Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa

Background

Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure.

Methods and Findings

Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I—presented epitopes. One sequence polymorphism at position 303 of p24 Gag (T_Gag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control.

Conclusions

These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells.     

Maternal lineages in native Canadian equine populations and their relationship to the Nordic and Mountain and Moorland pony breeds

A 378-bp section of the mitochondrial displacement loop was used to estimate genetic diversity in the native Canadian equine populations. The inclusion of 10 Mountain and Moorland, 3 Nordic pony breeds, 2 feral populations, and 5 horse breeds were also investigated as they may have influenced the development (or rejuvenation) of the native Canadian populations. A total of 281 samples were sequenced, which produced 75 haplotypes derived from 54 informative sites. On further investigation, 36 of these 75 haplotypes were found to be previously unreported. Overall, total diversity was lowest in the feral Sable Island population with a haplotype diversity (0.27 ± 0.12), nucleotide diversity (0.0007 ± 0.0004), and pairwise difference of 0.286 ± 0.317. This is not surprising due to the geographic isolation of this population. Haplotype diversity was highest (1.00 ± 0.13) in the New Forest population, pairwise difference was highest (8.061 ± 4.028) in the Icelandic breed, whereas nucleotide diversity was highest in the Exmoor breed (0.0209 ± 0.0025). Within the Canadian populations, haplotype diversity was highest in the Newfoundland pony (0.96 ± 0.08), whereas pairwise difference and nucleotide diversity was highest in the Canadian horse (7.090 ± 3.581 and 0.0188 ± 0.0042, respectively). Three different estimates of genetic distances were used to examine the phylogenetic relationships amongst these populations. All 3 estimates produced similar topologies. In general, the native Canadian populations were highly represented in the D clade, with particular emphasis in the D1 and D2 clades. This is an important factor when considering the phylogenetic conservation of these Canadian equine populations.     

Lack of association between HLA class II alleles and in vitro replication capacities of recombinant viruses encoding HIV-1 subtype C Gag-protease from chronically infected individuals

It is unknown whether favorable HLA class II alleles may attenuate HIV-1 through selection pressure in a manner similar to that of protective HLA class I alleles. We investigated the relationship between HLA class II alleles and in vitro replication capacities of recombinant viruses encoding HIV-1 subtype C Gag-protease from chronically infected individuals. No associations were found between individual alleles and lower replication capacity, suggesting no significant HIV-1 attenuation by HLA class II-restricted Gag-specific CD4(+) T cell immune pressure.     

Effects of food quality on tissue-specific isotope ratios in the mussel <Emphasis Type="Italic">Perna perna</Emphasis>

Investigations into trophic ecology and aquatic food web resolution are increasingly accomplished through stable isotope analysis. The incorporation of dietary and metabolic changes over time results in variations in isotope signatures and turnover rates of producers and consumers at tissue, individual, population and species levels. Consequently, the elucidation of trophic relationships in aquatic systems depends on establishing standard isotope values and tissue turnover rates for the level in question. This study investigated the effect of diet and food quality on isotopic signatures of four mussel tissues: adductor muscle, gonad, gill and mantle tissue from the brown mussel Perna perna. In the laboratory, mussels were fed one of the two isotopically distinct diets for 3 months. Although not all results were significant, overall δ¹³C ratios in adductor, mantle and gill tissues gradually approached food source signatures in both diets. PERMANOVA analyses revealed significant changes over time in tissue δ¹³C (mantle and gill) with both diets and in δ¹⁵N (all tissues) and C:N ratios (mantle and gill) for one diet only. The percentage of replaced carbon isotopes were calculated for the 3 month period and differed among tissues and between diets. The tissue with the highest and lowest amount of replaced isotopes over 81 days were mantle tissue on the kelp diet (33.89%) and adductor tissue on the fish food diet (4.14%), respectively. Percentages could not be calculated for any tissue in either diet for δ¹⁵N due to the lack of significant change in tissue nitrogen. Fractionation patterns in tissues for both diets can be linked to nutritional stress, suggesting that consumer isotopic signatures are strongly dependent on food quality, which can significantly affect the degree of isotopic enrichment within a trophic level.     

The sphingosine 1-phosphate receptor 5 and sphingosine kinases 1 and 2 are localised in centrosomes: Possible role in regulating cell division

We show here that the endogenous sphingosine 1-phosphate 5 receptor (S1P₅, a G protein coupled receptor (GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 and SK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where they may participate in regulating mitosis. The centrosome is a site for active GTP–GDP cycling involving the G-protein, G_i and tubulin, which are required for spindle pole organization and force generation during cell division. Therefore, the presence of S1P₅ (which normally functions as a plasma membrane guanine nucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P₅ may function as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. The addition of S1P to cells inhibits trafficking of S1P₅ to the centrosome, suggesting a dynamic shuttling endocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that the centrosomal S1P₅ receptor might function as an intracellular target of S1P linked to regulation of mitosis.     

New aspects of sphingosine 1-phosphate signaling in mammalian cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid phosphate that binds to cell surface G-protein-coupled receptors (GPCR), but also can elicit intracellular actions. The role of S1P in cancer has been an area of significant interest and we have focused our research on two aspects that are of importance with respect to cancer. First, we have investigated how cross talk between S1P and growth factors might affect the pathophysiology of cancer cells. In this regard, we have demonstrated that S1P receptors function to re-programme the spatial signaling specificity of receptor tyrosine kinases and vice versa to modulate cell responses. Second, we have investigated spatial/temporal aspects of intracellular S1P signaling and how this might be de-regulated in cancer. This has involved studies on: (i) the interaction of sphingosine kinase 1 (which catalyses the phosphorylation of sphingosine to produce S1P) and phospholipase D in the Golgi apparatus linked to regulation of cell survival and (ii) the novel regulatory interaction between sphingosine kinase 1 and 2 and centrosomal S1P₅ receptor linked to the regulation of mitosis in mammalian cells including MDA-MB-231 breast cancer cells. Therefore, we have focused on novel aspects of spatial and temporal S1P signaling that might enable this bioactive lipid phosphate to exhibit normal and aberrant function in health and disease respectively.     

Recurrent distal 7q11.23 deletion including HIP1 and YWHAG identified in patients with intellectual disabilities, epilepsy, and neurobehavioral problems

We report 26 individuals from ten unrelated families who exhibit variable expression and/or incomplete penetrance of epilepsy, learning difficulties, intellectual disabilities, and/or neurobehavioral abnormalities as a result of a heterozygous microdeletion distally adjacent to the Williams-Beuren syndrome region on chromosome 7q11.23. In six families with a common recurrent ∼1.2 Mb deletion that includes the Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) genes and that is flanked by large complex low-copy repeats, we identified sites for nonallelic homologous recombination in two patients. There were no cases of this ∼1.2 Mb distal 7q11.23 deletion copy number variant identified in over 20,000 control samples surveyed. Three individuals with smaller, nonrecurrent deletions (∼180–500 kb) that include HIP1 but not YWHAG suggest that deletion of HIP1 is sufficient to cause neurological disease. Mice with targeted mutation in the Hip1 gene (Hip1^−/−) develop a neurological phenotype characterized by failure to thrive, tremor, and gait ataxia. Overall, our data characterize a neurodevelopmental and epilepsy syndrome that is likely caused by recurrent and nonrecurrent deletions, including HIP1. These data do not exclude the possibility that YWHAG loss of function is also sufficient to cause neurological phenotypes. Based on the current knowledge of Hip1 protein function and its proposed role in AMPA and NMDA ionotropic glutamate receptor trafficking, we believe that HIP1 haploinsufficiency in humans will be amenable to rational drug design for improved seizure control and cognitive and behavioral function.     

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6–16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6–16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6–16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.     

The sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole induces proteasomal degradation of sphingosine kinase 1 in mammalian cells

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.