Upregulation of mRNA Expression of ADGRD1/GPR133 and ADGRG7/GPR128 in SARS-CoV-2-Infected Lung Adenocarcinoma Calu-3 Cells.

Adhesion G protein-coupled receptors (aGPCRs) play an important role in neurodevelopment, immune defence and cancer; however, their role throughout viral infections is mostly unexplored. We have been searching for specific aGPCRs involved in SARS-CoV-2 infection of mammalian cells. In the present study, we infected human epithelial cell lines derived from lung adenocarcinoma (Calu-3) and colorectal carcinoma (Caco-2) with SARS-CoV-2 in order to analyse changes in the level of mRNA encoding individual aGPCRs at 6 and 12 h post infection. Based on significantly altered mRNA levels, we identified four aGPCR candidates-ADGRB3/BAI3, ADGRD1/GPR133, ADGRG7/GPR128 and ADGRV1/GPR98. Of these receptors, ADGRD1/GPR133 and ADGRG7/GPR128 showed the largest increase in mRNA levels in SARS-CoV-2-infected Calu-3 cells, whereas no increase was observed with heat-inactivated SARS-CoV-2 and virus-cleared conditioned media. Next, using specific siRNA, we downregulated the aGPCR candidates and analysed SARS-CoV-2 entry, replication and infectivity in both cell lines. We observed a significant decrease in the amount of SARS-CoV-2 newly released into the culture media by cells with downregulated ADGRD1/GPR133 and ADGRG7/GPR128. In addition, using a plaque assay, we observed a reduction in SARS-CoV-2 infectivity in Calu-3 cells. In summary, our data suggest that selected aGPCRs might play a role during SARS-CoV-2 infection of mammalian cells.

Helquat dyes targeting G-quadruplexes as a new class of anti-HIV-1 inhibitors.

The secondary structure of nucleic acids containing quartets of guanines, termed G-quadruplexes, is known to regulate the transcription of many genes. Several G-quadruplexes can be formed in the HIV-1 long terminal repeat promoter region and their stabilization results in the inhibition of HIV-1 replication. Here, we identified helquat-based compounds as a new class of anti-HIV-1 inhibitors that inhibit HIV-1 replication at the stage of reverse transcription and provirus expression. Using Taq polymerase stop and FRET melting assays, we have demonstrated their ability to stabilize G-quadruplexes in the HIV-1 long-terminal repeat sequence. Moreover, these compounds were not binding to the general G-rich region, but rather to G-quadruplex-forming regions. Finally, docking and molecular dynamics calculations indicate that the structure of the helquat core greatly affects the binding mode to the individual G-quadruplexes. Our findings can provide useful information for the further rational design of inhibitors targeting G-quadruplexes in HIV-1.

LC/MS analysis and deep sequencing reveal the accurate RNA composition in the HIV-1 virion.

The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m1A profiling and deep sequencing. We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.

Design, Synthesis, and Biological Evaluation of Isothiosemicarbazones with Antimycobacterial Activity.

A series of benzaldehyde and salicylaldehyde-S-benzylisothiosemicarbazones was synthesized and tested against 12 different strains of mycobacteria, Gram-positive and Gram-negative bacteria, and the significant selectivity toward mycobacteria was proved. Twenty-eight derivatives were evaluated for the inhibition of isocitrate lyase, which is a key enzyme of the glyoxylate cycle necessary for latent tuberculosis infection, and their iron-chelating properties were investigated. Two derivatives, 5-bromosalicylaldehyde-S-(4-fluorobenzyl)-isothiosemicarbazone and salicylaldehyde-S-(4-bromobenzyl)-isothiosemicarbazone, influenced the isocitrate lyase activity and caused a better inhibition at 10 µmol/L than 3-nitropropionic acid, a standard inhibitor. The compounds were also found to act as exogenous chelators of iron, which is an obligate cofactor for many mycobacterial enzymes. Due to their low cytotoxicity, together with the activity against isocitrate lyase and the ability to sequester iron ions, the compounds belong to potential antibiotics with the main effect on mycobacteria.

Impaired human immunodeficiency virus type 1 replicative fitness in atypical viremic non-progressor individuals.

BACKGROUND: Progression rates from initial HIV-1 infection to advanced AIDS vary significantly among infected individuals. A distinct subgroup of HIV-1-infected individuals-termed viremic non-progressors (VNP) or controllers-do not seem to progress to AIDS, maintaining high CD4+ T cell counts despite high levels of viremia for many years. Several studies have evaluated multiple host factors, including immune activation, trying to elucidate the atypical HIV-1 disease progression in these patients; however, limited work has been done to characterize viral factors in viremic controllers. METHODS: We analyzed HIV-1 isolates from three VNP individuals and compared the replicative fitness, near full-length HIV-1 genomes and intra-patient HIV-1 genetic diversity with viruses from three typical (TP) and one rapid (RP) progressor individuals. RESULTS: Viremic non-progressors and typical patients were infected for >10 years (range 10-17 years), with a mean CD4+ T-cell count of 472 cells/mm3 (442-529) and 400 cells/mm3 (126-789), respectively. VNP individuals had a less marked decline in CD4+ cells (mean -0.56, range -0.4 to -0.7 CD4+/month) than TP patients (mean -10.3, -8.2 to -13.1 CD4+/month). Interestingly, VNP individuals carried viruses with impaired replicative fitness, compared to HIV-1 isolates from the TP and RP patients (p < 0.05, 95% CI). Although analyses of the near full-length HIV-1 genomes showed no clear patterns of single-nucleotide polymorphisms (SNP) that could explain the decrease in replicative fitness, both the number of SNPs and HIV-1 population diversity correlated inversely with the replication capacity of the viruses (r = -0.956 and r = -0.878, p < 0.01, respectively). CONCLUSION: It is likely that complex multifactorial parameters govern HIV-1 disease progression in each individual, starting with the infecting virus (phenotype, load, and quasispecies diversity) and the intrinsic ability of the host to respond to the infection. Here we analyzed a subset of viremic controller patients and demonstrated that similar to the phenomenon observed in patients with a discordant response to antiretroviral therapy (i.e., high CD4+ cell counts with detectable plasma HIV-1 RNA load), reduced viral replicative fitness seems to be linked to slow disease progression in these antiretroviral-naïve individuals.

Synthesis and evaluation of 2-pyridinylpyrimidines as inhibitors of HIV-1 structural protein assembly.

In an effort to identify an HIV-1 capsid assembly inhibitor with improved solubility and potency, we synthesized two series of pyrimidine analogues based on our earlier lead compound N-(4-(ethoxycarbonyl)phenyl)-2-(pyridine-4-yl)quinazoline-4-amine. In vitro binding experiments showed that our series of 2-pyridine-4-ylpyrimidines had IC50 values higher than 28µM. Our series of 2-pyridine-3-ylpyrimidines exhibited IC50 values ranging from 3 to 60µM. The congeners with a fluoro substituent introduced at the 4-N-phenyl moiety, along with a methyl at C-6, represent potent HIV capsid assembly inhibitors binding to the C-terminal domain of the capsid protein.

Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds.

Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations.

Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor.

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.