Motor, mood, and memory impairments persist during remission periods in chronic colitis and are influenced by neuroinflammation and sex.

Ulcerative colitis is a chronic pathology characterized by relapsing-remitting phases of intestinal inflammation. Additionally, some patients develop neuropsychiatric disorders, such as depression and anxiety, or cognitive deficits. We aimed to investigate whether the development of chronic colitis elicits memory, locomotion, and mood impairments. It further examined whether these impairments are influenced by the relapsing-remitting phases of the colitis or by sex. Here, we used a chronic colitis model in male and female rats, induced with sodium dextran sulfate, mirroring the phases of human ulcerative colitis. Our results revealed that the severity of colitis was slightly higher in males than females. Chronic colitis triggered motor and short-term memory deficits and induced anxiety- and depression-like behaviors that remained throughout the development of the disease. There are also sex differences under control or inflammatory conditions. Therefore, in both situations, females compared to males displayed: (i) slightly lower locomotion, (ii) increased anxiety-like behaviors, (iii) similar depression-like behaviors, and (iv) similar short-term memory deficit. Additionally, under control conditions, the mRNA levels of IL-1ß, IL-6, and TNF-α were higher in the female hippocampus. In both sexes, when chronic colitis was established, the neuroinflammation was evidenced by increased mRNA levels of these three cytokines in the hippocampus and in the motor and prefrontal cortices. Interestingly, this neuroinflammation was slightly greater in males. In summary, we show that the development of chronic colitis caused persistent behavioral abnormalities, highlighting sex differences, and that could be a consequence, at least in part, of the increase in IL-1ß, IL-6, and TNF-α in the brain.

Special Issue "Aquaporins in Brain Disease".

Water is an abundant and important component of the human brain, the homeostasis of which is rigorously controlled [...].

Inflammatory bowel disease induces pathological α-synuclein aggregation in the human gut and brain.

AIMS: According to Braak's hypothesis, it is plausible that Parkinson's disease (PD) originates in the enteric nervous system (ENS) and spreads to the brain through the vagus nerve. In this work, we studied whether inflammatory bowel diseases (IBDs) in humans can progress with the emergence of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. METHODS: We have analysed the gut and the ventral midbrain from subjects previously diagnosed with IBD and form a DSS-based rat model of gut inflammation in terms of α-syn pathology. RESULTS: Our data support the existence of pathogenic α-syn in both the gut and the brain, thus reinforcing the potential role of the ENS as a contributing factor in PD aetiology. Additionally, we have analysed the effect of a DSS-based rat model of gut inflammation to demonstrate (i) the appearance of P-α-syn inclusions in both Auerbach's and Meissner's plexuses (gut), (ii) an increase in α-syn expression in the ventral mesencephalon (brain) and (iii) the degeneration of nigral dopaminergic neurons, which all are considered classical hallmarks in PD. CONCLUSION: These results strongly support the plausibility of Braak's hypothesis and emphasise the significance of peripheral inflammation and the gut-brain axis in initiating α-syn aggregation and transport to the substantia nigra, resulting in neurodegeneration.

Reelin Protects against Colon Pathology via p53 and May Be a Biomarker for Colon Cancer Progression.

Previous observations made in human and mouse colons suggest that reelin protects the colon from pathology. In this study, we evaluated reelin expression during the transition from either colitis or precancerous lesions to colon cancer and tried to elucidate reelin regulation under these transition processes. Samples of healthy and pathological colons from humans and mice treated with either azoxymethane/dextran sulfate sodium (DSS) or azoxymethane alone were used. The relative abundances of reelin, DNMT-1 and ApoER2 mRNAs were determined by PCR in the colon samples cited above and in the tissue adjacent to mouse colon polyps and adenocarcinomas. In both, humans and mice, reelin mRNA abundance increased significantly in ulcerative colitis and slightly in polyps and decreased in adenomas and adenocarcinomas. Reelin expression was higher in the tissue adjacent to the colon adenocarcinoma and lower in the lesion itself. The reelin expression changes may result, at least in part, from those in DNMT-1 and appear to be independent of ApoER2. Lack of reelin downregulated p-Akt and p53 in healthy colon and prevented their increases in the inflamed colon, whereas it increased GSK-3ß in DSS-untreated mice. In conclusion, reelin mRNA abundance depends on the severity of the colon pathology, and its upregulation in response to initial injuries might prevent the beginning of colon cancer, whereas reelin repression favors it. Increased p53 expression and activation may be involved in this protection. We also propose that changes in colon reelin abundance could be used to predict colon pathology progression.

Acute Colon Inflammation Triggers Primary Motor Cortex Glial Activation, Neuroinflammation, Neuronal Hyperexcitability, and Motor Coordination Deficits.

