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. HCV-infected cells display a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. 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 pools by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without affecting replication. Reducing PI4P levels via knockdown of PI4 kinase IIIb led to the same effect. We then examined the role of PI4P binding proteins GOLPH3, FAPP1 and FAPP2 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. 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

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.

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.