Validation of digital droplet PCR assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA for clinical studies in HIV-1 cure research.

BACKGROUND: Cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA (usRNA) are important virological parameters for monitoring HIV-1 persistence and activation of latent HIV-1. Assays fully validated by CLIA and/or GCLP standards are needed for future clinical trials that seek to evaluate treatments directed towards HIV-1 cure. OBJECTIVES: To determine performance characteristics of sensitive, moderate-throughput, digital droplet PCR (ddPCR) assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA that can detect a broad range of HIV-1 M-group subtypes. STUDY DESIGN: To evaluate linearity, limit of detection, precision, and accuracy of each assay, contrived specimens were analyzed in a background of uninfected PBMC. Detection breadth was evaluated by in silico analysis of primer and probes sets and analysis of material harvested from PBMC infected in vitro with various HIV-1 subtypes. A cohort of clinical specimens from viremic and virologically suppressed individuals was analyzed to demonstrate applicability to clinical research. RESULTS: The empirically determined limit of detection of these assays was 29, 7, and 60 copies per million PBMC for HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA, respectively. The assays detect a broad range of HIV-1 M-group subtypes. Finally, analysis of clinical specimens demonstrate that these assays can detect low levels of cell-associated HIV-1 DNA, HIV-1 usRNA, and HIV-1 2-LTR circle and correlate with clinical histories and viral loads of untreated and antiretroviral treated individuals. CONCLUSIONS: We report the clinical validation of three HIV reservoir assays with broad HIV-1 coverage for future cure studies.

Austere Diagnosis and Reduction of Anterior Shoulder Dislocations: 10-Year Review of a Ski Patrol-Based Program with Emergency Medical Technicians.

INTRODUCTION: Glenohumeral dislocations present a challenging management dilemma in austere settings where patient transport time may be prolonged. Expeditious reduction is preferable, but treatment is commonly expected to take place within a hospital or comparable facility. Through a novel shoulder injury program, professional ski patrollers trained as emergency medical technicians (EMTs) have diagnosed and reduced anterior shoulder dislocations using biomechanical techniques without sedation or analgesia for over 20 y. Summary records have been maintained to track the performance and safety of this program. METHODS: Five hundred forty-six records of winter sports-related shoulder injuries from 2009-10 to 2019-20 were retrospectively analyzed to determine the assessment performance and dislocation reduction efficacy of EMTs, with the intent of ascertaining whether EMT-trained practitioners can reliably and safely diagnose and reduce anterior shoulder dislocations without premedication in a remote and resource-limited setting. RESULTS: EMTs identified anterior shoulder dislocations with 98% sensitivity and 96% diagnostic accuracy. The overall success rate of reduction attempts was 86%, or 88% when limited to confirmed anterior dislocations. Two fracture-dislocations and 4 misdiagnoses were manipulated. No instances of iatrogenic harm were identified, and no patients who underwent successful reductions required ambulance transportation. CONCLUSIONS: With appropriate education and within a structured program, EMT-trained practitioners can reliably and safely diagnose and reduce anterior shoulder dislocations using biomechanical techniques without premedication in remote and resource-limited environments. Implementation of similar programs in austere settings has the potential to improve patient care. Further, using biomechanical reduction techniques may reduce reliance on procedural sedation irrespective of care setting.

A Pan-respiratory Antiviral Chemotype Targeting a Transient Host Multiprotein Complex.

We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease. One Sentence Summary: A host-targeted drug to treat all respiratory viruses without viral resistance development.

Reflections on the physical, executive developmental and systems applied framework in child neuropsychological rehabilitation.

This paper describes the influence of the Physical, Executive, Developmental and Systems (PEDS) framework on the delivery of community-based child neuropsychological rehabilitation and how it has been enhanced by the proliferation of neuroscientific, neuropsychological and psychosocial research and evidence-base in childhood brain injury and rehabilitation over the past decade. The paper signposts to some of the key models, theories and concepts currently shaping service delivery. Application of the PEDS framework in a clinical case is described.

Prediction of Neural Diameter From Morphology to Enable Accurate Simulation.

