The Temporal Neurogenesis Patterning of Spinal p3-V3 Interneurons into Divergent Subpopulation Assemblies.

Neuronal diversity provides the spinal cord with the functional flexibility required to perform complex motor tasks. Spinal neurons arise during early embryonic development with the establishment of spatially and molecularly discrete progenitor domains that give rise to distinct, but highly heterogeneous, postmitotic interneuron (IN) populations. Our previous studies have shown that Sim1-expressing V3 INs, originating from the p3 progenitor domain, are anatomically and physiologically divergent. However, the developmental logic guiding V3 subpopulation diversity remains elusive. In specific cases of other IN classes, neurogenesis timing can play a role in determining the ultimate fates and unique characteristics of distinctive subpopulations. To examine whether neurogenesis timing contributes to V3 diversity, we systematically investigated the temporal neurogenesis profiles of V3 INs in the mouse spinal cord. Our work uncovered that V3 INs were organized into either early-born [embryonic day 9.5 (E9.5) to E10.5] or late-born (E11.5-E12.5) neurogenic waves. Early-born V3 INs displayed both ascending and descending commissural projections and clustered into subgroups across dorsoventral spinal laminae. In contrast, late-born V3 INs became fate-restricted to ventral laminae and displayed mostly descending and local commissural projections and uniform membrane properties. Furthermore, we found that the postmitotic transcription factor, Sim1, although expressed in all V3 INs, exclusively regulated the dorsal clustering and electrophysiological diversification of early-born, but not late-born, V3 INs, which indicates that neurogenesis timing may enable newborn V3 INs to interact with different postmitotic differentiation pathways. Thus, our work demonstrates neurogenesis timing as a developmental mechanism underlying the postmitotic differentiation of V3 INs into distinct subpopulation assemblies.SIGNIFICANCE STATEMENT Interneuron (IN) diversity empowers the spinal cord with the computation flexibility required to perform appropriate sensorimotor control. As such, uncovering the developmental logic guiding spinal IN diversity is fundamental to understanding the development of movement. In our current work, through a focus on the cardinal spinal V3 IN population, we investigated the role of neurogenesis timing on IN diversity. We uncovered that V3 INs are organized into early-born [embryonic day 9.5 (E9.5) to E10.5] or late-born (E11.5-E12.5) neurogenic waves, where late-born V3 INs display increasingly restricted subpopulation fates. Next, to better understand the consequences of V3 neurogenesis timing, we investigated the time-dependent functions of the Sim1 transcription factor, which is expressed in postmitotic V3 INs. Interestingly, Sim1 exclusively regulated the diversification of early-born, but not late-born, V3 INs. Thus, our current work indicates neurogenesis timing can modulate the functions of early postmitotic transcription factors and, thus, subpopulation fate specifications.

A Phenotype-Genotype Codon Model for Detecting Adaptive Evolution.

A central objective in biology is to link adaptive evolution in a gene to structural and/or functional phenotypic novelties. Yet most analytic methods make inferences mainly from either phenotypic data or genetic data alone. A small number of models have been developed to infer correlations between the rate of molecular evolution and changes in a discrete or continuous life history trait. But such correlations are not necessarily evidence of adaptation. Here, we present a novel approach called the phenotype-genotype branch-site model (PG-BSM) designed to detect evidence of adaptive codon evolution associated with discrete-state phenotype evolution. An episode of adaptation is inferred under standard codon substitution models when there is evidence of positive selection in the form of an elevation in the nonsynonymous-to-synonymous rate ratio $\omega$ to a value $\omega > 1$. As it is becoming increasingly clear that $\omega > 1$ can occur without adaptation, the PG-BSM was formulated to infer an instance of adaptive evolution without appealing to evidence of positive selection. The null model makes use of a covarion-like component to account for general heterotachy (i.e., random changes in the evolutionary rate at a site over time). The alternative model employs samples of the phenotypic evolutionary history to test for phenomenological patterns of heterotachy consistent with specific mechanisms of molecular adaptation. These include 1) a persistent increase/decrease in $\omega$ at a site following a change in phenotype (the pattern) consistent with an increase/decrease in the functional importance of the site (the mechanism); and 2) a transient increase in $\omega$ at a site along a branch over which the phenotype changed (the pattern) consistent with a change in the site's optimal amino acid (the mechanism). Rejection of the null is followed by post hoc analyses to identify sites with strongest evidence for adaptation in association with changes in the phenotype as well as the most likely evolutionary history of the phenotype. Simulation studies based on a novel method for generating mechanistically realistic signatures of molecular adaptation show that the PG-BSM has good statistical properties. Analyses of real alignments show that site patterns identified post hoc are consistent with the specific mechanisms of adaptation included in the alternate model. Further simulation studies show that the covarion-like component of the PG-BSM plays a crucial role in mitigating recently discovered statistical pathologies associated with confounding by accounting for heterotachy-by-any-cause. [Adaptive evolution; branch-site model; confounding; mutation-selection; phenotype-genotype.].

