Residues Clustered in the Light-Sensing Knot of Phytochrome B are Necessary for Conformer-Specific Binding to Signaling Partner PIF3

The bHLH transcription factor, PHYTOCHROME INTERACTING FACTOR 3 (PIF3), interacts specifically with the photoactivated, Pfr, form of Arabidopsis phytochrome B (phyB). This interaction induces PIF3 phosphorylation and degradation in vivo and modulates phyB-mediated seedling deetiolation in response to red light. To identify missense mutations in the phyB N-terminal domain that disrupt this interaction, we developed a yeast reverse-hybrid screen. Fifteen individual mutations identified in this screen, or in previous genetic screens for Arabidopsis mutants showing reduced sensitivity to red light, were shown to also disrupt light-induced binding of phyB to PIF3 in in vitro co-immunoprecipitation assays. These phyB missense mutants fall into two general classes: Class I (eleven mutants) containing those defective in light signal perception, due to aberrant chromophore attachment or photoconversion, and Class II (four mutants) containing those normal in signal perception, but defective in the capacity to transduce this signal to PIF3. By generating a homology model for the three-dimensional structure of the Arabidopsis phyB chromophore-binding region, based on the crystal structure of Deinococcus radiodurans phytochrome, we predict that three of the four Class II mutated phyB residues are solvent exposed in a cleft between the presumptive PAS and GAF domains. This deduction suggests that these residues could be directly required for the physical interaction of phyB with PIF3. Because these three residues are also necessary for phyB-imposed inhibition of hypocotyl elongation in response to red light, they are functionally necessary for signal transfer from photoactivated phyB, not only to PIF3 and other related bHLH transcription factors tested here, but also to other downstream signaling components involved in regulating seedling deetiolation

Distinct roles for intracellular and extracellular lipids in hepatitis C virus infection

Hepatitis C is a chronic liver disease that contributes to progressive metabolic dysfunction. Infection of hepatocytes by hepatitis C virus (HCV) results in reprogramming of hepatic and serum lipids. However, the specific contribution of these distinct pools of lipids to HCV infection remains ill defined. In this study, we investigated the role of hepatic lipogenesis in HCV infection by targeting the rate-limiting step in this pathway, which is catalyzed by the acetyl-CoA carboxylase (ACC) enzymes. Using two structurally unrelated ACC inhibitors, we determined that blockade of lipogenesis resulted in reduced viral replication, assembly, and release. Supplementing exogenous lipids to cells treated with ACC inhibitors rescued HCV assembly with no effect on viral replication and release. Intriguingly, loss of viral RNA was not recapitulated at the protein level and addition of 2-bromopalmitate, a competitive inhibitor of protein palmitoylation, mirrored the effects of ACC inhibitors on reduced viral RNA without a concurrent loss in protein expression. These correlative results suggest that newly synthesized lipids may have a role in protein palmitoylation during HCV infection