A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis.

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.

Discovery of a first-in-class orally available HBV cccDNA inhibitor.

BACKGROUND & AIMS: The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. METHODS: A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. RESULTS: Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. CONCLUSIONS: We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. IMPACT AND IMPLICATIONS: Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.

Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication.

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.

Explainable machine learning improves interpretability in the predictive modeling of biological stream conditions in the Chesapeake Bay Watershed, USA.

Anthropogenic alterations have resulted in widespread degradation of stream conditions. To aid in stream restoration and management, baseline estimates of conditions and improved explanation of factors driving their degradation are needed. We used random forests to model biological conditions using a benthic macroinvertebrate index of biotic integrity for small, non-tidal streams (upstream area ≤200 km2) in the Chesapeake Bay watershed (CBW) of the mid-Atlantic coast of North America. We utilized several global and local model interpretation tools to improve average and site-specific model inferences, respectively. The model was used to predict condition for 95,867 individual catchments for eight periods (2001, 2004, 2006, 2008, 2011, 2013, 2016, 2019). Predicted conditions were classified as Poor, FairGood, or Uncertain to align with management needs and individual reach lengths and catchment areas were summed by condition class for the CBW for each period. Global permutation and local Shapley importance values indicated percent of forest, development, and agriculture in upstream catchments had strong impacts on predictions. Development and agriculture negatively influenced stream condition for model average (partial dependence [PD] and accumulated local effect [ALE] plots) and local (individual condition expectation and Shapley value plots) levels. Friedman's H-statistic indicated large overall interactions for these three land covers, and bivariate global plots (PD and ALE) supported interactions among agriculture and development. Total stream length and catchment area predicted in FairGood conditions decreased then increased over the 19-years (length/area: 66.6/65.4% in 2001, 66.3/65.2% in 2011, and 66.6/65.4% in 2019). Examination of individual catchment predictions between 2001 and 2019 showed those predicted to have the largest decreases in condition had large increases in development; whereas catchments predicted to exhibit the largest increases in condition showed moderate increases in forest cover. Use of global and local interpretative methods together with watershed-wide and individual catchment predictions support conservation practitioners that need to identify widespread and localized patterns, especially acknowledging that management actions typically take place at individual-reach scales.

Activation of the Liver X Receptor Pathway Inhibits HBV Replication in Primary Human Hepatocytes.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is ranked among the top health priorities worldwide. Accumulating evidence suggests that HBV infection and replication are closely associated with liver metabolism. The liver X receptors (LXRs), which belong to the superfamily of nuclear hormone receptors, are important physiological regulators of lipid and cholesterol metabolism. However, the association between the LXR pathway and HBV infection remains largely unclear. APPROACH AND RESULTS: In this study, the antiviral activity of LXR agonists was investigated using multiple HBV cellular models. We observed that in HBV-infected primary human hepatocytes (PHHs), synthetic LXR agonists (T0901317, GW3965, and LXR-623), but not an LXR antagonist (SR9238), potently inhibited HBV replication and gene expression, as demonstrated by substantial reductions in viral RNA, DNA, and antigen production following agonist treatment. However, covalently closed circular DNA (cccDNA) levels were not significantly reduced by the agonists. In addition, no rebound in viral replication was observed after treatment withdrawal, indicating a long-lasting inhibitory effect. These results suggest that LXR agonists decrease the transcriptional activity of cccDNA. In contrast, no significant anti-HBV effect was observed in HepG2-derived cell lines. Interestingly, LXR agonist treatment strongly reduced cholesterol 7α-hydroxylase 1 (CYP7A1) mRNA levels. Knockdown of CYP7A1 gene expression with small interfering RNA inhibited HBV activity in PHHs, suggesting CYP7A1 as a potential factor contributing to the antiviral effects of LXR agonists. CONCLUSIONS: We found that activation of the LXR pathway with synthetic LXR agonists could elicit potent anti-HBV activity in PHHs, possibly through sustained suppression of cccDNA transcription. Our work highlights the therapeutic potential of targeting the LXR pathway for the treatment of chronic HBV infection.

Using expert knowledge to support Endangered Species Act decision-making for data-deficient species.

