Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus infection.

ADP-ribosylation is a highly dynamic and fully reversible post-translational modification performed by poly(ADP-ribose) polymerases (PARPs) that modulates protein function, abundance, localization and turnover. Here we show that influenza A virus infection causes a rapid and dramatic upregulation of global ADP-ribosylation that inhibits viral replication. Mass spectrometry defined for the first time the global ADP-ribosylome during infection, creating an infection-specific profile with almost 4,300 modification sites on ~1,080 host proteins, as well as over 100 modification sites on viral proteins. Our data indicate that the global increase likely reflects a change in the form of ADP-ribosylation rather than modification of new targets. Functional assays demonstrated that modification of the viral replication machinery antagonizes its activity and further revealed that the anti-viral activity of PARPs and ADP-ribosylation is counteracted by the influenza A virus protein NS1, assigning a new activity to the primary viral antagonist of innate immunity. We identified PARP1 as the enzyme producing the majority of poly(ADP-ribose) present during infection. Influenza A virus replicated faster in cells lacking PARP1, linking PARP1 and ADP-ribosylation to the anti-viral phenotype. Together, these data establish ADP-ribosylation as an anti-viral innate immune-like response to viral infection antagonized by a previously unknown activity of NS1.

Lectin-Seq: A method to profile lectin-microbe interactions in native communities.

Soluble human lectins are critical components of innate immunity. Genetic models suggest that lectins influence host-resident microbiota, but their specificity for commensal and mutualist species is understudied. Elucidating lectins' roles in regulating microbiota requires an understanding of which microbial species they bind within native communities. To profile human lectin recognition, we developed Lectin-Seq. We apply Lectin-Seq to human fecal microbiota using the soluble mannose-binding lectin (MBL) and intelectin-1 (hItln1). Although each lectin binds a substantial percentage of the samples (10 to 20%), the microbial interactomes of MBL and hItln1 differ markedly in composition and diversity. MBL binding is highly selective for a small subset of species commonly associated with humans. In contrast, hItln1's interaction profile encompasses a broad range of lower-abundance species. Our data uncover stark differences in the commensal recognition properties of human lectins.

Human oral lectin ZG16B acts as a cell wall polysaccharide probe to decode host-microbe interactions with oral commensals.

The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.

Factors Influencing Treatment Adherence Among Drug-Sensitive Tuberculosis (DS-TB) Patients in Armenia: A Qualitative Study.

Purpose: Despite the availability of effective treatment, tuberculosis (TB) is still one of the leading causes of mortality around the globe. Poor adherence to treatment challenges TB management both globally and locally. Proper adherence to treatment contributes to successful outcomes and prevents the development of drug-resistant forms of TB. Patients and Methods: We conducted a qualitative study to identify and describe the factors that facilitated treatment adherence among drug-sensitive TB (DS-TB) patients in Armenia. Sixteen in-depth interviews (IDIs) with former DS-TB patients, two IDIs with family members of former DS-TB patients, and a focus group discussion with healthcare providers who manage DS-TB patients were conducted. The educational and ecological assessment component of the PRECEDE-PROCEED model was applied as a conceptual framework to guide the interview content and data analysis. Results: Former patients' awareness of TB and its treatment, beliefs about TB, trust in TB healthcare providers, and a sense of responsibility were the most common factors that predisposed them to complete the treatment. Support received from providers, family, and friends, a desire to avoid TB-associated stigma, and good tolerance of TB medications were the main reinforcing factors. Enabling factors included a relatively simple regimen of TB treatment and accessibility and affordability of TB services. Conclusion: The findings of the study provide new perspectives on factors that facilitate adherence to long-term therapies, such as TB. Interventions that aim to invoke a patient's sense of responsibility and positive beliefs about TB as well as engage families might promote the successful completion of treatment.

High-Throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain.

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen that identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib inhibits SARS-CoV-2 and MERS-CoV Mac1 but not the closest human homologue, MacroD2. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for the screening of large compound libraries to identify improved macrodomain inhibitors and to explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

ADP-ribose and analogues bound to the deMARylating macrodomain from the bat coronavirus HKU4.

Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.

Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 Macrodomain Affect Neurovirulence in Mice.

Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADP-ribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-κB-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice.IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADP-ribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis.

