Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization

AbstractHIV-1 genomic RNA (gRNA) dimerization is important for viral infectivity and is regulated by proteolytic processing of the Gag precursor protein (Pr55gag) under the direction of the viral protease. The processing occurs in successive steps and, to date, the step associated with formation of a wild-type (WT) level of gRNA dimers has not been identified. The primary cleavage divides Pr55gag into two proteins. The C-terminal polypeptide is termed NCp15 (NCp7–p1–p6) because it contains the nucleocapsid protein (NC), a key determinant of gRNA dimerization and packaging. To examine the importance of precursor polypeptides NCp15 and NCp9 (NCp7–p1), we introduced mutations that prevented the proteolytic cleavages responsible for the appearance of NCp9 or NCp7. Using native Northern blot analysis, we show that gRNA dimerization was impaired when both the secondary (p1–p6) and tertiary (p7–p1) cleavage sites of NCp15 were abolished, but unaffected when only one or the other site was abolished. Though processing to NCp9 therefore suffices for a WT level of gRNA dimerization, we also show that preventing cleavage at the p7–p1 site abolished HIV-1 replication. To identify the minimum level of protease activity compatible with a WT level of gRNA dimers, we introduced mutations Thr26Ser and Ala28Ser in the viral protease to partially inactivate it, and we prepared composite HIV-1 resulting from the cotransfection of various ratios of WT and protease-inactive proviral DNAs. The results reveal that a 30% processing of Pr55gag into mature capsid proteins (CA/CA-p2) yielded a WT level of gRNA dimers, while a 10% Pr55gag processing hardly increased gRNA dimerization above the level seen in protease-inactive virions. We found that full gRNA dimerization required less than 50% WT NC in complementation asssays. Finally, we show that if we destroy alpha helix 1 of the capsid protein (CA), gRNA dimerization is impaired to the same extent as when the viral protease is inactivated. Cotransfection studies show that this CA mutation, in contrast to the NC-disabling mutations, has a dominant negative effect on HIV-1 RNA dimerization, viral core formation, and viral replication. This represents the first evidence that a capsid mutation can affect HIV-1 RNA dimerization

Novel Staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of HIV-1 genomic RNA

Human immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA.Fil: Abrahamyan, Levon G.. Davis Jewish General Hospital; CanadáFil: Chatel Chaix, Laurent. Davis Jewish General Hospital; CanadáFil: Ajamian, Lara. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Milev, Miroslav P.. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Monette, Anne. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Clément, Jean François. Davis Jewish General Hospital; CanadáFil: Song, Rujun. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Lehmann, Martin. Davis Jewish General Hospital; CanadáFil: DesGroseillers, Luc. University Of Montreal; CanadáFil: Laughrea, Michael. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Boccaccio, Graciela Lidia. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Mouland, Andrew J.. Mc Gill University; Canadá. Davis Jewish General Hospital; Canad

Suramin Alleviates Glomerular Injury and Inflammation in the Remnant Kidney

Background: Recently, we demonstrated that suramin, a compound that inhibits the interaction of multiple cytokines/ growth factors with their receptors, inhibits activation and proliferation of renal interstitial fibroblasts, and attenuates the development of renal interstitial fibrosis in the murine model of unilateral ureteral obstruction (UUO). However, it remains unclear whether suramin can alleviate glomerular and vascular lesions, which are not typical pathological changes in the UUO model. So we tested the efficacy of suramin in the remnant kidney after 5/6 nephrectomy, a model characterized by the slow development of glomerulosclerosis, vascular sclerosis, tubulointerstitial fibrosis and renal inflammation, mimicking human disease. Methods/Findings: 5/6 of normal renal mass was surgically ablated in male rats. On the second week after surgery, rats were randomly divided into suramin treatment and non-treatment groups. Suramin was given at 10 mg/kg once per week for two weeks. In the remnant kidney of mice receiving suramin, glomerulosclerosis and vascular sclerosis as well as inflammation were ameliorated. Suramin also attenuated tubular expression of two chemokines, monocyte chemoattractant protein-1 and regulated upon expression normal T cell expressed and secreted (RANTES). After renal mass ablation, several intracellular molecules associated with renal fibrosis, including NF-kappaB p65, Smad-3, signal transducer and activator of transcription-3 and extracellular regulated kinase 1/2, are phosphorylated; suramin treatment inhibited thei

