scholarly journals In Vitro Quantification of the Effects of IP6 and Other Small Polyanions on Immature HIV-1 Particle Assembly and Core Stability

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Alžběta Dostálková ◽  
Filip Kaufman ◽  
Ivana Křížová ◽  
Barbora Vokatá ◽  
Tomáš Ruml ◽  
...  
Keyword(s):  
Life Cycle ◽  
Host Cell ◽  
Therapeutic Potential ◽  
Core Stability ◽  
Dependent Manner ◽  
Particle Assembly ◽  
Inositol Hexaphosphate ◽  
Host Cell Factors ◽  
Hiv 1

ABSTRACT Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles. IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

scholarly journals A Structural Perspective of the Role of IP6 in Immature and Mature Retroviral Assembly

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1853
Author(s):  
Martin Obr ◽  
Florian K. M. Schur ◽  
Robert A. Dick
Keyword(s):  
Life Cycle ◽  
Binding Site ◽  
Core Stability ◽  
The Family ◽  
In Vitro Assembly ◽  
Retroviral Assembly ◽  
Immature Virus ◽  
Hiv 1

The small cellular molecule inositol hexakisphosphate (IP6) has been known for ~20 years to promote the in vitro assembly of HIV-1 into immature virus-like particles. However, the molecular details underlying this effect have been determined only recently, with the identification of the IP6 binding site in the immature Gag lattice. IP6 also promotes formation of the mature capsid protein (CA) lattice via a second IP6 binding site, and enhances core stability, creating a favorable environment for reverse transcription. IP6 also enhances assembly of other retroviruses, from both the Lentivirus and the Alpharetrovirus genera. These findings suggest that IP6 may have a conserved function throughout the family Retroviridae. Here, we discuss the different steps in the viral life cycle that are influenced by IP6, and describe in detail how IP6 interacts with the immature and mature lattices of different retroviruses.

scholarly journals Structure, Function, and Interactions of the HIV-1 Capsid Protein

Life ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 100
Author(s):  
Eric Rossi ◽  
Megan E. Meuser ◽  
Camille J. Cunanan ◽  
Simon Cocklin
Keyword(s):  
Life Cycle ◽  
Capsid Protein ◽  
Adaptor Proteins ◽  
Restriction Factors ◽  
Gag Polyprotein ◽  
Immunodeficiency Virus ◽  
Host Cell Factors ◽  
Hiv 1

The capsid (CA) protein of the human immunodeficiency virus type 1 (HIV-1) is an essential structural component of a virion and facilitates many crucial life cycle steps through interactions with host cell factors. Capsid shields the reverse transcription complex from restriction factors while it enables trafficking to the nucleus by hijacking various adaptor proteins, such as FEZ1 and BICD2. In addition, the capsid facilitates the import and localization of the viral complex in the nucleus through interaction with NUP153, NUP358, TNPO3, and CPSF-6. In the later stages of the HIV-1 life cycle, CA plays an essential role in the maturation step as a constituent of the Gag polyprotein. In the final phase of maturation, Gag is cleaved, and CA is released, allowing for the assembly of CA into a fullerene cone, known as the capsid core. The fullerene cone consists of ~250 CA hexamers and 12 CA pentamers and encloses the viral genome and other essential viral proteins for the next round of infection. As research continues to elucidate the role of CA in the HIV-1 life cycle and the importance of the capsid protein becomes more apparent, CA displays potential as a therapeutic target for the development of HIV-1 inhibitors.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

scholarly journals Restoration of Retinoid Sensitivity by MDR1 Ribozymes in Retinoic Acid–Resistant Myeloid Leukemic Cells

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2452-2458 ◽  
Author(s):  
Hiromichi Matsushita ◽  
Masahiro Kizaki ◽  
Hiroyuki Kobayashi ◽  
Hironori Ueno ◽  
Akihiro Muto ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cellular Proliferation ◽  
Therapeutic Potential ◽  
Acquired Resistance ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
P Glycoprotein ◽  
All Trans Retinoic Acid ◽  
Mdr1 Mrna

Complete remission is achieved in a high proportion of patients with acute promyelocytic leukemia (APL) after all-trans retinoic acid (RA) treatment, but most patients relapse and develop RA-resistant APL. We have previously reported that both RA-resistant HL-60 (HL-60R) and APL cells express P-glycoprotein and MDR1 transcripts; and these cells differentiate to mature granulocytes after culture with RA and P-glycoprotein antagonist. Ribozymes have been shown to be able to intercept a target RNA by catalytic activity. To address the role of MDR1 in overcoming RA-resistance in APL cells, we investigated the biologic effects of ribozymes against the MDR1 transcript in HL-60R cells. These ribozymes efficiently cleaved MDR1 mRNA at a specific site in vitro. The 196 MDR1 ribozyme was cloned into an expression vector, and stably transfected (HL-60R/196Rz) cells were obtained. Expression of MDR1 transcripts was decreased in HL-60R/196Rz cells compared with parental HL-60R and empty vector-transfected (HL-60R/neo) cells. Interestingly, RA inhibited cellular proliferation and induced differentiation of HL-60R/196Rz cells in a dose-dependent manner, suggesting reversal of drug resistance in HL-60R cells by the MDR1 ribozyme. These data are direct evidence that P-glycoprotein/MDR1 is responsible in part for acquired resistance to RA in myeloid leukemic cells. The MDR1 ribozyme may be a useful tool for investigating the biology of retinoid resistance and may have therapeutic potential for patients with RA-resistant APL.

scholarly journals Phosphoserine acidic cluster motifs in the cytoplasmic domains of transmembrane proteins bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

2018 ◽  
Author(s):  
Rajendra Singh ◽  
Charlotte Stoneham ◽  
Christopher Lim ◽  
Xiaofei Jia ◽  
Javier Guenaga ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Transmembrane Proteins ◽  
Adaptor Protein ◽  
Cellular Protein ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Cytoplasmic Domains ◽  
Clathrin Adaptor ◽  
Hiv 1

AbstractProtein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease Furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of Furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro. The sites of these serines vary among mammals in a manner consistent with host-pathogen conflict, yet the Serinc3-PSAC seems dispensible for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu, Furin, and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

scholarly journals In VitroExposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa

2016 ◽  
Vol 60 (9) ◽  
pp. 5459-5466 ◽  
Author(s):  
Guillermo Villegas ◽  
Giulia Calenda ◽  
Shimin Zhang ◽  
Olga Mizenina ◽  
Kyle Kleinbeck ◽  
...  
Keyword(s):  
Phase 1 ◽  
Lavage Fluid ◽  
Dependent Manner ◽  
Phase 1 Trial ◽  
Cervical Mucosa ◽  
Cervicovaginal Lavage ◽  
Dose Dependent ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACTOur recent phase 1 trial demonstrated that PC-1005 gel containing 50 μM MIV-150, 14 mM zinc acetate dihydrate, and carrageenan (CG) applied daily vaginally for 14 days is safe and well tolerated. Importantly, cervicovaginal lavage fluid samples (CVLs) collected 4 or 24 h after the last gel application inhibited HIV-1 and human papillomavirus (HPV) in cell-based assays in a dose-dependent manner (MIV-150 for HIV-1 and CG for HPV). Herein we aimed to determine the anti-HIV and anti-herpes simplex virus 2 (anti-HSV-2) activity of PC-1005 in human cervical explants afterin vitroexposure to the gel and to CVLs from participants in the phase 1 trial. Single HIV-1BaLinfection and HIV-1BaL–HSV-2 coinfection explant models were utilized. Coinfection with HSV-2 enhanced tissue HIV-1BaLinfection.In vitroexposure to PC-1005 protected cervical mucosa against HIV-1BaL(up to a 1:300 dilution) in single-challenge and cochallenge models. CG gel (PC-525) provided some barrier effect against HIV-1BaLat the 1:100 dilution in a single-challenge model but not in the cochallenge model. Both PC-1005 and PC-525 at the 1:100 dilution inhibited HSV-2 infection, pointing to a CG-mediated protection. MIV-150 and CG in CVLs inhibited HIV (single-challenge or cochallenge models) and HSV-2 infections in explants in a dose-dependent manner (P< 0.05). Stronger inhibition of HIV-1 infection by CVLs collected 4 h after the last gel administration was observed compared to infection detected in the presence of baseline CVLs. The anti-HIV and anti-HSV-2 activity of PC-1005 gelin vitroand CVLs in human ectocervical explants supports the further development of PC-1005 gel as a broad-spectrum on-demand microbicide.

scholarly journals Design, synthesis and biological evaluation of a series of CNS penetrant HDAC inhibitors structurally derived from amyloid-β probes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Myeong A Choi ◽  
Sun You Park ◽  
Hye Yun Chae ◽  
Yoojin Song ◽  
Chiranjeev Sharma ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Hdac Inhibitors ◽  
Amyloid Β ◽  
Biological Evaluation ◽  
Pharmacokinetic Studies ◽  
Dependent Manner ◽  
Reference Drug ◽  
Novel Scaffold