Therapeutic profile of a latent heat eyelid warming device with temperature setting variation.

PURPOSE: To compare the effects on ocular temperature and tear film parameters following a single application of a latent heat eyelid warming device at a range of temperature settings. METHODS: Fifteen subjects were enrolled in a prospective, investigator-masked, randomised, cross-over trial. On separate days, participants were randomised to 10-minute application of a research latent heat device (Laboratoires Théa) at device temperature settings of 45 °C, 50 °C and 55 °C. Outer eyelid and corneal temperatures, tear film lipid layer grade, and non-invasive tear film breakup time (NIBUT) were measured at baseline and immediately after 10 min of device application. RESULTS: Baseline measurements did not differ between treatment groups (all p > 0.05). Ocular temperatures, lipid layer grade and non-invasive tear film stability rose significantly following device application in all treatment groups (all p < 0.05). The 55 °C setting effected a mean ocular surface temperature rise in the order of +4 °C from baseline, which was 1.46 and 1.26 times greater than at the 45 °C and 50 °C temperature settings, respectively (all p < 0.05). Similarly, improvements in mean non-invasive tear film stability from baseline in the order of +7 s were observed, which were 2.43 and 1.66 times greater than those at the lower temperature settings of 45 °C and 50 °C, respectively (all p < 0.05). CONCLUSIONS: At all temperature settings, the latent heat device resulted in clinically and statistically significant increases in ocular temperature, lipid layer grade, and non-invasive tear film stability. However, the 55 °C setting proved to be most effective at raising ocular temperature (in the order of +4 °C from baseline) and improving tear film stability.

Ion-Pairing with Triethylammonium Acetate Improves Solid-Phase Extraction of ADP-Ribosylated Peptides.

ADP-ribosylation refers to the post-translational modification of protein substrates with monomers or polymers of the small molecule ADP-ribose. ADP-ribosylation is enzymatically regulated and plays roles in cellular processes including DNA repair, nucleic acid metabolism, cell death, cellular stress responses, and antiviral immunity. Recent advances in the field of ADP-ribosylation have led to the development of proteomics approaches to enrich and identify endogenous ADP-ribosylated peptides by liquid chromatography tandem mass spectrometry (LC-MS/MS). A number of these methods rely on reverse-phase solid-phase extraction as a critical step in preparing cellular peptides for further enrichment steps in proteomics workflows. The anionic ion-pairing reagent trifluoroacetic acid (TFA) is typically used during reverse-phase solid-phase extraction to promote retention of tryptic peptides. Here we report that TFA and other carboxylate ion-pairing reagents are inefficient for reverse-phase solid-phase extraction of ADP-ribosylated peptides. Substitution of TFA with cationic ion-pairing reagents, such as triethylammonium acetate (TEAA), improves recovery of ADP-ribosylated peptides. We further demonstrate that substitution of TFA with TEAA in a proteomics workflow specific for identifying ADP-ribosylated peptides increases identification rates of ADP-ribosylated peptides by LC-MS/MS.

Structural analyses of NudT16-ADP-ribose complexes direct rational design of mutants with improved processing of poly(ADP-ribosyl)ated proteins.

ADP-ribosylation is a post-translational modification that occurs on chemically diverse amino acids, including aspartate, glutamate, lysine, arginine, serine and cysteine on proteins and is mediated by ADP-ribosyltransferases, including a subset commonly known as poly(ADP-ribose) polymerases. ADP-ribose can be conjugated to proteins singly as a monomer or in polymeric chains as poly(ADP-ribose). While ADP-ribosylation can be reversed by ADP-ribosylhydrolases, this protein modification can also be processed to phosphoribosylation by enzymes possessing phosphodiesterase activity, such as snake venom phosphodiesterase, mammalian ectonucleotide pyrophosphatase/phosphodiesterase 1, Escherichia coli RppH, Legionella pneumophila Sde and Homo sapiens NudT16 (HsNudT16). Our studies here sought to utilize X-ray crystallographic structures of HsNudT16 in complex with monomeric and dimeric ADP-ribose in identifying the active site for binding and processing free and protein-conjugated ADP-ribose into phosphoribose forms. These structural data guide rational design of mutants that widen the active site to better accommodate protein-conjugated ADP-ribose. We identified that several HsNudT16 mutants (Δ17, F36A, and F61S) have reduced activity for free ADP-ribose, similar processing ability against protein-conjugated mono(ADP-ribose), but improved catalytic efficiency for protein-conjugated poly(ADP-ribose). These HsNudT16 variants may, therefore, provide a novel tool to investigate different forms of ADP-ribose.