Selective thyroid hormone receptor ß agonists with oxadiazolone acid isosteres.

Use of the oxadiazolone acid isostere in triiodothyronine analogs yielded potent and selective agonists for the thyroid hormone receptor ß. Selected examples showed good in-vivo efficacy in a rat hypercholesterolemic model. One compound was further profiled in a diet-induced mouse model of nonalcoholic steatohepatitis (NASH) and showed robust target engagement and significant histological improvements in both liver steatosis and fibrosis.

Phenomenological Load on Model Parameters Can Lead to False Biological Conclusions.

When a substitution model is fitted to an alignment using maximum likelihood, its parameters are adjusted to account for as much site-pattern variation as possible. A parameter might therefore absorb a substantial quantity of the total variance in an alignment (or more formally, bring about a substantial reduction in the deviance of the fitted model) even if the process it represents played no role in the generation of the data. When this occurs, we say that the parameter estimate carries phenomenological load (PL). Large PL in a parameter estimate is a concern because it not only invalidates its mechanistic interpretation (if it has one) but also increases the likelihood that it will be found to be statistically significant. The problem of PL was not identified in the past because most off-the-shelf substitution models make simplifying assumptions that preclude the generation of realistic levels of variation. In this study, we use the more realistic mutation-selection framework as the basis of a generating model formulated to produce data that mimic an alignment of mammalian mitochondrial DNA. We show that a parameter estimate can carry PL when 1) the substitution model is underspecified and 2) the parameter represents a process that is confounded with other processes represented in the data-generating model. We then provide a method that can be used to identify signal for the process that a given parameter represents despite the existence of PL.

Shifting Balance on a Static Mutation-Selection Landscape: A Novel Scenario of Positive Selection.

A version of the mechanistic mutation-selection (MutSel) model that accounts for temporal dynamics at a site is presented. This is used to show that the rate ratio dN/dS at a site can be transiently >1 even when fitness coefficients are fixed or the fitness landscape is static. This occurs whenever a site drifts away from its fitness peak and is then forced back by selection, a process reminiscent of shifting balance. Shifting balance is strongest when the substitution process is not dominated by selection or drift, but admits interplay between the two. Under this condition, site-specific changes in dN/dS were inferred in 78-100% of trials, and positive selection (i.e., dN/dS>1) in 10-40% of trials, when sequence alignments generated under MutSel were fitted to two popular phenomenological branch-site models. These results demonstrate that positive selection can occur without a change in fitness regime, and that this is detectable by branch-site models. In addition, MutSel is used to show that a site can be occupied by a sub-optimal amino acid for long periods on a fixed landscape when selection is stringent. This has implications for the interpretation of constant-but-different site patterns typically attributed to changes in fitness. Furthermore, a version of MutSel with episodic changes in fitness coefficients is used to illustrate systematic differences between parameters used to generate data under MutSel and their counterparts estimated by a simple codon model. Motivated by a discrepancy in the literature, interpretation of dN/dS in the context of MutSel is also discussed.

The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production.

The hepatitis C virus (HCV) p7 protein is required for infectious virus production via its role in assembly and ion channel activity. Although NMR structures of p7 have been reported, the location of secondary structural elements and orientation of the p7 transmembrane domains differ among models. Furthermore, the p7 structure-function relationship remains unclear. Here, extensive mutagenesis, coupled with infectious virus production phenotyping and molecular modeling, demonstrates that the N-terminal helical region plays a previously underappreciated yet critical functional role, especially with respect to E2/p7 cleavage efficiency. Interrogation of specific N-terminal helix residues identified as having p7-specific defects and predicted to point toward the channel pore, in a context of independent E2/p7 cleavage, further supports p7 as a structurally plastic, minimalist ion channel. Together, our findings indicate that the p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production.

A diverse range of gene products are effectors of the type I interferon antiviral response.

The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.

A genetically humanized mouse model for hepatitis C virus infection.

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.

Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor.

EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.).

Functional subpopulations of V3 interneurons in the mature mouse spinal cord.

V3 interneurons (INs) are a major group of excitatory commissural interneurons in the spinal cord, and they are essential for producing a stable and robust locomotor rhythm. V3 INs are generated from the ventral-most progenitor domain, p3, but migrate dorsally and laterally during postmitotic development. At birth, they are located in distinctive clusters in the ventral horn and deep dorsal horn. To assess the heterogeneity of this genetically identified group of spinal INs, we combined patch-clamp recording and anatomical tracing with cluster analysis. We examined electrophysiological and morphological properties of mature V3 INs identified by their expression of tdTomato fluorescent proteins in Sim1(Cre/+); Rosa(floxstop26TdTom) mice. We identified two V3 subpopulations with distinct intrinsic properties and spatial distribution patterns. Ventral V3 INs, primarily located in lamina VIII, possess a few branching processes and were capable of generating rapid tonic firing spikes. By contrast, dorsal V3 INs exhibited a more complex morphology and relatively slow average spike frequency with strong adaptation, and they also displayed large sag voltages and post-inhibitory rebound potentials. Our data suggested that hyperpolarization-activated cation channel currents and T-type calcium channel currents may account for some of the membrane properties of V3 INs. Finally, we observed that ventral and dorsal V3 INs were active in different ways during running and swimming, indicating that ventral V3 INs may act as premotor neurons and dorsal V3 INs as relay neurons mediating sensory inputs. Together, we detected two physiologically and topographically distinct subgroups of V3 INs, each likely playing different roles in locomotor activities.

Different requirements for scavenger receptor class B type I in hepatitis C virus cell-free versus cell-to-cell transmission.

Hepatitis C virus (HCV) is believed to initially infect the liver through the basolateral side of hepatocytes, where it engages attachment factors and the coreceptors CD81 and scavenger receptor class B type I (SR-BI). Active transport toward the apical side brings the virus in close proximity of additional entry factors, the tight junction molecules claudin-1 and occludin. HCV is also thought to propagate via cell-to-cell spread, which allows highly efficient virion delivery to neighboring cells. In this study, we compared an adapted HCV genome, clone 2, characterized by superior cell-to cell spread, to its parental genome, J6/JFH-1, with the goal of elucidating the molecular mechanisms of HCV cell-to-cell transmission. We show that CD81 levels on the donor cells influence the efficiency of cell-to-cell spread and CD81 transfer between neighboring cells correlates with the capacity of target cells to become infected. Spread of J6/JFH-1 was blocked by anti-SR-BI antibody or in cells knocked down for SR-BI, suggesting a direct role for this receptor in HCV cell-to-cell transmission. In contrast, clone 2 displayed a significantly reduced dependence on SR-BI for lateral spread. Mutations in E1 and E2 responsible for the enhanced cell-to-cell spread phenotype of clone 2 rendered cell-free virus more susceptible to antibody-mediated neutralization. Our results indicate that although HCV can lose SR-BI dependence for cell-to-cell spread, vulnerability to neutralizing antibodies may limit this evolutionary option in vivo. Combination therapies targeting both the HCV glycoproteins and SR-BI may therefore hold promise for effective control of HCV dissemination.

Design and synthesis of lactam-thiophene carboxylic acids as potent hepatitis C virus polymerase inhibitors.

Herein we report the successful incorporation of a lactam as an amide replacement in the design of hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead compound has a favorable pharmacokinetic profile in rats and dogs.

A human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo.