Many questions relevant to conservation decision-making are characterized by extreme uncertainty due to lack of empirical data and complexity of the underlying ecologic processes, leading to a rapid increase in the use of structured protocols to elicit expert knowledge. Published ecologic applications often employ a modified Delphi method, where experts provide judgments anonymously and mathematical aggregation techniques are used to combine judgments. The Sheffield elicitation framework (SHELF) differs in its behavioral approach to synthesizing individual judgments into a fully specified probability distribution for an unknown quantity. We used the SHELF protocol remotely to assess extinction risk of three subterranean aquatic species that are being considered for listing under the U.S. Endangered Species Act. We provided experts an empirical threat assessment for each known locality over a video conference and recorded judgments on the probability of population persistence over four generations with online submission forms and R-shiny apps available through the SHELF package. Despite large uncertainty for all populations, there were key differences between species' risk of extirpation based on spatial variation in dominant threats, local land use and management practices, and species' microhabitat. The resulting probability distributions provided decision makers with a full picture of uncertainty that was consistent with the probabilistic nature of risk assessments. Discussion among experts during SHELF's behavioral aggregation stage clearly documented dominant threats (e.g., development, timber harvest, animal agriculture, and cave visitation) and their interactions with local cave geology and species' habitat. Our virtual implementation of the SHELF protocol demonstrated the flexibility of the approach for conservation applications operating on budgets and time lines that can limit in-person meetings of geographically dispersed experts.

Uso del Conocimiento Experto para Respaldar la Toma de Decisiones del Acta de Especies en Peligro para Especies con Información Deficiente Resumen Muchas preguntas relevantes para la toma de decisiones de conservación se caracterizan por una incertidumbre extrema causada por la falta de información empírica y por la complejidad de los procesos ecológicos subyacentes. Esto lleva a un rápido incremento en el uso de protocolos estructurados para obtener conocimiento de los expertos en el tema. Las aplicaciones ecológicas publicadas con frecuencia emplean un método Delphi modificado, en el cual los expertos proporcionan dictámenes anónimamente y luego se usan técnicas de agregación matemática para combinar estos dictámenes. El marco de trabajo de obtención Sheffield (SHELF) difiere en su enfoque conductual para sintetizar los dictámenes individuales en una distribución de probabilidad completamente especificada para una cantidad desconocida. Usamos el protocolo SHELF remotamente para evaluar el riesgo de extinción de tres especies acuáticas subterráneas que están siendo consideradas para ser incluidas en el Acta de Especies en Peligro de los E.U.A. Les proporcionamos a los expertos una evaluación empírica de la amenaza para cada localidad conocida durante una videoconferencia y registramos los dictámenes sobre la probabilidad de la persistencia poblacional durante cuatro generaciones por medio de formularios enviados en línea y las apps R-shiny disponibles a través del paquete SHELF. A pesar de la gran incertidumbre para todas las poblaciones, hubo diferencias importantes entre el riesgo de extirpación de las especies con base en la variación espacial en las amenazas dominantes, el uso del suelo local y las prácticas de manejo, y el microhábitat de las especies. Las distribuciones resultantes de la probabilidad proporcionaron al órgano decisorio un cuadro completo de la incertidumbre que fue consistente con la naturaleza probabilística de las evaluaciones de riesgo. Las discusiones entre los expertos durante la fase de agregación conductual de SHELF documentaron claramente las amenazas dominantes (p. ej.: desarrollo, extracción de madera, agricultura animal y visitas a las cuevas) y sus interacciones con la geología de las cuevas locales y el hábitat de la especie. Nuestra implementación virtual del protocolo SHELF demostró la flexibilidad del enfoque para las aplicaciones de la conservación que operan con presupuestos y líneas de tiempo que pueden limitar las reuniones en persona de expertos dispersados geográficamente.

PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization.

RG7834 is a potent, orally bioavailable small-molecule inhibitor of hepatitis B virus (HBV) gene expression that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three-hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies that phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down, suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. In addition, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusion: We identify PAPD5 and PAPD7 as cellular host factors required for HBV RNA stabilization and as therapeutic targets for the HBV cure.

Direct Inhibition of Hepatitis B e Antigen by Core Protein Allosteric Modulator.

Hepatitis B e antigen (HBeAg) is an important immunomodulator for promoting host immune tolerance during chronic hepatitis B (CHB) infection. In patients with CHB, HBeAg loss and seroconversion represent partial immune control of CHB infection and are regarded as valuable endpoints. However, the current approved treatments have only a limited efficacy in achieving HBeAg seroconversion in HBeAg-positive patients. Hepatitis B virus (HBV) core protein has been recognized as an attractive antiviral target, and two classes of core protein allosteric modulator (CpAM) have been discovered: the phenylpropenamides (PPAs) and the heteroaryldihydropyrimidines (HAPs). However, their differentiation and potential therapeutic benefit beyond HBV DNA inhibition remain to be seen. Here, we show that in contrast to PPA series compound AT-130, a HAP CpAM, HAP_R01, reduced HBeAg levels in multiple in vitro and in vivo HBV experimental models. Mechanistically, we found that HAP_R01 treatment caused the misassembly of capsids formed by purified HBeAg in vitro. In addition, HAP_R01 directly reduces HBeAg levels by inducing intracellular precore protein misassembly and aggregation. Using a HAP_R01-resistant mutant, we found that HAP_R01-mediated HBeAg and core protein reductions were mediated through the same mechanism. Furthermore, HAP_R01 treatment substantially reduced serum HBeAg levels in an HBV mouse model. Conclusion: Unlike PPA series compound AT-130, HAP_R01 not only inhibits HBV DNA levels but also directly reduces HBeAg through induction of its misassembly. HAP_R01, as well as other similar CpAMs, has the potential to achieve higher anti-HBeAg seroconversion rates than currently approved therapies for patients with CHB. Our findings also provide guidance for dose selection when designing clinical trials with molecules from HAP series.

