Identification of the P-TEFb complex-interacting domain of Brd4 as an inhibitor of HIV-1 replication by functional cDNA library screening in MT-4 cells

AbstractWe conducted a phenotypic cDNA screening using a T cell line-based assay to identify human genes that render cells resistant to human immunodeficiency virus type 1 (HIV-1). We isolated potential HIV-1 resistance genes, including the carboxy terminal domain (CTD) of bromodomain-containing protein 4 (Brd4). Expression of GFP-Brd4-CTD was tolerated in MT-4 and Jurkat cells in which HIV-1 replication was markedly inhibited. We provide direct experimental data demonstrating that Brd4-CTD serves as a specific inhibitor of HIV-1 replication in T cells. Our method is a powerful tool for the identification of host factors that regulate HIV-1 replication in T cells

Genotype and clinical characteristics of patients with Wolfram syndrome and WFS1-related disorders

Objective: Wolfram syndrome (WFS) is an autosomal recessive disorder associated with juvenile-onset diabetes mellitus, optic atrophy, diabetes insipidus, and sensorineural hearing loss. We sought to elucidate the relationship between genotypic and phenotypic presentations of Wolfram syndrome which would assist clinicians in classifying the severity and prognosis of Wolfram syndrome more accurately.Approach: Patient data from the Washington University International Registry and Clinical Study for Wolfram Syndrome and patient case reports were analyzed to select for patients with two recessive mutations in the WFS1 gene. Mutations were classified as being either nonsense/frameshift variants or missense/in-frame insertion/deletion variants. Missense/in-frame variants were further classified as transmembrane or non-transmembrane based on whether they affected amino acid residues predicted to be in transmembrane domains of WFS1. Statistical analysis was performed using Wilcoxon rank-sum tests with multiple test adjustment applied via the Bonferonni correction.Results: A greater number of genotype variants correlated with earlier onset and a more severe presentation of Wolfram syndrome. Secondly, non-sense and frameshift variants had more severe phenotypic presentations than missense variants, as evidenced by diabetes mellitus and optic atrophy emerging significantly earlier in patients with two nonsense/frameshift variants compared with zero or one nonsense/frameshift variants. In addition, the number of transmembrane in-frame variants demonstrated a statistically significant dose-effect on age of onset of diabetes mellitus and optic atrophy among patients with either one or two in-frame variants.Summary/Conclusion: The results contribute to our current understanding of the genotype-phenotype relationship of Wolfram syndrome, suggesting that alterations in coding sequences result in significant changes in the presentation and severity of Wolfram. The impact of these findings is significant, as the results will aid clinicians in predicting more accurate prognoses and pave the way for personalized treatments for Wolfram syndrome

Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-δ1 pleckstrin homology domain results in infectious pseudovirion production

The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-δ1 pleckstrin homology (PH) domain. PH–Gag–Pol PM targeting and viral production efficiencies were improved compared with Gag–Pol, consistent with the estimated increases in Gag–PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH–Gag–Pol and Gag–Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH–Gag–Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag

Repeated Intravaginal Inoculation of Zika Virus Protects Cynomolgus Monkeys from Subcutaneous Superchallenge

Zika virus (ZIKV) outbreaks in Central and South America caused severe public health problems in 2015 and 2016. These outbreaks were finally contained through several methods, including mosquito control using insecticides and repellents. Additionally, the development of herd immunity in these countries might have contributed to containing the epidemic. While ZIKV is mainly transmitted by mosquito bites and mucosal transmission via bodily fluids, including the semen of infected individuals, has also been reported. We evaluated the effect of mucosal ZIKV infection on continuous subcutaneous challenges in a cynomolgus monkey model. Repeated intravaginal inoculations of ZIKV did not induce detectable viremia or clinical symptoms, and all animals developed a potent neutralizing antibody, protecting animals from the subsequent subcutaneous superchallenge. These results suggest that viral replication at mucosal sites can induce protective immunity without causing systemic viremia or symptoms