Neuroinflammation underlies neurodegenerative diseases. Herein, we test whether acute colon inflammation activates microglia and astrocytes, induces neuroinflammation, disturbs neuron intrinsic electrical properties in the primary motor cortex, and alters motor behaviors. We used a rat model of acute colon inflammation induced by dextran sulfate sodium. Inflammatory mediators and microglial activation were assessed in the primary motor cortex by PCR and immunofluorescence assays. Electrophysiological properties of the motor cortex neurons were determined by whole-cell patch-clamp recordings. Motor behaviors were examined using open-field and rotarod tests. We show that the primary motor cortex of rats with acute colon inflammation exhibited microglial and astrocyte activation and increased mRNA abundance of interleukin-6, tumor necrosis factor-alpha, and both inducible and neuronal nitric oxide synthases. These changes were accompanied by a reduction in resting membrane potential and rheobase and increased input resistance and action potential frequency, indicating motor neuron hyperexcitability. In addition, locomotion and motor coordination were impaired. In conclusion, acute colon inflammation induces motor cortex microglial and astrocyte activation and inflammation, which led to neurons' hyperexcitability and reduced motor coordination performance. The described disturbances resembled some of the early features found in amyotrophic lateral sclerosis patients and animal models, suggesting that colon inflammation might be a risk factor for developing this disease.

Fecal Volatile Organic Compounds and Microbiota Associated with the Progression of Cognitive Impairment in Alzheimer's Disease.

Metabolites produced by an altered gut microbiota might mediate the effects in the brain. Among metabolites, the fecal volatile organic compounds (VOCs) are considered to be potential biomarkers. In this study, we examined both the VOCs and bacterial taxa in the feces from healthy subjects and Alzheimer's disease (AD) patients at early and middle stages. Remarkably, 29 fecal VOCs and 13 bacterial genera were differentiated from the healthy subjects and the AD patients. In general, higher amounts of acids and esters were found in in the feces of the AD patients and terpenes, sulfur compounds and aldehydes in the healthy subjects. At the early stage of AD, the most relevant VOCs with a higher abundance were short-chain fatty acids and their producing bacteria, Faecalibacterium and Lachnoclostridium. Coinciding with the development of dementia in the AD patients, parallel rises of heptanoic acid and Peptococcus were observed. At a more advanced stage of AD, the microbiota and volatiles shifted towards a profile in the feces with increases in hexanoic acid, Ruminococcus and Blautia. The most remarkable VOCs that were associated with the healthy subjects were 4-ethyl-phenol and dodecanol, together with their possible producers Clostridium and Coprococcus. Our results revealed a VOCs and microbiota crosstalk in AD development and their profiles in the feces were specific depending on the stage of AD. Additionally, some of the most significant fecal VOCs identified in our study could be used as potential biomarkers for the initiation and progression of AD.

Perturbing HIV-1 Ribosomal Frameshifting Frequency Reveals a cis Preference for Gag-Pol Incorporation into Assembling Virions.

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks" scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a "weighted Goldilocks" scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.

Evaluation of aquaporins in the cerebrospinal fluid in patients with idiopathic normal pressure hydrocephalus.

Brain aquaporin 1 (AQP1) and AQP4 are involved in cerebrospinal fluid (CSF) homeostasis and might participate in the origin of hydrocephalus. Studies have shown alterations of perivascular AQP4 expression in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD). Due to the overlapping of clinical signs between iNPH and certain neurological conditions, mainly AD, specific biomarkers might improve the diagnostic accuracy for iNPH. The goal of the present study was to analyze and quantify the presence of AQP1 and AQP4 in the CSF of patients with iNPH and AD to determine whether these proteins can be used as biomarkers of iNPH. We examined AQP1 and AQP4 protein levels in the CSF of 179 participants (88 women) classified into 5 groups: possible iNPH (81 participants), hydrocephalus associated with other neurological disorders (13 participants), AD (41 participants), non-AD dementia (32 participants) and healthy controls (12 participants). We recorded each participant's demographic and clinical variables and indicated, when available in the clinical history, the record of cardiovascular and respiratory complications. An ELISA showed virtually no AQP content in the CSF. Information on the vascular risk factors (available for 61 patients) confirmed some type of vascular risk factor in 86% of the patients with possible iNPH and 58% of the patients with AD. In conclusion, the ELISA analysis showed insufficient sensitivity to detect the presence of AQP1 and AQP4 in CSF, ruling out the possible use of these proteins as biomarkers for diagnosing iNPH.

Galectin-3 Deletion Reduces LPS and Acute Colitis-Induced Pro-Inflammatory Microglial Activation in the Ventral Mesencephalon.