Dimerization of human immunodeficiency virus type 1 genome : dimer maturation process and role of the 5' untranslated region in dimerization

Human Immunodeficiency Virus type I genome consists of two identical RNA molecules that are non-covalently linked to form a dimer. HIV-1 immature and mature genomic RNA (gRNA) dimers were found in protease defective (PR -) and wild type virions, respectively, and the 5'untranslated region (5' UTR) was shown to play key roles during the genome dimerization process; but the dimerization mechanism still remains to be clarified My research project is to characterize the dimerization process and the role of 5' UTR in genome dimerization in virions produced by tissue culture cells. I'll firstly show the dimer maturation processes of HIV-1 gRNA isolated from newly released to grown-up (≥10h old) wild type, PR-, and SL1 defective (DeltaIDS) virions respectively. The results showed that HIV-1 gRNA dimer maturation process was protease-dependent and involved multiple steps: from low to high dimerization level and dimer thermostability, and from low dimer mobility to intermediate and high mobility. PR- virions did not freeze gRNA conformation in the primordial nascent state and gRNA changed from monomeric in newly released virions to half dimeric in grown-up virions, which showed that genome was packaged in the form of monomeric RNA or fragile dimers, more thermolabile than immature dimers in grown-up PR- virions. DeltaDIS inhibited gRNA dimerization by about 50% in newly released virions, though grown-up DeltaDIS gRNA was fully dimeric, which indicated that the DIS played the initiation role in gRNA dimerization in HIV-1 virions. The gRNA dimerization rate in PR- or DeltaDIS virions was much slower than that in wild type virions. These results show for the first time the whole process of dimer maturation after virion release, the gRNA conformation rearrangement in PR- virions, and the initiation role of the DIS in HIV-1 virions. Next, I'll provide a rather systematic search for the contribution of different regions in 5' UTR to HIV-1 gRNA dimerization by studying selected mutations singly or together with defective SL1. The results showed that the 5'trans-activation response element (5'TAR) was directly involved in gRNA dimerization, and a long distance base-pairing interaction between a sequence in U5 region (nts105-1l5) and another around the initiation codon of the gag gene (nts334-344) was structurally contributive to gRNA dimerization. Deletions of sequences around the 3'end of Primer Binding Site (PBS) stem-loop moderately decreased gRNA dimerization level. Other sequences in 5' UTR except DIS/SL1, which was previously known to play important roles, didn't show any systematic role. Here the results suggested that the absence of inhibition on gRNA dimerization level with defective DIS might be the compensation of the direct role of 5'TAR; and wild type-like dimerization level of DeltaTAR must be the direct contribution of the DIS

A Piezoelectric Energy Harvester with Bending–Torsion Vibration in Low-Speed Water

This paper presents a piezoelectric energy harvester using an eccentric cylinder undergoing bending–torsion vibration in low-speed water. It can harvest energy from water using vortex-induced vibration (VIV). A distributed parameter beam model with respect to the motion of the piezoelectric beam was established based on Euler–Bernoulli beam theory. The governing coupled equations of the harvester system were derived by Lagrange’s equations. The optimal configurations and work conditions of harvesters were numerically analyzed according to the above mathematical models. Experiments were designed and performed to verify the numerical results. The numerical results were in good agreement with the experiment results, which verifies the validity of the mathematical models. The harvester with bending–torsion vibration generated an output power of 0.3978 mW, which is 1.99 times of that of the harvester with a solid-cylinder tip undergoing bending only

Energy-Harvesting Performances of Two Tandem Piezoelectric Energy Harvesters with Cylinders in Water

This paper presents a new energy-harvesting system with two identical piezoelectric energy harvesters in a tandem configuration. Each harvester consists of a piezoelectric beam and a circular cylinder. Experiments are performed to investigate the energy-harvesting performances of this system in water. It can be found that their energy-harvesting performances are all different from that of the single harvester (without an upstream or downstream harvester). The experimental results show that the water speed and the spacing ratio have significant effects on the energy-harvesting performances of the two tandem harvesters. The output power of the upstream harvester first increases, and then decreases with the water speed increasing. The maximum output power of 167.8 μW is achieved at the water speed of 0.306 m/s and the spacing ratio (L/D) of 2.5. Increasing the water speed results in an increase in the energy performance of the downstream harvester. Compared with the single harvester, the performance of the downstream harvester is weakened in the low water speed range, but enhanced in the higher water speed range. Further, the output power of 533 μW is obtained by the downstream harvester at the water speed of 0.412 m/s and the spacing ratio of 1.7, which is 29 times more than that of the single harvester. The results indicate the superiority of the two tandem harvesters in energy-harvesting performance

