The Torsin Activator LULL1 Is Required for Efficient Growth of Herpes Simplex Virus 1.

UNLABELLED: TorsinA is a membrane-tethered AAA+ ATPase implicated in nuclear envelope dynamics as well as the nuclear egress of herpes simplex virus 1 (HSV-1). The activity of TorsinA and the related ATPase TorsinB strictly depends on LAP1 and LULL1, type II transmembrane proteins that are integral parts of the Torsin/cofactor AAA ring, forming a composite, membrane-spanning assembly. Here, we use CRISPR/Cas9-mediated genome engineering to create single- and double knockout (KO) cell lines of TorA and TorB as well as their activators, LAP1 and LULL1, to investigate the effect on HSV-1 production. Consistent with LULL1 being the more potent Torsin activator, a LULL1 KO reduces HSV-1 growth by one order of magnitude, while the deletion of other components of the Torsin system in combination causes subtle defects. Notably, LULL1 deficiency leads to a 10-fold decrease in the number of viral genomes per host cell without affecting viral protein production, allowing us to tentatively assign LULL1 to an unexpected role that precedes HSV-1 nuclear egress. IMPORTANCE: In this study, we conduct the first comprehensive genetic and phenotypic analysis of the Torsin/cofactor system in the context of HSV-1 infection, establishing LULL1 as the most important component of the Torsin system with respect to viral production.

Arresting a Torsin ATPase reshapes the endoplasmic reticulum.

Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures.

Novel N-substituted benzimidazole CXCR4 antagonists as potential anti-HIV agents.

The lead optimization of a series of N-substituted benzimidazole CXCR4 antagonists is described. Side chain modifications and stereochemical optimization led to substantial improvements in potency and protein shift to afford compounds with low nanomolar anti-HIV activity.

Substituted tetrahydro-beta-carbolines as potential agents for the treatment of human papillomavirus infection.

The identification and optimization of a series of substituted tetrahydro-beta-carbolines with potent activity against human papillomavirus is described. Structure-activity studies focused on the substitution pattern and chirality of the beta-carboline ring system are discussed. Optimization of these parameters led to compounds with antiviral activities in the low nanomolar range.

Amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines as CXCR4 antagonists with potent activity against HIV-1.

Several novel amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines were synthesized which had potent activity against HIV-1. The synthetic approaches adopted allowed for variation of the substitution pattern and resulting changes in antiviral activity are highlighted. This led to the identification of compounds with low and sub-nanomolar anti-HIV-1 activity.

Pelger-Huët anomaly and Greenberg skeletal dysplasia: LBR-associated diseases of cholesterol metabolism.

Lamin B Receptor (LBR) is an inner nuclear membrane protein associated with the rare human diseases Pelger-Huët anomaly and Greenberg skeletal dysplasia. A new study has used CRISPR/Cas9-mediated genetic manipulations in a human cell system to determine that the molecular etiology of these previously poorly understood disorders is a defect in cholesterol synthesis due to loss of LBR-associated sterol C14 reductase activity. The study furthermore determined that disease-associated LBR point mutations reduce sterol C14 reductase activity by decreasing the affinity of LBR for the reducing agent NADPH. Moreover, two disease-associated LBR truncation mutants were found to be highly unstable at the protein level and are rapidly turned over by a novel nuclear membrane-based protein quality control pathway. Thus, truncated LBR variants can now be used as model substrates for further investigations of nuclear protein quality control to uncover possible implications for other disease-associated nuclear envelopathies.

Imidazopyridazine hepatitis C virus polymerase inhibitors. Structure-activity relationship studies and the discovery of a novel, traceless prodrug mechanism.

By reducing the basicity of the core heterocycle in a series of HCV NS5B inhibitors, the hERG liability was reduced. The SAR was then systematically explored in order to increase solubility and enable dose escalation while retaining potency. During this exploration, a facile decarboxylation was noted and was exploited as a novel prodrug mechanism. The synthesis and characterization of these prodrugs and their utilization in chronic toxicity studies are presented.

Discovery of a potent boronic acid derived inhibitor of the HCV RNA-dependent RNA polymerase.

A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild-type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a subgenomic replication system for a series of non-nucleoside boron-containing HCV RNA-dependent RNA polymerase (NS5B) inhibitors are described. A summary of the discovery of 3 (GSK5852), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included.

Ultra-potent P1 modified arylsulfonamide HIV protease inhibitors: the discovery of GW0385.

A novel series of P1 modified HIV protease inhibitors was synthesized and evaluated for in vitro antiviral activity against wild-type virus and protease inhibitor-resistant viruses. Optimization of the P1 moiety resulted in compounds with femtomolar enzyme activities and cellular antiviral activities in the low nanomolar range culminating in the identification of clinical candidate GW0385.

Pyrazolopyridine antiherpetics: SAR of C2' and C7 amine substituents.

A novel series of potent pyrazolo[1,5-a]pyridine inhibitors of herpes simplex virus 1 replication have been identified. Several complimentary synthetic methods were developed to allow facile access to a diverse set of analogs from common late stage intermediates. Detailed examination of the amine substituents at the C2' position of the pyrimidine and C7 position of the core pyrazolopyridine is described. The antiviral data suggests that non-polar amines are preferred for optimal activity. Additionally, the 2' position has been shown to require an NH group to retain activity levels similar to that of the gold standard acyclovir.

Synthesis of novel substituted 2-phenylpyrazolopyridines with potent activity against herpesviruses.

Herpesviruses are a significant source of human disease; amongst these herpes simplex virus 1 (HSV-1) and HSV-2 are very prevalent and cause recurrent infections. We recently identified a pyrazolo[1,5-a]pyridine scaffold that showed promising activity against HSV-1 and HSV-2 in Vero cell antiviral assays. Here, we describe the synthesis and anti-herpetic activity of several 3-pyrimidinyl-2-phenylpyrazolo[1,5-a]pyridines with differing 2-phenyl substitution patterns. Approaches to rapidly access a number of analogs with different 2-phenyl substitution patterns are outlined. Several of the compounds described have comparable activity to acyclovir against HSV-1 and HSV-2.

Statistical analysis of inappropriate results from current Hb screening methods for blood donors.

BACKGROUND: The objective was to apply statistical analysis to the false passes and fails that occur with the primary and secondary Hb-screening methods used at blood-donor sessions. STUDY DESIGN AND METHODS: Venous samples from 1513 potential donors who had undergone primary CuSO4 screening using capillary blood (Hb cut-offs: women, 125 g/L; men, 135 g/L) were tested at the session by a secondary method (HemoCue; cut-offs: women, 120 g/L; men, 130 g/L) and again at the base laboratory using another system (Beckman Coulter General S system), which generated the "true" Hb value. RESULTS: False-pass and -fail rates for women and men, respectively, were 11.2 and 6.3 percent (women) and 5.2 and 1.8 percent (men) for CuSO4; 1.9 and 3.7 percent (women) and 1.5 and 0.4 percent (men) for HemoCue; and 2.7 and 2.4 percent (women) and 1.8 and 0.2 percent (men) for a combined procedure that mimicked current practice of only testing CuSO4 fails by HemoCue. CONCLUSION: CuSO4 Hb screening gives large numbers of false passes, particularly in women. Using venous samples, the majority correctly pass at the lower HemoCue cut-offs. The current dual-testing policy appears convenient for donor sessions, but because small percentages of false passes and fails represent large numbers of donors, every effort should be made to improve the accuracy of Hb screening.