Publications

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-<i>C</i>-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-<i>C</i>-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-<i>C</i>-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-<i>C</i>-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-<i>C</i>-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.<b>IMPORTANCE</b> This study found that the nucleoside analog 7-deaza-2'-<i>C</i>-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-<i>C</i>-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

This study was undertaken to demonstrate the unique specificity of the chemokine receptor CXCR4 antagonist AMD3100. Calcium flux assays with selected chemokine/cell combinations, affording distinct chemokine receptor specificities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in different cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not affect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium fluxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunodeficiency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis).

Abstract The antiviral activity of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdUrd), acyclovir and other antiherpetic nucleosides depends on a selective phosphorylation by the herpesvirus-induced thymidine kinase in the infected cells. Viruses not encoding a specific thymidine kinase (TK) activity are resistant to the action of these nucleoside analogues. The nucleoside monophosphates are as such poorly taken up by the cells. In order to circumvent the necessity of intracellular phosphorylation, we synthesized four core oligonucleotides bearing a biological active nucleoside at the 2′end. It was hypothesized that these core oligonucleotides, like core 2–5A itself, would be taken up within the cell and that, following intracellular 2′-5′ phosphodiesterase cleavage, the 5′-monophosphate of the active product would be formed. In these circumstances, activity against TK- strains could be expected.

No abstract is provided for this article.

The chemokine receptors CCR5 and CXCR4 function as the principal coreceptors for human immunodeficiency virus type 1 (HIV-1). Coreceptor function has also been demonstrated for a variety of related receptors in vitro. The relative contributions of CCR5, CXCR4, and other putative coreceptors to HIV-1 disease in vivo have yet to be defined. In this study, we used sequential primary isolates and recombinant strains of HIV-1 to demonstrate that CXCR4-using (X4) viruses emerging in association with disease progression are highly pathogenic in ex vivo lymphoid tissues compared to CXCR4-independent viruses. Furthermore, synthetic receptor antagonists that specifically block CXCR4-mediated entry dramatically suppressed the depletion of CD4(+) T cells by recombinant and clinically derived X4 HIV-1 isolates. Moreover, in vitro specificity for the additional coreceptors CCR3, CCR8, BOB, and Bonzo did not augment cytopathicity or diminish sensitivity toward CXCR4 antagonists in lymphoid tissues. These data provide strong evidence to support the concept that adaptation to CXCR4 specificity in vivo accelerates HIV-1 disease progression. Thus, therapeutic intervention targeting the interaction of HIV-1 gp120 with CXCR4 may be highly valuable for suppressing the pathogenic effects of late-stage viruses.

In this study, a novel series of diarylpyrimidine derivatives with Fsp³-enriched spirocycles were designed and synthesized to further explore the chemical space of the hydrophobic channel of the NNRTI-binding pocket. The biological evaluation results showed that most of the compounds displayed effective inhibitory potency against the HIV-1 wild-type strain, with EC₅₀ values ranging from micromolar to submicromolar levels. Among them, TT6 turned out to be the most effective inhibitor with an EC₅₀ value of 0.17 μM, demonstrating up to 47 times more active than that of reference drug 3TC (EC₅₀ = 8.01 μM). More encouragingly, TT6 was found to potently inhibit the HIV-1 mutant strain K103N with an EC₅₀ value of 0.69 μM, being about 6-fold more potent than 3TC (EC₅₀ = 3.68 μM) and NVP (EC₅₀ = 4.62 μM). Furthermore, TT6 exhibited the most potent inhibitory activity toward HIV-1 reverse transcriptase with an IC₅₀ value of 0.33 μM. Additionally, molecular simulation studies were conducted to investigate the binding modes between TT6 and NNRTI-binding pocket, which may provide valuable clues for the follow-up structural optimizations.