Peptide-triggered conformational switch in HIV-1 RRE RNA complexes

We have used NMR spectroscopy to determine the solution structure of a complex between an oligonucleotide derived from stem IIB of the Rev responsive element (RRE-IIB) of HIV-1 mRNA and an in vivo selected, high affinity binding Arg-rich peptide. The peptide binds in a partially alpha-helical conformation into a pocket within the RNA deep groove. Comparison with the structure of a complex between an alpha-helical Rev peptide and RRE-IIB reveals that the sequence of the bound peptide determines the local conformation of the RRE peptide binding site. A conformational switch of an unpaired uridine base was revealed; this points out into the solvent in the Rev peptide complex, but it is stabilized inside the RNA deep groove by stacking with an Arg side chain in the selected peptide complex. The conformational switch has been visualized by NMR chemical shift mapping of the uridine H5/H6 atoms during a competition experiment in which Rev peptide was displaced from RRE-IIB by the higher affinity binding selected peptide.     

Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function

The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5′ leader Ψ elements plus poorly defined additional features. We previously defined minimal 5′ leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5′ leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5′ leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination.     

Functional Magnetic Resonance Imaging Connectivity Analyses Reveal Efference-Copy to Primary Somatosensory Area,BA2

Some theories of motor control suggest efference-copies of motor commands reach somatosensory cortices. Here we used functional magnetic resonance imaging to test these models. We varied the amount of efference-copy signal by making participants squeeze a soft material either actively or passively. We found electromyographical recordings, an efference-copy proxy, to predict activity in primary somatosensory regions, in particular Brodmann Area (BA) 2. Partial correlation analyses confirmed that brain activity in cortical structures associated with motor control (premotor and supplementary motor cortices, the parietal area PF and the cerebellum) predicts brain activity in BA2 without being entirely mediated by activity in early somatosensory (BA3b) cortex. Our study therefore provides valuable empirical evidence for efference-copy models of motor control, and shows that signals in BA2 can indeed reflect an input from motor cortices and suggests that we should interpret activations in BA2 as evidence for somatosensory-motor rather than somatosensory coding alone.     

Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8^−/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and validated evidence for the existence of the protease web, a network that affects the activity of most proteases and thereby influences the functional state of the proteome and cell activity.     

Adaptation of CD8 T Cell Responses to Changing HIV-1 Sequences in a Cohort of HIV-1 Infected Individuals Not Selected for a Certain HLA Allele

HIV evades CD8 T cell mediated pressure by viral escape mutations in targeted CD8 T cell epitopes. A viral escape mutation can lead to a decline of the respective CD8 T cell response. Our question was what happened after the decline of a CD8 T cell response and - in the case of viral escape – if a new CD8 T cell response towards the mutated antigen could be generated in a population not selected for certain HLA alleles. We studied 19 antiretroviral-naïve HIV-1 infected individuals with different disease courses longitudinally. A median number of 12 (range 2-24) CD8 T cell responses towards Gag and Nef were detected per study subject. A total of 30 declining CD8 T cell responses were studied in detail and viral sequence analyses showed amino acid changes in 25 (83%) of these. Peptide titration assays and definition of optimal CD8 T cell epitopes revealed 12 viral escape mutations with one de-novo response (8%). The de-novo response, however, showed less effector functions than the original CD8 T cell response. In addition we identified 4 shifts in immunodominance. For one further shift in immunodominance, the mutations occurred outside the optimal epitope and might represent processing changes. Interestingly, four adaptations to the virus (the de-novo response and 3 shifts in immunodominance) occurred in the group of chronically infected progressors. None of the subjects with adaptation to the changing virus carried the HLA alleles B57, B*58:01 or B27. Our results show that CD8 T cell responses adapt to the mutations of HIV. However it was limited to only 20% (5 out of 25) of the epitopes with viral sequence changes in a cohort not expressing protective HLA alleles.     

Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA

A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure–activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding.     

Functional Redundancy in HIV-1 Viral Particle Assembly

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.     