ELTA: Enzymatic Labeling of Terminal ADP-Ribose.

ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2'-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates.

ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication.

Alphaviruses are plus-strand RNA viruses that cause encephalitis, rash, and arthritis. The nonstructural protein (nsP) precursor polyprotein is translated from genomic RNA and processed into four nsPs. nsP3 has a highly conserved macrodomain (MD) that binds ADP-ribose (ADPr), which can be conjugated to protein as a posttranslational modification involving transfer of ADPr from NAD+ by poly ADPr polymerases (PARPs). The nsP3MD also removes ADPr from mono ADP-ribosylated (MARylated) substrates. To determine which aspects of alphavirus replication require nsP3MD ADPr-binding and/or hydrolysis function, we studied NSC34 neuronal cells infected with chikungunya virus (CHIKV). Infection induced ADP-ribosylation of cellular proteins without increasing PARP expression, and inhibition of MARylation decreased virus replication. CHIKV with a G32S mutation that reduced ADPr-binding and hydrolase activities was less efficient than WT CHIKV in establishing infection and in producing nsPs, dsRNA, viral RNA, and infectious virus. CHIKV with a Y114A mutation that increased ADPr binding but reduced hydrolase activity, established infection like WT CHIKV, rapidly induced nsP translation, and shut off host protein synthesis with reduced amplification of dsRNA. To assess replicase function independent of virus infection, a transreplicase system was used. Mutant nsP3MDs D10A, G32E, and G112E with no binding or hydrolase activity had no replicase activity, G32S had little, and Y114A was intermediate to WT. Therefore, ADP ribosylation of proteins and nsP3MD ADPr binding are necessary for initiation of alphavirus replication, while hydrolase activity facilitates amplification of replication complexes. These observations are consistent with observed nsP3MD conservation and limited tolerance for mutation.

Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry.

ADP-ribosylation is a posttranslational modification that involves the conjugation of monomers and polymers of the small molecule ADP-ribose onto amino acid side chains. A family of ADP-ribosyltransferases catalyzes the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD+) onto a variety of amino acid side chains including aspartate, glutamate, lysine, arginine, cysteine, and serine. The monomeric form of the modification mono(ADP-ribosyl)ation (MARylation) is reversed by a number of enzymes including a family of MacroD-type macrodomain-containing mono(ADP-ribose) (MAR) hydrolases. Though it has been inferred from various chemical tests that these enzymes have specificity for MARylated aspartate and glutamate residues in vitro, the amino acid and site specificity of different family members are often not unambiguously defined. Here we describe a mass spectrometry-based assay to determine the site specificity of MAR hydrolases in vitro.

Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation.

Recently we characterized the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the macrodomain of nonstructural protein 3 (nsP3MD) of chikungunya virus. Using recombinant viruses with targeted mutations in the macrodomain, we demonstrated that hydrolase function is important for viral replication in cultured neuronal cells and for neurovirulence in mice. Here, we describe the general cell culture and animal model infection protocols for alphaviruses and the technical details for biochemical characterization of the MAR hydrolase activity of nsP3MD mutants and the preparation of recombinant viruses incorporating those mutations through site-directed mutagenesis of an infectious cDNA virus clone.

Postnatal home visitation: Lessons from country programs operating at scale.