UNLABELLED: Endstage liver disease caused by chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation in the Western world. However, immediate reinfection of the grafted donor liver by circulating virus is inevitable and liver disease progresses much faster than the original disease. Standard antiviral therapy is not well tolerated and usually ineffective in liver transplant patients, whereas anti-HCV immunotherapy is hampered by the extreme genetic diversity of the virus and its ability to spread by way of cell-cell contacts. We generated a human monoclonal antibody against scavenger receptor class B type I (SR-BI), monoclonal antibody (mAb)16-71, which can efficiently prevent infection of Huh-7.5 hepatoma cells and primary hepatocytes by cell-culture-derived HCV (HCVcc). Using an Huh7.5 coculture system we demonstrated that mAb16-71 interferes with direct cell-to-cell transmission of HCV. Finally we evaluated the in vivo efficacy of mAb16-71 in "human liver urokinase-type plasminogen activator, severe combined immune deficiency (uPA-SCID) mice" (chimeric mice). A 2-week anti-SR-BI therapy that was initiated 1 day before viral inoculation completely protected all chimeric mice from infection with serum-derived HCV of different genotypes. Moreover, a 9-day postexposure therapy that was initiated 3 days after viral inoculation (when viremia was already observed in the animals) suppressed the rapid viral spread observed in untreated control animals. After cessation of anti-SR-BI-specific antibody therapy, a rise of the viral load was observed. CONCLUSION: Using in vitro cell culture and human liver-chimeric mouse models, we show that a human mAb targeting the HCV coreceptor SR-BI completely prevents infection and intrahepatic spread of multiple HCV genotypes. This strategy may be an efficacious way to prevent infection of allografts following liver transplantation in chronic HCV patients, and may even hold promise for the prevention of virus rebound during or following antiviral therapy.

Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics.

Hepatitis C virus induces interferon-λ and interferon-stimulated genes in primary liver cultures.

UNLABELLED: Hepatitis C virus (HCV) replication in primary liver cells is less robust than that in hepatoma cell lines, suggesting that innate antiviral mechanisms in primary cells may limit HCV replication or spread. Here we analyzed the expression of 47 genes associated with interferon (IFN) induction and signaling following HCV infection of primary human fetal liver cell (HFLC) cultures from 18 different donors. We report that cell culture-produced HCV (HCVcc) induced expression of Type III (λ) IFNs and of IFN-stimulated genes (ISGs). Little expression of Type I IFNs was detected. Levels of IFNλ and ISG induction varied among donors and, often, between adapted and nonadapted HCV chimeric constructs. Higher levels of viral replication were associated with greater induction of ISGs and of λ IFNs. Gene induction was dependent on HCV replication, as ultraviolet light-inactivated virus was not stimulatory and an antiviral drug, 2'-C-methyladenosine, reduced induction of λ IFNs and ISGs. The level of IFNλ protein induced was sufficient to inhibit HCVcc infection of naïve cultures. CONCLUSION: Together, these results indicate that despite its reported abilities to blunt the induction of an IFN response, HCV infection is capable of inducing antiviral cytokines and pathways in primary liver cell cultures. Induction of ISGs and λ IFNs may limit the growth and spread of HCV in primary cell cultures and in the infected liver. HCV infection of HFLC may provide a useful model for the study of gene induction by HCV in vivo.

Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells.

UNLABELLED: Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well-characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV-infected cells. V protein-transduced HFLC supported enhanced (10 to 100-fold) levels of HCV infection relative to untransduced or control vector-transduced HFLC. Infection was assessed by measurement of virus-driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV-infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein-transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV-inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein-transduced cultures. CONCLUSION: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population.

Genetic analysis of the carboxy-terminal region of the hepatitis C virus core protein.

Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.

Identification of a dominant negative inhibitor of human zinc finger antiviral protein reveals a functional endogenous pool and critical homotypic interactions.

The zinc finger antiviral protein (ZAP) is a host factor with potent antiviral activity when overexpressed in cells. ZAP blocks replication of the prototype alphavirus Sindbis virus (SINV) at a step at or before translation of the incoming viral genome. The mechanism of ZAP anti-SINV activity and the determinants of its antiviral function, however, have not been defined. Here, we have identified a dominant negative inhibitor of human ZAP. Rat ZAP with a cysteine-to-arginine mutation at position 88 (rZAPC88R), previously reported as a nonfunctional form of ZAP, increases SINV growth in cells. These results led us to discover a previously undetectable pool of endogenous functional ZAP within human cells. Investigation of the mechanism of dominant negative inhibition, combined with a comprehensive mutational analysis of the antiviral factor, revealed that homotypic associations are required for ZAP function in limiting SINV propagation.

Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis.

Viruses of the Flaviviridae family, including hepatitis C, dengue and bovine viral diarrhoea, are responsible for considerable morbidity and mortality worldwide. Recent advances in our understanding of virion assembly have uncovered commonalities among distantly related members of this family. We discuss the emerging hypothesis that physical virion components are not alone in forming the infectious particle, but that non-structural proteins are intimately involved in orchestrating morphogenesis. Pinpointing the roles of Flaviviridae proteins in virion production could reveal new avenues for antiviral therapeutics.