Functional Analyses of LONELY GUY Cytokinin-Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis

Cytokinins play crucial roles in diverse aspects of plant growth and development. Spatiotemporal distribution of bioactive cytokinins is finely regulated by metabolic enzymes. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice (Oryza sativa) shoot meristems. In this work, nine Arabidopsis thaliana LOG genes (At LOG1 to LOG9) were predicted as homologs of rice LOG. Seven At LOGs, which are localized in the cytosol and nuclei, had enzymatic activities equivalent to that of rice LOG. Conditional overexpression of At LOGs in transgenic Arabidopsis reduced the content of N⁶-(Δ²-isopentenyl)adenine (iP) riboside 5′-phosphates and increased the levels of iP and the glucosides. Multiple mutants of At LOGs showed a lower sensitivity to iP riboside in terms of lateral root formation and altered root and shoot morphology. Analyses of At LOG promoter:β-glucuronidase fusion genes revealed differential expression of LOGs in various tissues during plant development. Ectopic overexpression showed pleiotropic phenotypes, such as promotion of cell division in embryos and leaf vascular tissues, reduced apical dominance, and a delay of leaf senescence. Our results strongly suggest that the direct activation pathway via LOGs plays a pivotal role in regulating cytokinin activity during normal growth and development in Arabidopsis.     

Relationship of HIV-1 Envelope V2 and V3 Sequences of the Primary Isolates to the Viral Phenotype

We examined the relationship between the amino acid sequences of the V2 and V3 regions of the envelope protein and the biological properties of ten human immunodeficiency virus type 1 (HIV-1) primary isolates. The infectivity, cytopathic effect (CPE), and syncytium forming activity of these primary isolates were tested against three T cell lines (CEM, MT2, and MOLT4/CL.8 cells), CD8-depleted peripheral blood mononuclear cells (PBMC), and primary monocyte-derived macrophages (MDM) from seronegative donors. In addition to the viral groups which had the syncytium inducing/T-cell line tropic (SI/TT) phenotype or non-syncytium inducing/non-T cell line tropic (NSI/NT) phenotype (including the NSI/macrophage tropic (NSI/MT) phenotype), there was a group of viruses that infected one or two T cell lines and PBMC but could not mediate syncytium formation. We therefore classified this group of viruses as a non-syncytium inducing/partial T-cell line tropic (NSI/pTT) virus. To investigate the relationship between these viral phenotypes and the sequence variability of the V2 and V3 regions of the envelope, we cloned the viral gene segment and sequenced the individual isolates. The sequence data suggested that the SI/TT type changes in the V3 sequence alone mediate a partial T cell line tropism and mild cytopathic effect and that an isolate became more virulent (SI/TT phenotype) if there were additional changes in the V2 or other regions. On the other hand, sequence changes in the V2 region alone could not mediate phenotypic changes but some additional changes in the other variable regions (for example, V3) might be required for the phenotypic changes in combination with changes in V2. These findings also suggested that amino acid changes in both the V2 and V3 region are required for the development of virulent variants of HIV-1 that outgrow during advanced stages of the disease.     

Conformational Plasticity in the HIV-1 Fusion Peptide Facilitates Recognition by Broadly Neutralizing Antibodies

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Genetic Analyses of HIV-1 env Sequences Demonstrate Limited Compartmentalization in Breast Milk and Suggest Viral Replication within the Breast That Increases with Mastitis

The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na⁺) concentration. The association between milk Na⁺ and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P = 0.621), but Na⁺ was correlated with greater genetic distance between milk and blood HIV-1 populations (P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.Breastfeeding accounts for 30 to 50% of mother-to-child-transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) (38). MTCT through breastfeeding occurs primarily in sub-Saharan Africa, where the use of artificial infant formula is often not feasible because of cost and the associated infant mortality from infections due to the use of unsafe water and the lack of the protective effects of breast milk (19, 38, 51). Numerous strategies to reduce postnatal HIV-1 infection of infants while preserving the advantages of breastfeeding have been evaluated, including maternal use of combination antiretroviral therapy or infant antiretroviral prophylaxis during the period of breastfeeding (5, 25, 26, 30, 40). Understanding the biologic events that increase the concentration of HIV-1 in breast milk is critical to the development and evaluation of interventions to reduce postnatal MTCT.The risk of MTCT is strongly associated with the concentration of HIV-1 in breast milk (28, 46, 47). Although breast milk HIV-1 RNA concentrations correlate with those in plasma, levels in milk are typically 2 log₁₀ lower (15, 24, 43). This suggests that HIV-1 in blood and milk may not mix freely, likely because of the closure of tight junctions between mammary alveolar cells that occurs once milk production is established and before weaning (16). Thus, HIV-1 may evolve in the breast without substantial mixing with blood, i.e., evolving viral variants would become compartmentalized—a phenomenon that has been observed in the central nervous system (50) and in some studies of the genital tract (10, 44, 57). Compartmentalization of HIV-1 variants has been detected in the breast milk of a small number of women (3, 4), but other data suggest that compartmentalization in breast milk may be uncommon (22).Breast inflammation (mastitis) occurs frequently during lactation, most commonly without symptoms. Mastitis is associated with elevations in HIV-1 RNA levels in milk (15, 31, 47, 55), an increase in the number of inflammatory cells in milk, and opening of tight junctions in the mammary epithelium that allows passage of subcellular blood components, of which sodium (Na⁺) serves as a marker (15, 16, 36, 47, 55). Greater permeability of mammary epithelia may allow the passage of free virus from the blood into breast milk, which would result in the mixing of HIV-1 subpopulations from blood and milk. Alternatively, inflammation in the breast may induce replication of virus by HIV-1-infected cells within the breast, which would result in divergence between milk and blood HIV-1 subpopulations. Here we describe detailed genetic analyses of HIV-1 subpopulations in the blood and breast milk to determine whether mastitis affects the structure of these populations and to gain understanding of the processes that may lead to increased concentrations of HIV-1 in milk.     

Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning.     

Limited Nucleotide Changes in the Rev Response Element (RRE) during HIV-1 Infection Alter Overall Rev-RRE Activity and Rev Multimerization

HIV-1 Rev and the Rev response element (RRE) enable a critical step in the viral replication cycle by facilitating the nuclear export of intron-containing mRNAs, yet their activities have rarely been analyzed in natural infections. This study characterized their genetic and functional variation in a small cohort of HIV-infected individuals. Multiple Rev and RRE sequences were obtained using single-genome sequencing (SGS) of plasma samples collected within 6 months after seroconversion and at a later time. This allowed the identification of cognate sequences that were linked in vivo in the same viral genome and acted together as a functional unit. Phylogenetic analyses of these sequences indicated that 4/5 infections were founded by a single transmission event. Rev and RRE variants from each time point were subjected to functional analysis as both cognate pairs and as individual components. While a range of Rev-RRE activities were seen, the activity of cognate pairs from a single time point clustered to a discrete level, which was termed the set point. In 3/5 patients, this set point changed significantly over the time period studied. In all patients, RRE activity was more sensitive to sequence variation than Rev activity and acted as the primary driver of the cognate set point. Selected patient RREs were also shown to have differences in Rev multimerization using gel shift binding assays. Thus, rather than acting as a simple on-off switch or maintaining a constant level of activity throughout infection, the Rev-RRE system can fluctuate, presumably to control replication.     

Systematic Analyses of rpm-1 Suppressors Reveal Roles for ESS-2 in mRNA Splicing in Caenorhabditis elegans

The PHR (Pam/Highwire/RPM-1) family of ubiquitin E3 ligases plays conserved roles in axon patterning and synaptic development. Genetic modifier analysis has greatly aided the discovery of the signal transduction cascades regulated by these proteins. In Caenorhabditis elegans, loss of function in rpm-1 causes axon overgrowth and aberrant presynaptic morphology, yet the mutant animals exhibit little behavioral deficits. Strikingly, rpm-1 mutations strongly synergize with loss of function in the presynaptic active zone assembly factors, syd-1 and syd-2, resulting in severe locomotor deficits. Here, we provide ultrastructural evidence that double mutants, between rpm-1 and syd-1 or syd-2, dramatically impair synapse formation. Taking advantage of the synthetic locomotor defects to select for genetic suppressors, previous studies have identified the DLK-1 MAP kinase cascade negatively regulated by RPM-1. We now report a comprehensive analysis of a large number of suppressor mutations of this screen. Our results highlight the functional specificity of the DLK-1 cascade in synaptogenesis. We also identified two previously uncharacterized genes. One encodes a novel protein, SUPR-1, that acts cell autonomously to antagonize RPM-1. The other affects a conserved protein ESS-2, the homolog of human ES2 or DGCR14. Loss of function in ess-2 suppresses rpm-1 only in the presence of a dlk-1 splice acceptor mutation. We show that ESS-2 acts to promote accurate mRNA splicing when the splice site is compromised. The human DGCR14/ES2 resides in a deleted chromosomal region implicated in DiGeorge syndrome, and its mutation has shown high probability as a risk factor for schizophrenia. Our findings provide the first functional evidence that this family of proteins regulate mRNA splicing in a context-specific manner.