No abstract

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype–phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café‐au‐lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan‐like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1‐patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi‐exon deletion, providing genetic evidence that p.Arg1809Cys is a loss‐of‐function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype–phenotype correlation will affect counseling and management of a significant number of patients.

bSterile alpha motif domain-and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.

Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. In addition to its role as a receptor for LukED, CCR5 is the major coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and has been extensively studied. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. In contrast, the second ECL of CCR5 is necessary but not sufficient for HIV-1 infectivity. The analysis of CCR5 point mutations showed that glycine-163 is critical for HIV-1 infectivity, while arginine-274 and aspartic acid-276 are critical for LukED cytotoxicity. Point mutations in ECL2 diminished both HIV-1 infectivity and LukED cytotoxicity. Treatment of cells with LukED did not interfere with CCR5-tropic HIV-1 infectivity, demonstrating that LukED and the viral envelope glycoprotein use nonoverlapping sites on CCR5. Analysis of point mutations in LukE showed that amino acids 64 to 69 in the rim domain are required for CCR5 targeting and cytotoxicity. Taking the results together, this study identified the molecular basis by which LukED targets CCR5, highlighting the divergent molecular interactions evolved by HIV-1 and LukED to interact with CCR5.

e Sterile alpha motif-and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways. Human immunodeficiency virus type 1 (HIV-1) replicates primarily in activated CD4 ϩ T cells, while showing poor reproductive capacity in monocytes, macrophages, dendritic cells, and resting CD4 ϩ T cells (1-10). Sterile alpha motif-and histidine/ aspartic acid domain-containing protein 1 (SAMHD1) is responsible for blocking HIV-1 replication in such cells (5,(11)(12)(13), reportedly by acting as a dGTP-stimulated deoxynucleotide triphosphohydrolase that hydrolyzes deoxynucleoside triphosphates (dNTPs), thus decreasing the amounts of dNTPs available for reverse transcription (3,4,(14)(15)(16)(17)(18)(19).Nucleoside reverse transcriptase inhibitors (NRTIs) are nucleoside analogs and key components of antiretroviral therapies (20-26). They generally lack a 3=-OH group and thus act as chain terminators upon incorporation into viral DNA by reverse transcriptase (RT) (26-29). However, 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) retains a 3=-OH group, acts primarily by blocking RT translocation following incorporation of EFdA monophosphate (MP) into the template-primer, and has picomolar antiviral potency (30-37). NRTIs are administered as nucleosides and are phosphorylated to their active forms by cellular kinases (38). Hence, they compete with dNTPs for activation by cellular kinases, and their incorporation by RT is influenced by the cellular concentrations of dNTPs, which compete with NRTI triphosphates (TPs) at the RT active site (39,40). Amie et al. (19) recently reported that SAMHD1 does not significantly hydrolyze dideoxynucleoside triphosphates (ddNTPs) or zidovudine (AZT)-TP and that depletion of SAMHD1 in monocytic THP-1 cells decreased the potency of these NRTIs in a pseudotype-based assay. Strong evidence that the decreased potency of these NRTIs was due to increased amounts of competing dNTPs was presented. Our parallel independent study confirmed their data, extended the number of NRTIs studied, validated the results with fully infectious HIV-1, and found an unexpected disparity in the effects of SAMHD1 on the deoxyribosylthymine (dT) analogs AZT and stavudine (d4T). We demonstrate that this is due to differences in the activation of AZT and d4T, highlighting the importance of distinct metabolic pathways in NRTI activation, in addition to competition with dNTPs.We tested purified Escherichia coli-produced recombinant SAMHD1 for dGTP-regulated NRTI-TP hydrolysis (using dNTPs as a re...