Accurate neuron morphologies are paramount for computational model simulations of realistic neural responses. Over the last decade, the online repository NeuroMorpho.Org has collected over 140,000 available neuron morphologies to understand brain function and promote interaction between experimental and computational research. Neuron morphologies describe spatial aspects of neural structure; however, many of the available morphologies do not contain accurate diameters that are essential for computational simulations of electrical activity. To best utilize available neuron morphologies, we present a set of equations that predict dendritic diameter from other morphological features. To derive the equations, we used a set of NeuroMorpho.org archives with realistic neuron diameters, representing hippocampal pyramidal, cerebellar Purkinje, and striatal spiny projection neurons. Each morphology is separated into initial, branching children, and continuing nodes. Our analysis reveals that the diameter of preceding nodes, Parent Diameter, is correlated to diameter of subsequent nodes for all cell types. Branching children and initial nodes each required additional morphological features to predict diameter, such as path length to soma, total dendritic length, and longest path to terminal end. Model simulations reveal that membrane potential response with predicted diameters is similar to the original response for several tested morphologies. We provide our open source software to extend the utility of available NeuroMorpho.org morphologies, and suggest predictive equations may supplement morphologies that lack dendritic diameter and improve model simulations with realistic dendritic diameter.

A SARS-CoV-2 Nucleocapsid Variant that Affects Antigen Test Performance.

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

Addressing Antiretroviral Drug Resistance with Host-Targeting Drugs-First Steps towards Developing a Host-Targeting HIV-1 Assembly Inhibitor.

The concerning increase in HIV-1 resistance argues for prioritizing the development of host-targeting antiviral drugs because such drugs can offer high genetic barriers to the selection of drug-resistant viral variants. Targeting host proteins could also yield drugs that act on viral life cycle events that have proven elusive to inhibition, such as intracellular events of HIV-1 immature capsid assembly. Here, we review small molecule inhibitors identified primarily through HIV-1 self-assembly screens and describe how all act either narrowly post-entry or broadly on early and late events of the HIV-1 life cycle. We propose that a different screening approach could identify compounds that specifically inhibit HIV-1 Gag assembly, as was observed when a potent rabies virus inhibitor was identified using a host-catalyzed rabies assembly screen. As an example of this possibility, we discuss an antiretroviral small molecule recently identified using a screen that recapitulates the host-catalyzed HIV-1 capsid assembly pathway. This chemotype potently blocks HIV-1 replication in T cells by specifically inhibiting immature HIV-1 capsid assembly but fails to select for resistant viral variants over 37 passages, suggesting a host protein target. Development of such small molecules could yield novel host-targeting antiretroviral drugs and provide insight into chronic diseases resulting from dysregulation of host machinery targeted by these drugs.

Identification of an Antiretroviral Small Molecule That Appears To Be a Host-Targeting Inhibitor of HIV-1 Assembly.

Given the projected increase in multidrug-resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life cycle stages not targeted by drugs currently in use. Host-targeting compounds are of particular interest because they can offer a high barrier to resistance. Here, we report identification of two related small molecules that inhibit HIV-1 late events, a part of the HIV-1 life cycle for which potent and specific inhibitors are lacking. This chemotype was discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular host-catalyzed viral capsid assembly pathways. These compounds inhibit replication of HIV-1 in human T cell lines and peripheral blood mononuclear cells, and are effective against a primary isolate. They reduce virus production, likely by inhibiting a posttranslational step in HIV-1 Gag assembly. Notably, the compound colocalizes with HIV-1 Gag in situ; however, unexpectedly, selection experiments failed to identify compound-specific resistance mutations in gag or pol, even though known resistance mutations developed upon parallel nelfinavir selection. Thus, we hypothesized that instead of binding to Gag directly, these compounds localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that form during immature HIV-1 capsid assembly. Indeed, imaging of infected cells shows compound colocalized with two host enzymes found in assembly intermediates, ABCE1 and DDX6, but not two host proteins found in other complexes. While the exact target and mechanism of action of this chemotype remain to be determined, our findings suggest that these compounds represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly.IMPORTANCE The success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including virus-host interactions that promote assembly. This effort led to the identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations, likely by acting on assembly. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly. However, resistance selection did not result in compound-specific mutations in gag, suggesting that the chemotype does not directly target Gag. We hypothesize that this chemotype represents a first-in-class inhibitor of virus production that acts by targeting a virus-host complex important for HIV-1 Gag assembly.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules.IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies.

Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

UNLABELLED: The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. IMPORTANCE: The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues in the major homology region are required for assembly of retroviruses; however, when these residues are required during assembly is not clear. Here, we used biochemical and electron microscopic analyses to demonstrate that these conserved residues function after assembling HIV-1 Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane but before Gag has completed the assembly process. By revealing precisely when conserved residues in the major homology region are required during assembly, these studies resolve existing controversies and set the stage for future experiments aimed at a more complete understanding of how the major homology region functions.

How HIV-1 Gag assembles in cells: Putting together pieces of the puzzle.