Disentangling the potential effects of land-use and climate change on stream conditions.

Land-use and climate change are significantly affecting stream ecosystems, yet understanding of their long-term impacts is hindered by the few studies that have simultaneously investigated their interaction and high variability among future projections. We modeled possible effects of a suite of 2030, 2060, and 2090 land-use and climate scenarios on the condition of 70,772 small streams in the Chesapeake Bay watershed, United States. The Chesapeake Basin-wide Index of Biotic Integrity, a benthic macroinvertebrate multimetric index, was used to represent stream condition. Land-use scenarios included four Special Report on Emissions Scenarios (A1B, A2, B1, and B2) representing a range of potential landscape futures. Future climate scenarios included quartiles of future climate changes from downscaled Coupled Model Intercomparison Project - Phase 5 (CMIP5) and a watershed-wide uniform scenario (Lynch2016). We employed random forests analysis to model individual and combined effects of land-use and climate change on stream conditions. Individual scenarios suggest that by 2090, watershed-wide conditions may exhibit anywhere from large degradations (e.g., scenarios A1B, A2, and the CMIP5 25th percentile) to small degradations (e.g., scenarios B1, B2, and Lynch2016). Combined land-use and climate change scenarios highlighted their interaction and predicted, by 2090, watershed-wide degradation in 16.2% (A2 CMIP5 25th percentile) to 1.0% (B2 Lynch2016) of stream kilometers. A goal for the Chesapeake Bay watershed is to restore 10% of stream kilometers over a 2008 baseline; our results suggest meeting and sustaining this goal until 2090 may require improvement in 11.0%-26.2% of stream kilometers, dependent on land-use and climate scenario. These results highlight inherent variability among scenarios and the resultant uncertainty of predicted conditions, which reinforces the need to incorporate multiple scenarios of both land-use (e.g., development, agriculture, etc.) and climate change in future studies to encapsulate the range of potential future conditions.

A novel orally available small molecule that inhibits hepatitis B virus expression.

BACKGROUND & AIMS: The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). METHODS: RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. RESULTS: Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. CONCLUSION: We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. LAY SUMMARY: We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.

Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1.

A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs.IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

HBVcircle: A novel tool to investigate hepatitis B virus covalently closed circular DNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA. METHODS: Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI). RESULTS: We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system. CONCLUSION: Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery. LAY SUMMARY: To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.

Global landscape of HIV-human protein complexes.

Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages.

BACKGROUND: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases, which sulfonate proteins, glycoproteins, glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs), which sulfonate various small molecules including hormones, neurotransmitters, and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions, cell signaling, and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism, namely reverse transcription. METHODS: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions, MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels, and qPCR was used to measure viral RNA and DNA products. RESULTS: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs, and through siRNA knockdown experiments, we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription. CONCLUSIONS: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules, raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics.

Human host factors required for influenza virus replication.

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.

Tumor suppressor cylindromatosis (CYLD) controls HIV transcription in an NF-κB-dependent manner.

UNLABELLED: Characterizing the cellular factors that play a role in the HIV replication cycle is fundamental to fully understanding mechanisms of viral replication and pathogenesis. Whole-genome small interfering RNA (siRNA) screens have identified positive and negative regulators of HIV replication, providing starting points for investigating new cellular factors. We report here that silencing of the deubiquitinase cylindromatosis protein (CYLD), increases HIV infection by enhancing HIV long terminal repeat (LTR)-driven transcription via the NF-κB pathway. CYLD is highly expressed in CD4(+) T lymphocytes, monocyte-derived macrophages, and dendritic cells. We found that CYLD silencing increases HIV replication in T cell lines. We confirmed the positive role of CYLD silencing in HIV infection in primary human CD4(+) T cells, in which CYLD protein was partially processed upon activation. Lastly, Jurkat T cells latently infected with HIV (JLat cells) were more responsive to phorbol 12-myristate 13-acetate (PMA) reactivation in the absence of CYLD, indicating that CYLD activity could play a role in HIV reactivation from latency. In summary, we show that CYLD acts as a potent negative regulator of HIV mRNA expression by specifically inhibiting NF-κB-driven transcription. These findings suggest a function for this protein in modulating productive viral replication as well as in viral reactivation. IMPORTANCE: HIV transcription is regulated by a number of host cell factors. Here we report that silencing of the lysine 63 deubiquitinase CYLD increases HIV transcription in an NF-κB-dependent manner. We show that CYLD is expressed in HIV target cells and that its silencing increases HIV infection in transformed T cell lines as well as primary CD4(+) T cells. Similarly, reactivation of latent provirus was facilitated in the absence of CYLD. These data suggest that CYLD, which is highly expressed in CD4(+) T cells, can control HIV transcription in productive infection as well as during reactivation from latency.

