Role of phosphatidylinositol 4-phosphate (PI4P) and its binding protein GOLPH3 in hepatitis C virus secretion.

Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.

Glycogen metabolism is required for optimal cyanobacterial growth in the rapid light-dark cycle of low-Earth orbit.

Some designs for bioregenerative life support systems to enable human space missions incorporate cyanobacteria for removal of carbon dioxide, generation of oxygen, and treatment of wastewater, as well as providing a source of nutrition. In this study, we examined the effects of the short light-dark (LD) cycle of low-Earth orbit on algal and cyanobacterial growth, approximating conditions on the International Space Station, which orbits Earth roughly every 90 min. We found that growth of green algae was similar in both normal 12 h light:12 h dark (12 h:12 h LD) and 45':45' LD cycles. Three diverse strains of cyanobacteria were not only capable of growth in short 45':45' LD cycles, but actually grew better than in 12 h:12 h LD cycles. We showed that 45':45' LD cycles do not affect the endogenous 24 h circadian rhythms of Synechococcus elongatus. Using a dense library of randomly barcoded transposon mutants, we identified genes whose loss is detrimental for the growth of S. elongatus under 45':45' LD cycles. These include several genes involved in glycogen metabolism and the oxidative pentose phosphate pathway. Notably, 45':45' LD cycles did not affect the fitness of strains that carry mutations in the biological circadian oscillator or the clock input and output regulatory pathways. Overall, this study shows that cultures of cyanobacteria could be grown under natural sunlight of low-Earth orbit and highlights the utility of a functional genomic study in a model organism to better understand key biological processes in conditions that are relevant to space travel.

Genetic variations in nitric oxide synthase 1 adaptor protein are associated with sudden cardiac death in US white community-based populations.

BACKGROUND: The ECG QT interval is associated with risk of sudden cardiac death (SCD). A previous genome-wide association study demonstrated that allelic variants (rs10494366 and rs4657139) in the nitric oxide synthase 1 adaptor protein (NOS1AP), which encodes a carboxy-terminal PDZ ligand of neuronal nitric oxide synthase, are associated with the QT interval in white adults. The present analysis was conducted to validate the association between NOS1AP variants and the QT interval and to examine the association with SCD in a combined population of 19 295 black and white adults from the Atherosclerosis Risk In Communities Study and the Cardiovascular Health Study. METHODS AND RESULTS: We examined 19 tagging single-nucleotide polymorphisms in the genomic blocks containing rs10494366 and rs4657139 in NOS1AP. SCD was defined as a sudden pulseless condition of cardiac origin in a previously stable individual. General linear models and Cox proportional hazards regression models were used. Multiple single-nucleotide polymorphisms in NOS1AP, including rs10494366, rs4657139, and rs16847548, were significantly associated with adjusted QT interval in whites (P<0.0001). In whites, after adjustment for age, sex, and study, the relative hazard of SCD associated with each C allele at rs16847548 was 1.31 (95% confidence interval 1.10 to 1.56, P=0.002), assuming an additive model. In addition, a downstream neighboring single-nucleotide polymorphism, rs12567209, which was not correlated with rs16847548 or QT interval, was also independently associated with SCD in whites (relative hazard 0.57, 95% confidence interval 0.39 to 0.83, P=0.003). Adjustment for QT interval and coronary heart disease risk factors attenuated but did not eliminate the association between rs16847548 and SCD, and such adjustment had no effect on the association between rs12567209 and SCD. No significant associations between tagging single-nucleotide polymorphisms in NOS1AP and either QT interval or SCD were observed in blacks. CONCLUSIONS: In a combined analysis of 2 population-based prospective cohort studies, sequence variations in NOS1AP were associated with baseline QT interval and the risk of SCD in white US adults.

Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting.

To facilitate the genetic engineering of diverse cyanobacterial strains, we have modified broad-host-range RSF1010-based plasmids to improve transmissibility, increase copy number, and facilitate cloning. RSF1010-based plasmids replicate in diverse bacterial strains but produce low amounts of useable DNA for cloning. We previously engineered a mobAY25F mutation in RSF1010-based plasmids that improved cloning but decreased conjugation efficiency. Here, we engineered RSF1010-based plasmids to restore conjugation efficiency, which was demonstrated in three diverse laboratory strains of cyanobacteria. We then used an improved RSF1010-based plasmid in mating experiments with cultured samples of wild cyanobacteria. This plasmid, which confers antibiotic resistance and carries a yfp reporter gene, allowed selection of exconjugant cyanobacteria and facilitated the isolation of genetically tractable strains from mixed wild cultures. Improved RSF1010 vectors can be used for bioprospecting genetically tractable strains and are compatible with the CYANO-VECTOR cloning system, a versatile toolbox for constructing plasmids for cyanobacterial genetic engineering.

Phosphoinositides in the hepatitis C virus life cycle.

Eukaryotes possess seven different phosphoinositides (PIPs) that help form the unique signatures of various intracellular membranes. PIPs serve as docking sites for the recruitment of specific proteins to mediate membrane alterations and integrate various signaling cascades. The spatio-temporal regulation of PI kinases and phosphatases generates distinct intracellular hubs of PIP signaling. Hepatitis C virus (HCV), like other plus-strand RNA viruses, promotes the rearrangement of intracellular membranes to assemble viral replication complexes. HCV stimulates enrichment of phosphatidylinositol 4-phosphate (PI4P) pools near endoplasmic reticulum (ER) sites by activating PI4KIIIα, the kinase responsible for generation of ER-specific PI4P pools. Inhibition of PI4KIIIα abrogates HCV replication. PI4P, the most abundant phosphoinositide, predominantly localizes to the Golgi and plays central roles in Golgi secretory functions by recruiting effector proteins involved in transport vesicle generation. The PI4P effector proteins also include the lipid-transfer and structural proteins such as ceramide transfer protein (CERT), oxysterol binding protein (OSBP) and Golgi phosphoprotein 3 (GOLPH3) that help maintain Golgi-membrane composition and structure. Depletion of Golgi-specific PI4P pools by silencing PI4KIIIß, expression of dominant negative CERT and OSBP mutants, or silencing GOLPH3 perturb HCV secretion. In this review we highlight the role of PIPs and specifically PI4P in the HCV life cycle.