Parkinson's disease is a highly prevalent neurological disorder for which there is currently no cure. Therefore, the knowledge of risk factors as well as the development of new putative molecular targets is mandatory. In this sense, peripheral inflammation, especially the originated in the colon, is emerging as a predisposing factor for suffering this disease. We have largely studied the pleiotropic roles of galectin-3 in driving microglia-associated immune responses. However, studies aimed at elucidating the role of galectin-3 in peripheral inflammation in terms of microglia polarization are lacking. To achieve this, we have evaluated the effect of galectin-3 deletion in two different models of acute peripheral inflammation: intraperitoneal injection of lipopolysaccharide or gut inflammation induced by oral administration of dextran sodium sulfate. We found that under peripheral inflammation the number of microglial cells and the expression levels of pro-inflammatory mediators take place specifically in the dopaminergic system, thus supporting causative links between Parkinson's disease and peripheral inflammation. Absence of galectin-3 highly reduced neuroinflammation in both models, suggesting an important central regulatory role of galectin-3 in driving microglial activation provoked by the peripheral inflammation. Thus, modulation of galectin-3 function emerges as a promising strategy to minimize undesired microglia polarization states.

Aquaporin-4 Removal from the Plasma Membrane of Human Müller Cells by AQP4-IgG from Patients with Neuromyelitis Optica Induces Changes in Cell Volume Homeostasis: the First Step of Retinal Injury?

Aquaporin-4 (AQP4) is the target of the specific immunoglobulin G autoantibody (AQP4-IgG) produced in patients with neuromyelitis optica spectrum disorders (NMOSD). Previous studies demonstrated that AQP4-IgG binding to astrocytic AQP4 leads to cell-destructive lesions. However, the early physiopathological events in Müller cells in the retina are poorly understood. Here, we investigated the consequences of AQP4-IgG binding to AQP4 of Müller cells, previous to the inflammatory response, on two of AQP4's key functions, cell volume regulation response (RVD) and cell proliferation, a process closely associated with changes in cell volume. Experiments were performed in a human retinal Müller cell line (MIO-M1) exposed to complement-inactivated sera from healthy volunteers or AQP4-IgG positive NMOSD patients. We evaluated AQP4 expression (immunofluorescence and western blot), water permeability coefficient, RVD, intracellular calcium levels and membrane potential changes during hypotonic shock (fluorescence videomicroscopy) and cell proliferation (cell count and BrdU incorporation). Our results showed that AQP4-IgG binding to AQP4 induces its partial internalization, leading to the decrease of the plasma membrane water permeability, a reduction of swelling-induced increase of intracellular calcium levels and the impairment of RVD in Müller cells. The loss of AQP4 from the plasma membrane induced by AQP4-IgG positive sera delayed Müller cells' proliferation rate. We propose that Müller cell dysfunction after AQP4 removal from the plasma membrane by AQP4-IgG binding could be a non-inflammatory mechanism of retinal injury in vivo, altering cell volume homeostasis and cell proliferation and consequently, contributing to the physiopathology of NMOSD.

Proper E-cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer.

We reported that Disabled-2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E-cadherin. Dab2 depletion in Caco-2 cells led to E-cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium-induced colitis increased Dab2/E-cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E-cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E-cadherin delocalization. Epithelial Dab2 decrement preceded that of E-cadherin. We suggest that Dab2, by inhibiting E-cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.

Predictive Value of Serum Antibodies and Point Mutations of AQP4, AQP1 and MOG in A Cohort of Spanish Patients with Neuromyelitis Optica Spectrum Disorders.

The detection of IgG aquaporin-4 antibodies in the serum of patients with Neuromyelitis optica (NMO) has dramatically improved the diagnosis of this disease and its distinction from multiple sclerosis. Recently, a group of patients have been described who have an NMO spectrum disorder (NMOsd) and who are seronegative for AQP4 antibodies but positive for IgG aquaporin-1 (AQP1) or myelin oligodendrocyte glycoprotein (MOG) antibodies. The purpose of this study was to determine whether AQP1 and MOG could be considered new biomarkers of this disease; and if point mutations in the gDNA of AQP4, AQP1 and MOG genes could be associated with the etiology of NMOsd. We evaluated the diagnostic capability of ELISA and cell-based assays (CBA), and analyzed their reliability, specificity, and sensitivity in detecting antibodies against these three proteins. The results showed that both assays can recognize these antigen proteins under appropriate conditions, but only anti-AQP4 antibodies, and not AQP1 or MOG, appears to be a clear biomarker for NMOsd. CBA is the best method for detecting these antibodies; and serum levels of AQP4 antibodies do not correlate with the progression of this disease. So far, the sequencing analysis has not revealed a genetic basis for the etiology of NMOsd, but a more extensive analysis is required before definitive conclusions can be drawn.

Small and large intestine express a truncated Dab1 isoform that assembles in cell-cell junctions and co-localizes with proteins involved in endocytosis.

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location.

Reelin protects from colon pathology by maintaining the intestinal barrier integrity and repressing tumorigenic genes.

We previously reported that reelin, an extracellular matrix protein first known for its key role in neuronal migration, reduces the susceptibility to dextran sulphate sodium (DSS)-colitis. The aim of the current study was to determine whether reelin protects from colorectal cancer and how reelin defends from colon pathology. In the colon of wild-type and of mice lacking reelin (reeler mice) we have analysed the: i) epithelium cell renewal processes, ii) morphology, iii) Sox9, Cdx2, Smad5, Cyclin D1, IL-6 and IFNγ mRNA abundance in DSS-treated and untreated mice, and iv) development of azoxymethane/DSS-induced colorectal cancer, using histological and real time-PCR methodologies. The reeler mutation increases colitis-associated tumorigenesis, with increased tumours number and size. It also impairs the intestinal barrier because it reduces cell proliferation, migration, differentiation and apoptosis; decreases the number and maturation of goblet cells, and expands the intercellular space of the desmosomes. The intestinal barrier impairment might explain the increased susceptibility to colon pathology exhibited by the reeler mice and is at least mediated by the down-regulation of Sox9 and Cdx2. In response to DSS-colitis, the reeler colon increases the mRNA abundance of IL-6, Smad5 and Cyclin D1 and decreases that of IFNγ, conditions that might result in the increased colitis-associated tumorigenesis found in the reeler mice. In conclusion, the results highlight a role for reelin in maintaining intestinal epithelial cell homeostasis and providing resistance against colon pathology.

Reelin expression is up-regulated in mice colon in response to acute colitis and provides resistance against colitis.

Reelin is an extracellular matrix protein first known for its key role in neuronal migration. Studies in rodent small intestine suggested that reelin protects the organism from intestinal pathology. Here we determined in mice colon, by real time-PCR and immunological assays, the expression of the reelin signalling system; its response to dextran sulphate sodium (DSS) and the response of wild-type and reeler mice to DSS-treatment. DNA methylation was determined by bisulfite modification and sequencing of genomic DNA. In the colon mucosa reelin expression is restricted to the myofibroblasts, whereas both epithelial cells and myofibroblasts express reelin receptors (ApoER2 and VLDLR) and its effector protein Dab1. The muscle layer also expresses reelin. DSS-treatment reduces reelin expression in the muscle but it is activated in the mucosa. Activation of mucosal reelin is greater in magnitude and is delayed until after the activation of the myofibroblasts marker, α-SMA. This indicates that the DSS-induced reelin up-regulation results from changes in the reelin gene expression rather than from myofibroblasts proliferation. DSS-treatment does not modify Sp1 or Tbr1 mRNA abundance, but increases that of TGF-ß1 and ApoER2, decreases that of CASK and DNMT1 and it also decreases the reelin promoter methylation. Finally, the reeler mice exhibit higher inflammatory scores than wild-type mice, indicating that the mutation increases the susceptibility to DSS-colitis. In summary, this data are the first to demonstrate that mouse distal colon increases reelin production in response to DSS-colitis via a DNMT1-dependent hypo-methylation of the gene promoter region and that reelin provides protection against colitis.

Reelin-Dab1 signaling system in human colorectal cancer.

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-ß1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers. © 2016 Wiley Periodicals, Inc.

The Synaptojanins in the murine small and large intestine.

The expression of the phosphoinositides phosphatases Synaptojanins (Synjs) 1 and 2 has been shown in brain and in some peripheral tissues, but their expression in the intestine has not been reported. Herein we show that the small and large intestine express Synj1 and Synj2. Their mRNA levels, measured by RT-PCR, are not affected by development in the small intestine but in the colon they increase with age. Immunostaining assays reveal that both Synjs localize at the apical domain of the epithelial cells and at the lamina propria at sites also expressing the neuron marker calretinin. Synj2 staining at the lamina propria is fainter than that of Synj1. In colonocytes Synjs are at the apical membrane and cytosolic membrane vesicles. Synj2 is also at the mitochondria. Western blots reveal that the intestinal mucosa expresses at least two Synj1 (170- and 139-kDa) and two Synj2 (160- and 148-kDa) isoforms. The observations suggest that Synj1-170, Synj2-160, and Synj2-148 in colonocytes, might participate in processes that take place mainly at the apical domain of the epithelial cells whereas Synj1-139 in those at the enteric nervous system. Experimental colitis augments the mRNA abundance of both Synjs in colon but only Synj2 mRNA levels are increased in colon tumors. In conclusion, as far as we know, this is the first report showing expression, location and isoforms of Synj1 and Synj2 in the small and large intestine and that they might participate in intestinal pathology.

Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity.

UNLABELLED: Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCE: HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.

N-Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus' structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.

Structure and dynamics of the HIV-1 frameshift element RNA.

The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs.