Dual-Reporter SARS-CoV-2 Replicon for Screening Viral Polymerase Inhibitors.

To design a safe cellular system for testing inhibitors targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, a genetic construct was engineered containing viral cDNA with two blocks of reporter genes while the genes encoding structural S, E, and M proteins were absent. The first reporter block, consisting of Renilla luciferase and green fluorescent protein (Rluc-GFP), was located upstream of the SARS-CoV-2 5'-UTR. Meanwhile, the second block represented by firefly luciferase and red fluorescent protein (Fluc-RFP) was positioned downstream of the transcription regulatory sequence (TRS-N).

Post-Integrational DNA Repair of HIV-1 Is Associated with Activation of the DNA-PK and ATM Cellular Protein Kinases and Phosphorylation of Their Targets.

Integration of the DNA copy of HIV-1 genome into the cellular genome results in series of damages, repair of which is critical for successful replication of the virus. We have previously demonstrated that the ATM and DNA-PK kinases, normally responsible for repairing double-strand breaks in the cellular DNA, are required to initiate the HIV-1 DNA postintegrational repair, even though integration does not result in DNA double-strand breaks. In this study, we analyzed changes in phosphorylation status of ATM (pSer1981), DNA-PK (pSer2056), and their related kinase ATR (pSer428), as well as their targets: Chk1 (pSer345), Chk2 (pThr68), H2AX (pSer139), and p53 (pSer15) during the HIV-1 DNA postintegrational repair.

Role of I182, R187, and K188 Amino Acid Residues in the Catalytic Domain of HIV-1 Integrase in the Processes of Reverse Transcription and Integration.

Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer.

-Methylated Spermidine Derivatives: Convenient Syntheses and Antizyme-Related Effects.

The biogenic polyamines, spermidine (Spd) and spermine (Spm), are present at millimolar concentrations in all eukaryotic cells, where they participate in the regulation of vitally important cellular functions. Polyamine analogs and derivatives are a traditional and important instrument for the investigation of the cellular functions of polyamines, enzymes of their metabolism, and the regulation of the biosynthesis of antizyme-a key downregulator of polyamine homeostasis. Here, we describe convenient gram-scale syntheses of a set of -methylated analogs of Spd.

Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions.

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation.

DNA sequence-specific ligands. XVIII. Synthesis, physico-chemical properties; genetic, virological, and biochemical studies of fluorescent dimeric bisbenzimidazoles DBPA(n).

A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells.

Antiretroviral Hydrophobic Core Graft-Copolymer Nanoparticles: The Effectiveness against Mutant HIV-1 Strains and in Vivo Distribution after Topical Application.

Purpose: Developing and testing of microbicides for pre-exposure prophylaxis and post-exposure protection from HIV are on the list of major HIV/AIDS research priorities. To improve solubility and bioavailability of highly potent anti-retroviral drugs, we explored the use of a nanoparticle (NP) for formulating a combination of two water-insoluble HIV inhibitors.

Methods: The combination of a non-nucleoside HIV reverse transcriptase inhibitor (NNRTI), Efavirenz (EFV), and an inhibitor of HIV integrase, Elvitegravir (ELV) was stabilized with a graft copolymer of methoxypolyethylene glycol-polylysine with a hydrophobic core (HC) composed of fatty acids (HC-PGC).

A qPCR assay for measuring the post-integrational DNA repair in HIV-1 replication.

The post-integrational gap repair is a critical and poorly studied stage of the lentiviral life cycle. It might be performed by various cellular DNA repair pathways but the exact mechanism of the repair process has not yet been described. One of the reasons for that is the lack of a functional quantitative assay that could precisely measure the amount of integrated viral DNA that has completed the post-integrational gap repair stage.

Characterization of HIV-1 integrase interaction with human Ku70 protein and initial implications for drug targeting.

Human Ku70/Ku80 protein is known to influence HIV-1 replication. One of the possible reasons may be the protection of integrase from proteasomal degradation by Ku70 subunit. We demonstrated that recombinant HIV-1 integrase and Ku70 form a stable complex, while no interaction of Ku70 with integrase from prototype foamy virus was observed.

Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.

Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described.

Hydrophobic-core PEGylated graft copolymer-stabilized nanoparticles composed of insoluble non-nucleoside reverse transcriptase inhibitors exhibit strong anti-HIV activity.

Benzophenone-uracil (BPU) scaffold-derived candidate compounds are efficient non-nucleoside reverse transcriptase inhibitors (NNRTI) with extremely low solubility in water. We proposed to use hydrophobic core (methoxypolyethylene glycol-polylysine) graft copolymer (HC-PGC) technology for stabilizing nanoparticle-based formulations of BPU NNRTI in water. Co-lyophilization of NNRTI/HC-PGC mixtures resulted in dry powders that could be easily reconstituted with the formation of 150-250 nm stable nanoparticles (NP).

A convenient solid-phase method for the synthesis of novel oligonucleotide-folate conjugates.

We describe the preparation of two batches of a polymer support for the incorporation of folic acid into oligonucleotides. The method permits the regioselective attachment of a target nucleic acid sequence through its 3'-end to either the alpha-or gamma-carboxyl group of L-glutamic acid, respectively. The supports have been tested in solid-phase synthesis of oligonucleotide-folate conjugates for cell delivery studies.

The solution synthesis of antisense oligonucleotide-peptide conjugates directly linked via phosphoramide bond by using a fragment coupling approach.

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell-penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e.

Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides.

Hybridization properties of oligodeoxyxylonucleotides (OXNs) built from pyrimidine monomers with an inverted 3'-OH group of the furanose have been studied using the gel mobility shift, UV melting and circular dichroism (CD) spectroscopy methods. Pyrimidine OXNs form triple helices with complementary purine RNA in which one OXN is parallel and another is antiparallel with respect to the RNA target. Surprisingly, no duplex formation between the pyrimidine OXNs and purine RNAs is detected.

Optimisation of dendrimer-mediated gene transfer by anionic oligomers.

Background: The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. Their use, however, is limited since they generally lack the efficiency of gene transfer achieved with recombinant viral vectors such as adenovirus. Polyamidoamine (PAMAM) and phosphorus-containing dendrimers (P-dendrimers) are specific polymers with a defined spherical structure.