During the late stage of the viral life cycle, HIV-1 Gag assembles into a spherical immature capsid, and undergoes budding, release, and maturation. Here we review events involved in immature capsid assembly from the perspective of five different approaches used to study this process: mutational analysis, structural studies, assembly of purified recombinant Gag, assembly of newly translated Gag in a cell-free system, and studies in cells using biochemical and imaging techniques. We summarize key findings obtained using each approach, point out where there is consensus, and highlight unanswered questions. Particular emphasis is placed on reconciling data suggesting that Gag assembles by two different paths, depending on the assembly environment. Specifically, in assembly systems that lack cellular proteins, high concentrations of Gag can spontaneously assemble using purified nucleic acid as a scaffold. However, in the more complex intracellular environment, barriers that limit self-assembly are present in the form of cellular proteins, organelles, host defenses, and the absence of free nucleic acid. To overcome these barriers and promote efficient immature capsid formation in an unfavorable environment, Gag appears to utilize an energy-dependent, host-catalyzed, pathway of assembly intermediates in cells. Overall, we show how data obtained using a variety of techniques has led to our current understanding of HIV assembly.

A temporospatial map that defines specific steps at which critical surfaces in the Gag MA and CA domains act during immature HIV-1 capsid assembly in cells.

UNLABELLED: During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCE: While much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used biochemical and ultrastructural analyses to generate a temporospatial map showing the precise order in which four critical surfaces in Gag act during immature capsid formation in provirus-expressing cells. Because three of these surfaces make important contacts in the hexameric lattices that are found in the completed immature capsid, these data allow us to propose a model for the sequence of events leading to formation of the hexameric lattices. By providing a dynamic view of when and where critical Gag-Gag contacts form during the assembly process and how those contacts function in the nascent capsid, our study provides novel insights into how an immature capsid is built in infected cells.

HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly.

To produce progeny virus, human immunodeficiency virus type I (HIV-1) Gag assembles into capsids that package the viral genome and bud from the infected cell. During assembly of immature capsids, Gag traffics through a pathway of assembly intermediates (AIs) that contain the cellular adenosine triphosphatase ABCE1 (ATP-binding cassette protein E1). In this paper, we showed by coimmunoprecipitation and immunoelectron microscopy (IEM) that these Gag-containing AIs also contain endogenous processing body (PB)-related proteins, including AGO2 and the ribonucleic acid (RNA) helicase DDX6. Moreover, we found a similar complex containing ABCE1 and PB proteins in uninfected cells. Additionally, knockdown and rescue studies demonstrated that the RNA helicase DDX6 acts enzymatically to facilitate capsid assembly independent of RNA packaging. Using IEM, we localized the defect in DDX6-depleted cells to Gag multimerization at the plasma membrane. We also confirmed that DDX6 depletion reduces production of infectious HIV-1 from primary human T cells. Thus, we propose that assembling HIV-1 co-opts a preexisting host complex containing cellular facilitators such as DDX6, which the virus uses to catalyze capsid assembly.

HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag.

During HIV-1 assembly, Gag polypeptides multimerize to form an immature capsid and also package HIV-1 genomic RNA. Assembling Gag forms immature capsids by progressing through a stepwise pathway of assembly intermediates containing the cellular ATPase ABCE1, which facilitates capsid formation. The NC domain of Gag is required for ABCE1 binding, acting either directly or indirectly. NC is also critical for Gag multimerization and RNA binding. Previous studies of GagZip chimeric proteins in which NC was replaced with a heterologous leucine zipper that promotes protein dimerization but not RNA binding established that the RNA binding properties of NC are dispensable for capsid formation per se. Here we utilized GagZip proteins to address the question of whether the RNA binding properties of NC are required for ABCE1 binding and for the formation of ABCE1-containing capsid assembly intermediates. We found that assembly-competent HIV-1 GagZip proteins formed ABCE1-containing intermediates, while assembly-incompetent HIV-1 GagZip proteins harboring mutations in residues critical for leucine zipper dimerization did not. Thus, these data suggest that ABCE1 does not bind to NC directly or through an RNA bridge, and they support a model in which dimerization of Gag, mediated by NC or a zipper, results in exposure of an ABCE1-binding domain located elsewhere in Gag, outside NC. Additionally, we demonstrated that immature capsids formed by GagZip proteins are insensitive to RNase A, as expected. However, unexpectedly, immature HIV-1 capsids were almost as insensitive to RNase A as GagZip capsids, suggesting that RNA is not a structural element holding together immature wild-type HIV-1 capsids.

