Viral sequence classification has wide applications in clinical, epidemiological, structural and functional categorization studies. Most existing approaches rely on an initial alignment step followed by classification based on phylogenetic or statistical algorithms. Here we present an ultrafast alignment-free subtyping tool for human immunodeficiency virus type one (HIV-1) adapted from Prediction by Partial Matching compression. This tool, named COMET, was compared to the widely used phylogeny-based REGA and SCUEAL tools using synthetic and clinical HIV data sets (1 090 698 and 10 625 sequences, respectively). COMET's sensitivity and specificity were comparable to or higher than the two other subtyping tools on both data sets for known subtypes. COMET also excelled in detecting and identifying new recombinant forms, a frequent feature of the HIV epidemic. Runtime comparisons showed that COMET was almost as fast as USEARCH. This study demonstrates the advantages of alignment-free classification of viral sequences, which feature high rates of variation, recombination and insertions/deletions. COMET is free to use via an online interface.

The chemokine receptors CCR5 and CXCR4 are promising non-virus-encoded targets for human immunodeficiency virus (HIV) therapy. We describe a selection procedure to isolate mutant forms of RANTES (CCL5) with antiviral activity considerably in excess of that of the native chemokine. The phage-displayed library of randomly mutated and N-terminally extended variants was screened by using live CCR5-expressing cells, and two of the selected mutants, P1 and P2, were further characterized. Both were significantly more potent HIV inhibitors than RANTES, with P2 being the most active (50% inhibitory concentration of 600 pM in a viral coat-mediated cell fusion assay, complete protection of target cells against primary HIV type 1 strains at a concentration of 10 nM). P2 resembles AOP-RANTES in that it is a superagonist of CCR5 and potently induces receptor sequestration. P1, while less potent than P2, has the advantage of significantly reduced signaling activity via CCR5 (30% of that of RANTES). Additionally, both P1 and P2 exhibit not only significantly increased affinity for CCR5 but also enhanced receptor selectivity, retaining only trace levels of signaling activity via CCR1 and CCR3. The phage chemokine approach that was successfully applied here could be adapted to other chemokine-chemokine receptor systems and used to further improve the first-generation mutants reported in this paper.Despite the success of highly active antiretroviral therapy, new human immunodeficiency virus type 1 (HIV-1) inhibitors are still needed and among the most promising new approaches is the blockade of viral entry into target cells (20). HIV-1 entry into target cells is initially dependent on the interaction of its envelope glycoproteins with CD4 and a coreceptor, with the chemokine receptors CCR5 and CXCR4 being by far the most commonly used by HIV-1 (5).HIV entry is inhibited by the natural chemokine ligands of the coreceptors, including MIP-1␣ (CCL3), MIP-1␤ (CCL4), and RANTES (CCL5) for CCR5 (8) and SDF-1 (CXCL12) for CXCR4 (6,23). Certain N-terminal modifications have been shown to increase the anti-HIV activity of native chemokines (21,32,33,36), and the most potent of these molecules owe their anti-HIV activity to their ability to induce prolonged intracellular sequestration of coreceptors (18,31).Up until now, chemokine structure-activity relationships have been studied via either scanning or truncation mutagenesis (14,16,19,24), peptide scanning of primary sequence (22), or semirational design of chemokine analogues (21,32,36). A more-rapid, bioengineering-based approach for the selection of useful chemokine variants has yet to be described.We decided to apply current knowledge of the structureactivity relationship of chemokines and the mechanism by which they inhibit HIV entry (2,7,18) to the design of a phage display strategy for the discovery of N-terminally mutated RANTES variants with improved anti-HIV activity. Selection led to the isolation of around 40 clones that exhibited a consensus sequence, and two clones were chosen...

The emergence of human immunodeficiency virus type 1 resistance to raltegravir, an integrase strand transfer inhibitor, follows distinct and independent genetic pathways, among which the N155H and Q148HKR pathways are the most frequently encountered in treated patients. After prolonged viral escape, mutants of the N155H pathway are replaced by mutants of the Q148HKR pathway. We have examined the mechanisms driving this evolutionary pattern using an approach that assesses the selective advantage of site-directed mutant viruses as a function of drug concentration. These selective-advantage curves revealed that among single mutants, N155H had the highest and the widest (1 to 500 nM) selective-advantage profile. Despite the higher 50% inhibitory concentration, Q148H displayed a lower and narrower (10 to 100 nM) selective-advantage profile. Among double mutants, the highest and widest selective-advantage profile was seen with G140S؉Q148H. This finding likely explains why N155H can be selected early in the course of RAL resistance evolution in vivo but is later replaced by genotypes that include Q148HKR.

Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is mod-

The reactivation of telomerase reverse transcriptase (TERT) protein is the principal mechanism of telomere maintenance in cancer cells. Mutations in the TERT promoter (TERTp) are a common mechanism of TERT reactivation in many solid cancers, particularly those originating from slow-replicating tissues. They are associated with increased TERT levels, telomere stabilization, and cell immortalization and proliferation. Much effort has been invested in recent years in characterizing their prevalence in different cancers and their potential as biomarkers for tumor stratification, as well as assessing their molecular mechanism of action, but much remains to be understood. Notably, they appear late in cell transformation and are mutually exclusive with each other as well as with other telomere maintenance mechanisms, indicative of overlapping selective advantages and of a strict regulation of TERT expression levels. In this review, we summarized the latest literature on the role and prevalence of TERTp mutations across different cancer types, highlighting their biased distribution. We then discussed the need to maintain TERT levels at sufficient levels to immortalize cells and promote proliferation while remaining within cell sustainability levels. A better understanding of TERT regulation is crucial when considering its use as a possible target in antitumor strategies.Cells 2020, 9, 749 2 of 28 by copy number variants (CNV), TERT or TERTp structural variants, chromosomal rearrangements juxtaposing TERTp to enhancer elements, cellular and viral oncogenes such as Hepatitis B virus (HBV) X protein (HBx) or high-risk Human papillomavirus (HPV)16 and HPV18 E6 oncoprotein, and, last but not least, mutations within TERTp (31% of TERT-expressing cancers) ( Figure 1A) [10, 30-38] (reviewed in [3,4,9,18-20,39]). Increased TERTp methylation is typically recorded in >50% of TERT-expressing tumors and cell lines [10,[40][41][42][43][44][45][46][47]. Epigenetic regulation of TERTp is based on altered methylation patterns of specific regions. Hypomethylation of the region between −200 and −100 from the Translational Start site (TSS), encompassing the core promoter, enables binding of c-Myc and Sp-1, thus reactivating transcription. In contrast, the region spanning exon 1 (positions +1 to ±100 from the TSS) contains a binding site for the DNA insulator CTCF. Hypermethylation of this region disrupts binding of CTCF and therefore allows TERT transcription [41][42][43][44]. Similarly, the region between −600 and −200 from the TSS contains a second CTCF binding site and is partially hypermethylated in TERT-expressing cells [41][42][43][44].