Abstract To develop novel CNS penetrant HDAC inhibitors, a new series of HDAC inhibitors having benzoheterocycle were designed, synthesized, and biologically evaluated. Among the synthesized compounds, benzothiazole derivative 9b exhibited a remarkable anti-proliferative activity (GI50 = 2.01 μM) against SH-SY5Y cancer cell line in a dose and time-dependent manner, better than the reference drug SAHA (GI50 = 2.90 μM). Moreover, compound 9b effectively promoted the accumulation of acetylated Histone H3 and α-tubulin through inhibition of HDAC1 and HDAC6 enzymes, respectively. HDAC enzyme assay also confirmed that compound 9b efficiently inhibited HDAC1 and HDAC6 isoforms with IC50 values of 84.9 nM and 95.9 nM. Furthermore, compound 9b inhibited colony formation capacity of SH-SY5Y cells, which is considered a hallmark of cell carcinogenesis and metastatic potential. The theoretical prediction, in vitro PAMPA-BBB assay, and in vivo brain pharmacokinetic studies confirmed that compound 9b had much higher BBB permeability than SAHA. In silico docking study demonstrated that compound 9b fitted in the substrate binding pocket of HDAC1 and HDAC6. Taken together, compound 9b provided a novel scaffold for developing CNS penetrant HDAC inhibitors and therapeutic potential for CNS-related diseases.

scholarly journals Novel Intersubunit Interaction Critical for HIV-1 Core Assembly Defines a Potentially Targetable Inhibitor Binding Pocket

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Pierrick Craveur ◽  
Anna T. Gres ◽  
Karen A. Kirby ◽  
Dandan Liu ◽  
John A. Hammond ◽  
...  
Keyword(s):  
Viral Replication ◽  
Specific Interaction ◽  
Binding Pocket ◽  
Core Stability ◽  
Host Cells ◽  
Capsid Assembly ◽  
Dynamic Simulations ◽  
Virus Like Particle ◽  
Hiv 1

ABSTRACTHIV-1 capsid protein (CA) plays critical roles in both early and late stages of the viral replication cycle. Mutagenesis and structural experiments have revealed that capsid core stability significantly affects uncoating and initiation of reverse transcription in host cells. This has led to efforts in developing antivirals targeting CA and its assembly, although none of the currently identified compounds are used in the clinic for treatment of HIV infection. A specific interaction that is primarily present in pentameric interfaces in the HIV-1 capsid core was identified and is reported to be important for CA assembly. This is shown by multidisciplinary characterization of CA site-directed mutants using biochemical analysis of virus-like particle formation, transmission electron microscopy ofin vitroassembly, crystallographic studies, and molecular dynamic simulations. The data are consistent with a model where a hydrogen bond between CA residues E28 and K30′ from neighboring N-terminal domains (CANTDs) is important for CA pentamer interactions during core assembly. This pentamer-preferred interaction forms part of anN-terminaldomaininterface (NDI) pocket that is amenable to antiviral targeting.IMPORTANCEPrecise assembly and disassembly of the HIV-1 capsid core are key to the success of viral replication. The forces that govern capsid core formation and dissociation involve intricate interactions between pentamers and hexamers formed by HIV-1 CA. We identified one particular interaction between E28 of one CA and K30′ of the adjacent CA that appears more frequently in pentamers than in hexamers and that is important for capsid assembly. Targeting the corresponding site could lead to the development of antivirals which disrupt this interaction and affect capsid assembly.

scholarly journals Crocetin Alleviates Inflammation in MPTP-Induced Parkinson’s Disease Models through Improving Mitochondrial Functions

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Na Dong ◽  
Zhong Dong ◽  
Ying Chen ◽  
Xiaosu Gu
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Mitochondrial Permeability Transition ◽  
Therapeutic Potential ◽  
Adenine Nucleotide ◽  
Motor Deficits ◽  
Dependent Manner ◽  
Cyclophilin D ◽  
Mitochondrial Functions

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Crocetin, derived from saffron, exerts multiple pharmacological properties, such as anti-inflammatory, antioxidant, antifatigue, and anticancer effects. However, the effect of crocetin on PD remains unclear. In this study, we designed experiments to investigate the effect of crocetin against MPTP-induced PD models and the underlying mechanisms. Our results showed that crocetin treatment attenuates MPTP-induced motor deficits and protects dopaminergic neurons. Both in vivo and in vitro experiments demonstrated that crocetin treatment decreased the expression of inflammatory associated genes and inflammatory cytokines. Furthermore, crocetin treatment protected mitochondrial functions against MPP+ induced damage by regulating the mPTP (mitochondrial permeability transition pore) viability in the interaction of ANT (adenine nucleotide translocase) and Cyp D (Cyclophilin D) dependent manner. Therefore, our results demonstrate that crocetin has therapeutic potential in Parkinson’s disease.

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