Taking a developmental and systemic perspective on neuropsychological rehabilitation with children with brain injury and their families.

This paper reviews current approaches and models in the neuropsychological rehabilitation of children following Traumatic Brain Injury (TBI) and cites a large evidence base for a developmental and family systems perspective to neuropsychological rehabilitation of children post-TBI. The paper describes the application and efficacy of a systemic and developmental perspective to rehabilitation of children with TBI, with a clinical case example of this approach. The paper describes the development of a community-based child neuropsychology rehabilitation service integrating applied developmental neuropsychology and systems ideas as a way of working effectively with children with TBI and their families.

Intracellular destinies: degradation, targeting, assembly, and endocytosis of HIV Gag.

The HIV-1 Gag protein assembles into immature capsids when expressed in human cells. Although self-assembly of Gag was once thought to be sufficient to explain capsid formation, in the past decade it has become increasingly apparent that in cells, the pathway from Gag synthesis to assembled capsids is coordinated and facilitated by host factors. These cellular factors likely direct the trafficking, membrane targeting, and multimerization of Gag, and could also assist with encapsidation of viral RNA. While some of these factors have been identified, much remains to be learned about the mechanisms by which they act to promote capsid formation. Moreover, studies suggest that the amount of intracellular Gag undergoing assembly per se at any given time may be quite low, with the majority of Gag in some cell types undergoing degradation or representing Gag that remains cell-associated after assembly. If this model holds true, then defining the Gag subpopulations on which individual cellular factors act will be important for understanding the rqle of host factors. Towards this end, it will be important to find markers and features that can distinguish subpopulations of Gag destined for different outcomes so that these populations can be quantified and tracked separately both at the biochemical and microscopic level. Thus, the challenge for the future will be to understand which cellular factors act during the pathway from Gag synthesis to assembly, and exactly where and how they act in this pathway.

Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production.

In primate cells, assembly of a single HIV-1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV-1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation. In the next approximately 2 h, Gag-ABCE1 complexes increase in size to approximately that of immature capsids. Dissociation of ABCE1 from Gag correlates closely with Gag processing during virion maturation and occurs much less efficiently when the HIV-1 protease is inactivated. Finally, quantitative double-label immunogold electron microscopy reveals that ABCE1 is recruited to sites of assembling wild-type Gag at the plasma membrane but not to sites of an assembly-defective Gag mutant at the plasma membrane. Together these findings demonstrate that a population of Gag present at plasma membrane sites of assembly associates with ABCE1 throughout capsid formation until the onset of virus maturation, which is then followed by virus release. Moreover, the data suggest a linkage between Gag-ABCE1 dissociation and subsequent events of virion production.

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon.

Launching the Beet yellows virus (BYV) minireplicon by agrobacterial delivery resulted in an unexpectedly low number of infected cells per inoculated leaf. This effect was due to a strong RNA silencing response in the agroinfiltrated leaves. Strikingly, ectopic co-expression of p21, a BYV RNA silencing suppressor, increased minireplicon infectivity by three orders of magnitude. Mutational analysis demonstrated that this effect correlates with suppressor activity of p21. Five diverse, heterologous viral suppressors were also active in this system, providing a useful approach for a dramatic, up to 10,000-fold, increase of the efficiency of agroinfection. The minireplicon agroinfection assay was also used to identify a new suppressor, a homolog of BYV p21, derived from Grapevine leafroll-associated virus-2. In addition, we report preliminary data on the suppressor activity of the p10 protein of Grapevine virus A and show that this protein belongs to a family of Zn-ribbon-containing proteins encoded by filamentous plant RNA viruses from three genera. The members of this family are predicted to have RNA silencing suppressor activity.

Human implicit memory for irrelevant dimension values is similar to rats' incidental memory in simultaneous discrimination tasks.

Participants completed a category-learning task in which they needed to discover which of three stimulus dimensions (shape, color or size) was relevant. After meeting a learning criterion (nine of 10 consecutive correct responses), participants continued making categorization choices and response latencies associated with these trials were examined. In both Experiments 1 and 2, people responded reliably faster when correct responses matched the previous responses with respect to irrelevant dimension values. Thus, they demonstrated a form of incidental short-term memory analogous to that we previously reported in studies of rats. In Experiment 2, participants' explicit memory for irrelevant dimension values was assessed after category learning was complete. The results indicated that people were unaware of the irrelevant dimension values encountered on trials preceding surprise probe trials. This indicates that memory for the irrelevant dimension values was implicit (i.e. unconscious). The findings are discussed with respect to both human and non-human studies of hippocampus-independent memory and implicit memory.