自閉症におけるメチルコバラミン療法の予備的検討

我々は13例の自閉症患者でビタミンB_<12>(メチルコバラミン:Me-B_<12>)の治療効果をオープン試験で試みた.Me-B_<12>の治療開始時期は2歳3ヵ月から18歳で,Me-B_<12>を25～30μg/kg/day,6～25ヵ月間投与した.IQ/DQによる評価では,治療前は45±5.3(平均±標準誤差)であったが,治療後は52±6.0と有意な上昇が認められた(p=0.0199).CARS検査(小児自閉症評定尺度)では,治療前は35.9±1.6であったが,治療後は33.0±1.7(p=0.0008)と改善した.自然経過による改善とMe-B_<12>効果を鑑別するため,患者を年齢,知能でそれぞれ2群に分類し検討したところ,思春期群と低IQ群のCARSスコアーが,幼児・早期学童期群,高IQ群と同様に改善した.自閉症児において思春期は一般に症状改善に乏しく,症状の自然改善は高機能自閉症児に見られることから,この結果は, Me-B_<12>効果は自然経過とは異なると考えられた.検討はまだ予備的段階であるが,自閉症においてMe-B_<12>は治療薬として試みる価値があると考えられた.We conducted an open trial on the effect of methylcobalamin (Me-B_<12>) in 13 patients with autism. The patients' ages at the start of treatment ranged from 2 years and 3 months to 18 years. Me-B_<12> was administered at a dosage of 25-30 g/kg/day for 6-25 months. The intelligence and developmental quotient (IQ/DQ) after Me-B_<12>treatment was 52±6.0 (mean ± SE), which was significantly higher than before treatment (45±5.3) (p<0.05). The childhood autism rating scale (CARS) score improved significantly, from 35.9±1.6 before to 33.0±1.7 after treatment (p<0.001). To distinguish the effects of Me-B_<12> from natural developmental factors, the patients were divided into two subgroups, both according to age and according to intelligence. The CARS scores of the teenage and the low-IQ groups improved as much as those of the pre-early school age and the high-IQ groups. As adolescent autism patients usually show a decline in their condition, while some high function autism patients exhibit positive development, the improvement of CARS scores with Me-B_<12> treatment in the teenage and the low-IQ groups suggests that the Me-B_<12> effect is distinct from developmental factors. Although these data are very preliminary, Me-B_<12> is potentially beneficial in autism as an additional or alternative treatment

自閉症におけるメチルコバラミン療法の予備的検討

我々は13例の自閉症患者でビタミンB_(メチルコバラミン:Me-B_)の治療効果をオープン試験で試みた.Me-B_の治療開始時期は2歳3ヵ月から18歳で,Me-B_を25～30μg/kg/day,6～25ヵ月間投与した.IQ/DQによる評価では,治療前は45±5.3(平均±標準誤差)であったが,治療後は52±6.0と有意な上昇が認められた(p=0.0199).CARS検査(小児自閉症評定尺度)では,治療前は35.9±1.6であったが,治療後は33.0±1.7(p=0.0008)と改善した.自然経過による改善とMe-B_効果を鑑別するため,患者を年齢,知能でそれぞれ2群に分類し検討したところ,思春期群と低IQ群のCARSスコアーが,幼児・早期学童期群,高IQ群と同様に改善した.自閉症児において思春期は一般に症状改善に乏しく,症状の自然改善は高機能自閉症児に見られることから,この結果は, Me-B_効果は自然経過とは異なると考えられた.検討はまだ予備的段階であるが,自閉症においてMe-B_は治療薬として試みる価値があると考えられた.We conducted an open trial on the effect of methylcobalamin (Me-B_) in 13 patients with autism. The patients\u27 ages at the start of treatment ranged from 2 years and 3 months to 18 years. Me-B_ was administered at a dosage of 25-30 g/kg/day for 6-25 months. The intelligence and developmental quotient (IQ/DQ) after Me-B_treatment was 52±6.0 (mean ± SE), which was significantly higher than before treatment (45±5.3) (p from natural developmental factors, the patients were divided into two subgroups, both according to age and according to intelligence. The CARS scores of the teenage and the low-IQ groups improved as much as those of the pre-early school age and the high-IQ groups. As adolescent autism patients usually show a decline in their condition, while some high function autism patients exhibit positive development, the improvement of CARS scores with Me-B_ treatment in the teenage and the low-IQ groups suggests that the Me-B_ effect is distinct from developmental factors. Although these data are very preliminary, Me-B_ is potentially beneficial in autism as an additional or alternative treatment

Novel Postentry Inhibitor of Human Immunodeficiency Virus Type 1 Replication Screened by Yeast Membrane-Associated Two-Hybrid System▿

Human immunodeficiency virus (HIV) Gag protein targets to the plasma membrane and assembles into viral particles. In the next round of infection, the mature Gag capsids disassemble during viral entry. Thus, Gag plays a central role in the HIV life cycle. Using a yeast membrane-associated two-hybrid assay based on the SOS-RAS signaling system, we developed a system to measure the Gag-Gag interaction and isolated 6 candidates for Gag assembly inhibitors from a chemical library composed of 20,000 small molecules. When tested in the human MT-4 cell line and primary peripheral blood mononuclear cells, one of the candidates, 2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine (BMMP), displayed an inhibitory effect on HIV replication, although a considerably high dose was required. Unexpectedly, neither particle production nor maturation was inhibited by BMMP. Confocal microscopy confirmed that BMMP did not block Gag plasma membrane targeting. Single-round infection assays with envelope-pseudotyped and luciferase-expressing viruses revealed that BMMP inhibited HIV replication postentry but not simian immunodeficiency virus (SIV) or murine leukemia virus infection. Studies with HIV/SIV Gag chimeras indicated that the Gag capsid (CA) domain was responsible for the BMMP-mediated HIV postentry block. In vitro studies indicated that BMMP accelerated disassembly of HIV cores and, conversely, inhibited assembly of purified CA protein in a dose-dependent manner. Collectively, our data suggest that BMMP primarily targets the HIV CA domain and disrupts viral infection postentry, possibly through inducing premature disassembly of HIV cores. We suggest that BMMP is a potential lead compound to develop antiretroviral drugs bearing novel mechanisms of action