BACKGROUND: Newborn mortality remains unacceptably high in many countries. Postnatal home visits (PNHVs) have been endorsed as a strategy for delivery of postnatal care (PNC) to reduce newborn mortality as well as to improve maternal outcomes. This paper reports on a review of coverage-related performance of such programs implemented at scale through government health services in Bangladesh, Ethiopia, Ghana, India, Indonesia, Malawi, Myanmar, Nepal, Pakistan, Rwanda, Sri Lanka and Uganda. METHODS: We undertook a multi-country, mixed-method program review and used available survey and administrative data and key informant interviews to characterize performance of postnatal home visitation programs. In results presented in this paper, we have relied primarily on population-based surveys, notably Demographic and Health Surveys and Multi-Indicator Cluster Surveys. In addition, based on key informant interviews, we sought to understand the implementation challenges experienced delivering PNHVs, as well as responses to those challenges, in order to provide useful insights to countries to design home visitation programming when they can meet requirements for effective delivery at scale - and to identify other options when they cannot. RESULTS: Contact coverage of PNC within 48 hours of birth following home birth (the group most prioritized in these programs) is below 10% in most of the countries reviewed; in no country does it exceed 20%. Most country programs have been unable to achieve PNHV contact coverage that would have any meaningful impact on newborn or maternal mortality. Country responses to disappointing performance have varied: some continued programming unchanged, some suspended attempts to provide PNHVs, and others modified their strategies for providing postnatal care (PNC). CONCLUSIONS: Policymakers and program managers need to consider seriously context and local feasibility when determining whether and how to use a strategy like PNHVs. At the global level, we need more than evidence of effectiveness (as determined through proof-of-concept trials) as a basis for formulating recommendations for how governments should provide services. We must also give serious attention to what can be learned from experience implementing at scale and place greater importance on feasibility of implementation in the real world.

ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence.

Chikungunya virus (CHIKV), an Old World alphavirus, is transmitted to humans by infected mosquitoes and causes acute rash and arthritis, occasionally complicated by neurologic disease and chronic arthritis. One determinant of alphavirus virulence is nonstructural protein 3 (nsP3) that contains a highly conserved MacroD-type macrodomain at the N terminus, but the roles of nsP3 and the macrodomain in virulence have not been defined. Macrodomain is a conserved protein fold found in several plus-strand RNA viruses that binds to the small molecule ADP-ribose. Prototype MacroD-type macrodomains also hydrolyze derivative linkages on the distal ribose ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated proteins. Using mass spectrometry, we unambiguously defined its substrate specificity as mono(ADP-ribosyl)ated aspartate and glutamate but not lysine residues. Mutant viruses lacking hydrolase activity were unable to replicate in mammalian BHK-21 cells or mosquito Aedes albopictus cells and rapidly reverted catalytically inactivating mutations. Mutants with reduced enzymatic activity had slower replication in mammalian neuronal cells and reduced virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hydrolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice.

ENPP1 processes protein ADP-ribosylation in vitro.

ADP-ribosylation is a conserved post-translational protein modification that plays a role in all major cellular processes, particularly DNA repair, transcription, translation, stress response and cell death. Hence, dysregulation of ADP-ribosylation is linked to the physiopathology of several human diseases including cancers, diabetes and neurodegenerative disorders. Protein ADP-ribosylation can be reversed by the macrodomain-containing proteins PARG, TARG1, MacroD1 and MacroD2, which hydrolyse the ester bond known to link proteins to ADP-ribose as well as consecutive ADP-ribose subunits; targeting this bond can thus result in the complete removal of the protein modification or the conversion of poly(ADP-ribose) to mono(ADP-ribose). Recently, proteins containing the NUDIX domain - namely human NUDT16 and bacterial RppH - have been shown to process in vitro protein ADP-ribosylation through an alternative mechanism, converting it into protein-conjugated ribose-5'-phosphate (R5P, also known as pR). Though this protein modification was recently identified in mammalian tissues, its physiological relevance and the mechanism of generating protein phosphoribosylation are currently unknown. Here, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as the first known mammalian enzyme lacking a NUDIX domain to generate pR from ADP-ribose on modified proteins in vitro. Thus, our data show that at least two enzyme families - Nudix and ENPP/NPP - are able to metabolize protein-conjugated ADP-ribose to pR in vitro, suggesting that pR exists and may be conserved from bacteria to mammals. We also demonstrate the utility of ENPP1 for converting protein-conjugated mono(ADP-ribose) and poly(ADP-ribose) into mass spectrometry-friendly pR tags, thus facilitating the identification of ADP-ribosylation sites.

ADPr-ChAP: Mapping ADP-Ribosylation onto the Genome.

In this issue of Molecular Cell, Bartolomei et al. (2016) describe a chromatin affinity precipitation method using well-characterized ADP-ribose binding domains to provide the first genome-wide view of ADP-ribosylated chromatin. Here, we discuss its potential applications and the remaining challenges ahead.