ZASC1 stimulates HIV-1 transcription elongation by recruiting P-TEFb and TAT to the LTR promoter.

Transcription from the HIV-1 LTR promoter efficiently initiates but rapidly terminates because of a non-processive form of RNA polymerase II. This premature termination is overcome by assembly of an HIV-1 TAT/P-TEFb complex at the transactivation response region (TAR), a structured RNA element encoded by the first 59 nt of HIV-1 mRNA. Here we have identified a conserved DNA-binding element for the cellular transcription factor, ZASC1, in the HIV-1 core promoter immediately upstream of TAR. We show that ZASC1 interacts with TAT and P-TEFb, co-operating with TAT to regulate HIV-1 gene expression, and promoting HIV-1 transcriptional elongation. The importance of ZASC1 to HIV-1 transcription elongation was confirmed through mutagenesis of the ZASC1 binding sites in the LTR promoter, shRNAs targeting ZASC1 and expression of dominant negative ZASC1. Chromatin immunoprecipitation analysis revealed that ZASC1 recruits Tat and P-TEFb to the HIV-1 core promoter in a TAR-independent manner. Thus, we have identified ZASC1 as novel regulator of HIV-1 gene expression that functions through the DNA-dependent, RNA-independent recruitment of TAT/P-TEFb to the HIV-1 promoter.

Accounting for groundwater in stream fish thermal habitat responses to climate change.

Forecasting climate change effects on aquatic fauna and their habitat requires an understanding of how water temperature responds to changing air temperature (i.e., thermal sensitivity). Previous efforts to forecast climate effects on brook trout (Salvelinus fontinalis) habitat have generally assumed uniform air-water temperature relationships over large areas that cannot account for groundwater inputs and other processes that operate at finer spatial scales. We developed regression models that accounted for groundwater influences on thermal sensitivity from measured air-water temperature relationships within forested watersheds in eastern North America (Shenandoah National Park, Virginia, USA, 78 sites in nine watersheds). We used these reach-scale models to forecast climate change effects on stream temperature and brook trout thermal habitat, and compared our results to previous forecasts based upon large-scale models. Observed stream temperatures were generally less sensitive to air temperature than previously assumed, and we attribute this to the moderating effect of shallow groundwater inputs. Predicted groundwater temperatures from air-water regression models corresponded well to observed groundwater temperatures elsewhere in the study area. Predictions of brook trout future habitat loss derived from our fine-grained models. were far less pessimistic than those from prior models developed at coarser spatial resolutions. However, our models also revealed spatial variation in thermal sensitivity within and among catchments resulting in a patchy distribution of thermally suitable habitat. Habitat fragmentation due to thermal barriers therefore may have an increasingly important role for trout population viability in headwater streams. Our results demonstrate that simple adjustments to air-water temperature regression models can provide a powerful and cost-effective approach for predicting future stream temperatures while accounting for effects of groundwater.

Using ecological indicators and a decision support system for integrated ecological assessment at two national park units in the mid-Atlantic region, USA.

We implemented an integrated ecological assessment using a GIS-based decision support system model for Upper Delaware Scenic and Recreational River (UPDE) and Delaware Water Gap National Recreation Area (DEWA)-national park units with the mid-Atlantic region of the United States. Our assessment examined a variety of aquatic and terrestrial indicators of ecosystem components that reflect the parks' conservation purpose and reference condition. Our assessment compared these indicators to ecological thresholds to determine the condition of park watersheds. Selected indicators included chemical and physical measures of water quality, biologic indicators of water quality, and landscape condition measures. For the chemical and physical measures of water quality, we used a water quality index and each of its nine components to assess the condition of water quality in each watershed. For biologic measures of water quality, we used the Ephemeroptera, Plecoptera, Trichoptera aquatic macroinvertebrate index and, secondarily, the Hilsenhoff aquatic macroinvertebrate index. Finally, for the landscape condition measures of our model, we used percent forest and percent impervious surface. Based on our overall assessment, UPDE and DEWA watersheds had an ecological assessment score of 0.433 on a -1 to 1 fuzzy logic scale. This score indicates that, in general, the natural resource condition within watersheds at these parks is healthy or ecologically unimpaired; however, we had only partial data for many of our indicators. Our model is iterative and new data may be incorporated as they become available. These natural parks are located within a rapidly urbanizing landscape-we recommend that natural resource managers remain vigilant to surrounding land uses that may adversely affect natural resources within the parks.