A Novel Piezoelectric Energy Harvester Using the Macro Fiber Composite Cantilever with a Bicylinder in Water

A novel piezoelectric energy harvester equipped with two piezoelectric beams and two cylinders was proposed in this work. The energy harvester can convert the kinetic energy of water into electrical energy by means of vortex-induced vibration (VIV) and wake-induced vibration (WIV). The effects of load resistance, water velocity and cylinder diameter on the performance of the harvester were investigated. It was found that the vibration of the upstream cylinder was VIV which enhanced the energy harvesting capacity of the upstream piezoelectric beam. As for the downstream cylinder, both VIV and the WIV could be obtained. The VIV was found with small L/D, e.g., 2.125, 2.28, 2.5, and 2.8. Additionally, the WIV was stimulated with the increase of L/D (such as 3.25, 4, and 5.5). Due to the WIV, the downstream beam presented better performance in energy harvesting with the increase of water velocity. Furthermore, it revealed that more electrical energy could be obtained by appropriately matching the resistance and the diameter of the cylinder. With optimal resistance (170 kΩ) and diameter of the cylinder (30 mm), the maximum output power of 21.86 μW (sum of both piezoelectric beams) was obtained at a water velocity of 0.31 m/s

Modeling and Analysis of Upright Piezoelectric Energy Harvester under Aerodynamic Vortex-induced Vibration

This paper presents an upright piezoelectric energy harvester (UPEH) with cylinder extension along its longitudinal direction. The UPEH can generate energy from low-speed wind by bending deformation produced by vortex-induced vibrations (VIVs). The UPEH has the advantages of less working space and ease of setting up an array over conventional vortex-induced vibration harvesters. The nonlinear distributed modeling method is established based on Euler–Bernoulli beam theory and aerodynamic vortex-induced force of the cylinder is obtained by the van der Pol wake oscillator theory. The fluid–solid–electricity governing coupled equations are derived using Lagrange’s equation and solved through Galerkin discretization. The effect of cylinder gravity on the dynamic characteristics of the UPEH is also considered using the energy method. The influences of substrate dimension, piezoelectric dimension, the mass of cylinder extension, and electrical load resistance on the output performance of harvester are studied using the theoretical model. Experiments were carried out and the results were in good agreement with the numerical results. The results showed that a UPEH configuration achieves the maximum power of 635.04 μW at optimum resistance of 250 kΩ when tested at a wind speed of 4.20 m/s. The theoretical results show that the UPEH can get better energy harvesting output performance with a lighter tip mass of cylinder, and thicker and shorter substrate in its synchronization working region. This work will provide the theoretical guidance for studying the array of multiple upright energy harvesters

A Magnetically Coupled Electromagnetic Energy Harvester with Low Operating Frequency for Human Body Kinetic Energy

In this paper, a magnetically coupled electromagnetic energy harvester (MCEEH) is proposed for harvesting human body kinetic energy. The proposed MCEEH mainly consists of a pair of spring-connected magnets, coils, and a free-moving magnet. Specifically, the interaction force between the magnets is repulsive. The main feature of this structure is the use of a magnetic-spring structure to weaken the hardening response caused by the repulsive force. The magnetic coupling method enables the energy harvester system to harvest energy efficiently at low frequency. The MCEEH is experimentally investigated for improving energy harvesting efficiency. Under harmonic excitation with an acceleration of 0.5 g, the MCEEH reaches resonance frequency at 8.8 Hz and the maximum output power of the three coils are 5.2 mW, 2.8 mW, and 2.5 mW, respectively. In the case of hand-shaking excitation, the generator can obtain the maximum voltage of 0.6 V under the excitation acceleration of 0.2 g and the excitation frequency of 3.4 Hz. Additionally, a maximum instantaneous power can be obtained of about 26 mW from the human body’s kinetic energy

Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4+ T Cells

Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24+ HIV iCD4s that maintained cell surface CD4 (iCD4+), did not maintain CD4 (iCD4−) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4+ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4−) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells