as "noise," assumed to obscure meaningful signal and thus something to be minimized both analytically by researchers and functionally by the brain. Changes to this signal-to-noise ratio have been proposed as a possible neural mechanism behind the increased reaction-time variability (RTV) in attention deficit hyperactivity disorder (ADHD). However, not all variability is the same - in some cases, variability can have some underlying "statistical structure" that can be beneficial to information processing. The challenge lies in distinguishing meaningful variability from random noise. The edge-of-synchrony critical point, which describes a system poised between synchronous and asynchronous regimes, could be a good theoretical framework to study these different types of neural variability. In this study, we investigate whether changes in criticality and oscillatory dynamics preceded slower behavioral responses during a bimodal continuous performance task in ADHD. We find evidence that, prior to slower responses, neural dynamics shift toward criticality in both ADHD and control groups, suggesting that increase variability in ADHD and during attention lapses are related to structured variability and not necessarily random noise. Notably, these findings run counter predictions based on the proposed model and previous literature on neural noise in this population, challenging predictions of edge-of-synchrony criticality as a unifying account of neural variability and behavioral performance. Furthermore, this effect did not emerge at the between-subject level, underscoring the limitations of relying on between-subject correlations to infer neural mechanisms. Impact StatementOur findings add new perspective to the hypothesis that links neural variability to reaction time variability in adults with and without ADHD. We found that neural dynamics shift towards criticality prior to slow reaction times in adults with and without ADHD, but in ADHD, dynamics lie closer to criticality regardless of response type, suggesting a different "attractor" state.

neuroscience↗

bioRxiv2026

Mitochondrial Permeability Transition in Skeletal Muscle Phenocopies Muscle Alterations seen in Cancer Cachexia and other Wasting Conditions

Skeletal muscle in wasting conditions often exhibits atrophy, mitochondrial respiratory dysfunction, and fragmentation of the acetylcholine receptor (AChR) cluster at the endplate. The accompanying alterations of mitochondrial morphology suggest mitochondria may be involved in muscle pathology in these conditions. To address this gap, we tested an established pathological mechanism in ischemia-reperfusion injury and neurodegeneration but poorly studied in skeletal muscle: mitochondrial permeability transition (mPT). We tested if mPT recapitulated phenotypes common in wasting conditions, whether tumor-conditioned media (TCM) could promote mPT, and compared differentially expressed genes (DEGs) induced by mPT with DEGs observed in a mouse model of pancreatic cancer cachexia. Inducing mPT in mouse skeletal muscle bundles progressively altered mitochondrial cristae morphology, culminating in breach of the outer mitochondrial membrane. Inducing mPT in mouse muscle fibers increased mROS and Caspase 3 activity and caused atrophy. Inducing mPT caused a complex I mitochondrial respiratory impairment, increased lysosome-mitochondrion co-localization, and fragmented the AChR cluster at the muscle endplate. The Ca2+ threshold for mPT, mitochondrial calcein colocalization and mitochondrial membrane potential were reduced by TCM in skeletal muscle or C2C12 myoblasts, respectively. Knockout of the mPT-regulating protein CypD attenuated the reduction in Ca2+ threshold for mPT by TCM. Inhibitors of mPT attenuated atrophy with TCM in C2C12 and human primary myotubes. Finally, there was overlap between the DEGs of mPT and diaphragm muscle in a mouse model of pancreatic cancer cachexia during the muscle wasting phase. We conclude that mPT should be explored as a therapeutic target in muscle wasting disorders.

physiology↗

bioRxiv2025

A Population-Specific PARP1 Gene Variation Modulates PARP Trapping

Poly-(ADP-ribose) polymerase inhibitors (PARPi) block NAD+-binding pocket of PARP1, inhibiting PAR synthesis. However, they differ in their ability to retain PARP1 on damaged DNA to induce synthetic lethality in homologous recombination-deficient (HRD) cancer. Allosteric enzymatic activation requires destabilization of the helical domain (HD) of PARP1 and is indispensable for activation and chromatin retention induced by distinct PARPi. Here we report that the effect of the clinical PARPi talazoparib is robustly impacted by a common human polymorphism within the HD. PARP1V762 greatly enhanced talazoparib-driven allosteric retention on chromatin, prolonged XRCC1 recruitment, and enhanced cell killing. Talazoparib switches from Type-II PARPi behavior in PARP1A762 to allosteric, pro-retention Type-I behavior for PARP1V762. Thus, both PARPi efficacy and dose-limiting tolerability depends on PARP1 allele, motivating variant-guided cancer therapies. TEASEROne of the four FDA-approved PARPi drugs, talazoparib, is modulated by a PARP1 SNP that is widespread in the population.

cancer biology↗

bioRxiv2025

Long-Read Deep Sequencing Reveals High Rates of Multilineage Transmission and Rapid Viral Population Changes in Acute HIV Infection

Understanding the selective forces acting upon HIV early in infection is crucial to design prevention strategies. By leveraging deep sequencing and the short diagnostic intervals of the FRESH and RV217 cohorts (median 4 days) between the last-negative and first-positive RNA tests, we captured a precise and early snapshot of acute HIV infection. The frequency of multiple transmitted viruses of 38% in these as well as placebo recipients from the AMP trials was higher than previously published, with the true frequency likely to be higher. The relative abundance of lineages fluctuated substantially over time in two-thirds of the multilineage infections, generating uncertainty in identifying the specific viruses that were transmitted and founding the infection. Viral populations exhibited diversity and selection on the Gag and Env proteins at the earliest times examined, with sites inferred to be undergoing negative selection most evident. These data may help explain vaccination failures and provide new targets for prevention.

microbiology↗

bioRxiv2025

The IFN I response in tumor cells is shaped by PARP7-p300/CBP interactions through distinct loss- and gain-of-function mechanisms

PARP7, a mono-ADP-ribosyl (MAR) transferase, is a key suppressor of the type I interferon (IFN-I) IFN{beta} in various tumor cells and a validated drug target. This negative regulation is reversed by small-molecule inhibitors of PARP7 catalytic activity, resulting in increased IFN-{beta} expression. Yet, the mechanism of action of PARP7 inhibitors remains unclear because the relevant substrates of PARP7-mediated MARylation are unknown. Using an optimized analog- sensitive chemical genetic (ASCG) approach, we identified the co-activators, p300 and CBP, as nuclear PARP7 substrates. We identified an -helical domain in PARP7 essential for p300/CBP interaction, MARylation, and proteasome degradation. Disrupting PARP7-p300/CBP interaction prevents PARP7s suppression of IFN{beta} in colorectal cancer cells. p300/CBP reciprocally regulate PARP7s activity and nuclear localization. Intriguingly, treatment with PARP7 inhibitors increased IFN{beta} expression more than PARP7 knockout in a p300/CBP-dependent manner. Our findings suggest that in some contexts, IFN{beta} induction by PARP7 inhibitors occurs via two mechanisms: inhibiting MARylation of p300/CBP (loss-of-function) and stabilizing the PARP7- p300/CBP complex (gain-of-function). TeaserChemical genetics discovery of p300 and CBP as substrates of PARP7 that are essential for PARP7-mediated regulation of IFN{beta} via a dual mechanism.

cancer biology↗

bioRxiv2025

A family of E3 ligases extend K11 polyubiquitin on sites of MARUbylation

Ubiquitin (Ub) cooperation with other post-translational modifications provides a tiered opportunity for protein regulation. Small modifications to Ub such as phosphorylation, acetylation, or ADP-ribosylation have varying impacts on signaling. The Deltex family of E3 ligases was previously implicated in the ubiquitylation of ADP-ribose (ADPr) and ADPr-containing macromolecules. Our previous work found ester-linked mono-ADPr ubiquitylation (MARUbylation) on PARP7 and PARP10 in cells and that this mark is extended with K11 polyUb. We previously screened for E3 ligases that interact with PARP7 through three different approaches and identified six candidates, including the Deltex family member DTX2. One of these hits, RNF114, interacts with various other PARPs, leading us to hypothesize that RNF114 binds to sites of MARUbylation and extends K11 polyUb. Here, we show that DTX2 generates the initial MARUbe on PARP7 in cells, which depends on PARP7 catalytic activity. The MARUbe on PARP7 is extended with K11 polyUb by RNF114. To investigate the mechanism of RNF114 reader/writer function, we developed a click chemistry-inspired chemoenzymatic approach to create a novel fluorescent Ub-ADPr probe for studying its interaction with RNF114. Strikingly, we found that RNF114 has a weak affinity for ADPr and Ub separately but explicitly recognizes the linkage between Ub and ADPr present in MARUbylated species. We used AlphaFold3 modeling to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identified a tandem Di19-UIM module in RNF114 as a MARUbe-binding domain (MUBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we described a small family of MUBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call MARUbe-Targeted Ligases (MUTLs).

biochemistry↗

bioRxiv2024

Neural correlates of age-related changes in social decisions from episodic memory

Older adults are frequent victims of financial scams. Previous behavioral research suggests that this may be due to systematic biases in how they make decisions about whom to trust: for instance, Lempert et al. (2022) found that relative to younger adults, older adults were more likely to base decisions about whether to re-engage with someone on how generous that person looked, rather than on their memory for how they had previously behaved. Here, we aimed to identify the neural correlates of these age-dependent changes in social decision-making in order to clarify the mechanism by which they emerge. Using functional Magnetic Resonance Imaging (fMRI), we measured neural activity while a total of 86 participants - 45 younger and 41 older adults - learned about how much of a $10 endowment an individual, represented by a picture of their face, was willing to share with them in a dictator game. After this encoding phase, participants then made decisions about whom they wanted to play another round of the dictator game with. In line with previous findings, we found that older adults did not reliably prefer to re-engage with people who had proven themselves to be generous. This bias was the result of several factors: (1) older adults had worse associative memory for how much each person had shared, possibly due to an age-dependent decrease in neural activity in the medial temporal lobe (MTL) during encoding, (2) older adults had a stronger tendency to re-engage with familiar over novel faces regardless of their past behavior, and (3) while activity in value-responsive brain regions tracked with how generous a face looked across the age range, older adults were less able to inhibit the influence of these irrelevant perceptual features when it was necessary to do so. In line with this behavioral effect, younger adults showed greater activation in the inferior frontal gyrus (IFG) during choices that required suppressing irrelevant perceptual features in favor of associative memory. Taken together, our findings highlight age-dependent changes in both the ability to encode relevant information and to adaptively deploy it in service of social decisions.

neuroscience↗

bioRxiv2024

Discovery of ester-linked ubiquitylation of PARP10 mono-ADP-ribosylation in cells: a dual post-translational modification on Glu/Asp side chains

The prevailing view on post-translational modifications (PTMs) is that amino acid side chains in proteins are modified with a single PTM at any given time. However, a growing body of work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation, where specialized E3 ligases ubiquitylate targets for proteasomal degradation in an ADP-ribosylation-dependent manner. More recently, the DELTEX family of E3 ligases was reported to catalyze ubiquitylation of the 3- hydroxy group of the adenine-proximal ribose of free NAD+ and ADP-ribose in vitro, generating a non-canonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester (MARUbe). We term this process mono-ADP-ribosyl ubiquitylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that PARP10 MARUbylation is extended with K11-linked polyubiquitin chains. Finally, mechanistic studies using proteasomal and ubiquitin-activating enzyme inhibitors demonstrated that PARP10 MARUbylation leads to its proteasomal degradation, providing a functional role for this new PTM in regulating protein turnover.

cell biology↗

bioRxiv2024

A genetically encoded sensor for real-time monitoring of poly-ADP-ribosylation dynamics in-vitro and in cells

ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) groups to proteins, is a conserved post-translational modification (PTM) that occurs most prominently in response to DNA damage. ADP-ribosylation is a dynamic PTM regulated by writers (PARPs), erasers (ADPr hydrolases), and readers (ADPR binders). PARP1 is the primary DNA damage-response writer responsible for adding a polymer of ADPR to proteins (PARylation). Real-time monitoring of PARP1-mediated PARylation, especially in live cells, is critical for understanding the spatial and temporal regulation of this unique PTM. Here, we describe a genetically encoded FRET probe (pARS) for semi-quantitative monitoring of PARylation dynamics. pARS feature a PAR-binding WWE domain flanked with turquoise and Venus. With a ratiometric readout and excellent signal-to-noise characteristics, we show that pARS can monitor PARP1-dependent PARylation temporally and spatially in real-time. pARS provided unique insights into PARP1-mediated PARylation kinetics in vitro and high-sensitivity detection of PARylation in live cells, even under mild DNA damage. We also show that pARS can be used to determine the potency of PARP inhibitors in vitro and, for the first time, in live cells in response to DNA damage. The robustness and ease of use of pARS make it an important tool for the PARP field.

biochemistry↗

bioRxiv2024

PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-{beta} and IFN-{lambda} responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14s antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1), a DNA virus, vesicular stomatitis virus (VSV), a negative-sense RNA virus, and lymphocytic choriomeningitis virus (LCMV), an ambisense RNA virus, to infect A549 PARP14 knockout (KO) cells. While LCMV infection was unaffected, HSV-1 replication was increased in PARP14 KO cells and VSV replication was decreased. These results indicate that PARP14 restricts HSV-1 replication but enhances the replication of VSV. A PARP14 active site inhibitor had no impact on HSV-1 or VSV replication, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both proviral and antiviral functions targeting multiple viruses. IMPORTANCEThe antiviral response is largely regulated by post-translation modifications (PTM), including ADP-ribosylation. PARP14 is an ADP-ribosyltransferase that is upregulated by interferon and is under positive selection, indicating that it is involved in host-pathogen conflict. However, no anti-viral function has been described for PARP14. Here, we found that PARP14 represses both coronavirus and HSV-1 replication, demonstrating that PARP14 has antiviral functions. Surprisingly, we also found that PARP14 also has pro-viral functions, as it was critical for the efficient replication of VSV. These data indicate that PARP14 has both proviral and antiviral functions. Defining the mechanisms used by PARP14 to both repress and promote virus replication will provide new insights into how PARPs regulate virus infection.

microbiology↗

bioRxiv2023

Targeting the Hippo pathway in cancers via ubiquitination dependent TEAD degradation

The Hippo pathway is among the most frequently altered key signaling pathways in cancer. TEAD1-4 are essential transcription factors and key downstream effectors in the Hippo pathway in human cells. Here, we identified RNF146 as a ubiquitin ligase (E3) of TEADs, which negatively regulates their stability in cells through proteasome-mediated degradation. We show that RNF146-mediated TEAD ubiquitination is dependent on the TEAD PARylation state. We further validated the genetic interaction between RNF146 and the Hippo pathway in cancer cell lines and the model organism Drosophila melanogaster. Despite the RNF146 and proteasome-mediated degradation mechanisms, TEADs are stable proteins with a long half-life in cells. We demonstrate that degradation of TEADs can be greatly enhanced pharmacologically with heterobifunctional chemical inducers of protein degradation (CIDEs). These TEAD-CIDEs can effectively suppress activation of YAP/TAZ target genes in a dose-dependent manner and exhibit significant anti-proliferative effects in YAP/TAZ-dependent tumor cells, thus phenocopying the effect of genetic ablation of TEAD protein. Collectively, this study demonstrates that the ubiquitin-proteasome system plays an important role in regulating TEAD functions and provides a proof-of-concept demonstration that pharmacologically induced TEAD ubiquitination could be leveraged to target YAP/TAZ-driven cancers.

cancer biology↗

bioRxiv2023

Establishing a causal role for left ventrolateral prefrontal cortex in value-directed memory encoding with high-definition transcranial direct current stimulation

One critical approach for promoting the efficiency of memory is to adopt selective encoding strategies to prioritize more valuable information. Past neuroimaging studies have shown that value-directed modulation of verbal memory depends heavily on engagement of left-lateralized semantic processing regions, particularly in ventrolateral prefrontal cortex (VLPFC). In the present study, we used high-definition direct current stimulation (HD-tDCS) to seek evidence for a causal role of left VLPFC in supporting the memory advantage for high-value items. Three groups of healthy young adult participants were presented with lists of words to remember, with each word accompanied by an arbitrarily assigned point value. During the first session, all participants received sham stimulation as they encoded five lists of 30 words each. Two of these lists were immediately tested with free recall, with feedback given to allow participants to develop metacognitive insight and strategies to maximize their point total. The second session had the exact same structure as the first, but the groups differed in whether they received continued sham stimulation (N=22) or anodal stimulation of the left VLPFC (N=21) or right VLPFC (N=20). Those lists not tested with immediate recall were tested with recognition judgments after a one-day delay. Since no brain stimulation was applied during this Day 2 test, any performance differences can be attributed to the effects of stimulation on Day 1 encoding processes. Anodal stimulation of left VLPFC significantly boosted participants memory encoding selectivity. In comparison, no such effect was seen in participants who received right VLPFC or sham stimulation. Estimates of recollection- and familiarity-based responding revealed that left VLPFC stimulation specifically amplified the effects of item value on recollection. These results demonstrate a causal role for left VLPFC in the implementation of selective value-directed encoding strategies, putatively by boosting deep semantic processing of high-value words. Our findings also provide further evidence on the hemispheric lateralization of value-directed verbal memory encoding.

neuroscience↗

bioRxiv2022

Rapid Cell Type-Specific Nascent Proteome Labeling in Drosophila

Controlled protein synthesis is required to regulate gene expression and is often carried out in a cell type-specific manner. Protein synthesis is commonly measured by labeling the nascent proteome with amino acid analogs or isotope-containing amino acids. These methods have been difficult to implement in vivo as they require lengthy amino acid replacement procedures. O-propargyl-puromycin (OPP) is a puromycin analog that incorporates into nascent polypeptide chains. Through its terminal alkyne, OPP can be conjugated to a fluorophore-azide for directly visualizing nascent protein synthesis, or to a biotin-azide for capture and identification of newly-synthesized proteins. To achieve cell type-specific OPP incorporation, we developed phenylacetyl-OPP (PhAc-OPP), a puromycin analog harboring an enzyme-labile blocking group that can be removed by Penicillin G acylase (PGA). Here, we show that cell type-specific PGA expression in Drosophila can be used to achieve OPP labeling of newly-synthesized proteins in targeted cell populations within the brain. Following a brief 2-hour incubation of intact brains with PhAc-OPP, we observe robust imaging and affinity purification of OPP-labeled nascent proteins in PGA-targeted cell populations. We apply this method to show a pronounced age-related decline in neuronal protein synthesis in the fly brain, demonstrating the capability to quantitatively capture in vivo protein synthesis states using PhAc-OPP. This method, which we call POPPi (PGA-dependent OPP incorporation), should be applicable for rapidly visualizing protein synthesis and identifying nascent proteins synthesized under diverse physiological and pathological conditions with cellular specificity in vivo.

neuroscience↗

bioRxiv2020

A sort-seq approach to the development of single fluorescent protein biosensors

The utility of single fluorescent protein biosensors (SFPBs) in biological research is offset by the difficulty in engineering these tools. SFPBs generally consist of three basic components: a circularly permuted fluorescent protein, a ligand-binding domain, and a pair of linkers connecting the two domains. In the absence of predictive methods for biosensor engineering, most designs combining these three components will fail to produce allosteric coupling between ligand binding and fluorescence emission. Methods to construct libraries of biosensor designs with variations in the site of GFP insertion and linker sequences have been developed, however, our ability to construct new variants has exceeded our ability to test them for function. Here, we address this challenge by applying a massively parallel assay termed "sort-seq" to the characterization of biosensor libraries. Sort-seq combines binned fluorescence-activated cell sorting, next-generation sequencing, and maximum likelihood estimation to quantify the dynamic range of many biosensor variants in parallel. We applied this method to two common biosensor optimization tasks: choice of insertion site and optimization of linker sequences. The sort-seq assay applied to a maltose-binding protein domain-insertion library not only identified previously described high-dynamic-range variants but also discovered new functional insertion-sites with diverse properties. A sort-seq assay performed on a pyruvate biosensor linker library expressed in mammalian cell culture identified linker variants with substantially improved dynamic range. Machine learning models trained on the resulting data can predict dynamic range from linker sequence. This high-throughput approach will accelerate the design and optimization of SFPBs, expanding the biosensor toolbox.

biochemistry↗

bioRxiv2020

Coronavirus Infection and PARP Expression Dysregulate the NAD Metabolome: A Potentially Actionable Component of Innate Immunity

Poly-ADP-ribose polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using nicotinamide adenine dinucleotide (NAD) as the source of ADPR. While the well-known poly-ADP-ribosylating (PARylating) PARPs primarily function in the DNA damage response, many non-canonical mono-ADP-ribosylating (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust upregulation of several PARPs following infection with Murine Hepatitis Virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly upregulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while downregulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+. Finally, we show that NAMPT activation, NAM and NR dramatically decrease the replication of an MHV virus that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD, and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

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

Subordinate effect of -21M HLA-B dimorphism on NK cell repertoire diversity and function in HIV-1 infected individuals of African origin

Natural Killer (NK) cells play an important role in antiviral defence and their potent effector function identifies them as key candidates for immunotherapeutic interventions in chronic viral infections. Their remarkable functional agility is achieved by virtue of a wide array of germline encoded inhibitory and activating receptors ensuring a self-tolerant and tunable repertoire. NK cell diversity is generated by a combination of factors including genetic determinants and infections/environmental factors, which together shape the NK cell pool and functional potential. Recently a genetic polymorphism at position -21 of HLA-B, which influences the supply of HLA-E binding peptides and availability of HLA-E for recognition by the inhibitory NK cell receptor NKG2A, was shown to have a marked influence on NK cell functionality in healthy human cytomegalovirus (HCMV) seronegative Caucasian individuals. In this study, -21 methionine (M)-expressing alleles supplying HLA-E binding peptides were largely poor ligands for inhibitory killer immunoglobulin-like receptors (KIRs), and a bias to NKG2A-mediated education of functionally-potent NK cells was observed. Here, we investigated the effect of this polymorphism on the phenotype and functional capacity of NK cells in a cohort of African individuals with human immunodeficiency virus type 1 (HIV-1)/HCMV co-infection. A similarly profound influence of dimorphism at position -21 of HLA-B on NK cells was not evident in these subjects. They predominantly expressed African specific HLA-B and -C alleles that contribute a distinct supply of NKG2A and KIR ligands, and these genetic differences were compounded by the marked effect of HIV/HCMV coinfection on NK cell differentiation. Together, these factors resulted in a lack of correlation of the HLA-B -21 polymorphism with surface abundance of HLA-E and loss of the NK cell functional advantage in subjects with -21M HLA-B alleles. Instead our data suggest that during HIV/HCMV co-infection exposure of NK cells to an environment that displays altered HLA-E ligands drives adaptive NKG2C+ NK cell expansions influencing effector responses. Increased efforts to understand how NK cells are functionally calibrated to self-HLA during chronic viral infections will pave the way to developing targeted therapeutic interventions to overcome the current barriers to enhancing immune-based antiviral control.

immunology↗