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Removal of BFA allows the synthesis of PrPSc to continue.
The N terminus of ARF has been characterized as an effector domain in the generation of coated transport vesicles. Deletion of the first 17 amino acid residues results in a protein that lacks ARF activity, and synthetic peptides encoding the same residues were found to act as potent competitive inhibitors of the generation of coated vesicles from isolated Golgi membranes (). They are believed to compete for membrane-associated binding partners of ARF, which binds to the budding membrane in a saturable, site-specific manner ().
The apparently conflicting observations that poliovirus induces a secretory block but at the same time is inhibited by a drug that blocks transport suggest that viral genomic replication interacts with the secretory pathway in such a way as to disrupt its overall function. Simultaneously, it appears to recruit cellular components of the secretory pathway for its own use. In this study, we used a cell-free poliovirus replication system () to analyze this possible requirement. The system allows the faithful synthesis and processing of the polioviral polyprotein from input viral mRNA, genomic replication, and encapsidation of newly synthesized viral RNA to yield infectious particles (, , , , , , ). We have determined that poliovirus replication in the cell-free system is inhibited by 2 mM guanidine hydrochloride, a specific inhibitor of poliovirus replication in vivo (). Barton et al. () have reported that a soluble host cell fraction is required for the recovery of cell-free replication from the block induced by guanidine hydrochloride.
Total Synthesis of (+)-Brefeldin A - Organic Letters …
Brefeldin A is a reagent which causes the cis-medial and trans-Golgi cisternae to fuse with the ER and prevents exit of proteins from the ER-Golgi network (Doms et al., 1989).
Use of a scrapie-infected cell line which can support PrPSc production has shown that synthesis of PrPSc in the presence of agents which block N-linked glycosylation still allows the production of protease resistant PrPSc, albeit PrPSc produced in this fashion is unglycosylated (Taraboulos et al., 1990).
A simplified synthesis of (+)-brefeldin A - ScienceDirect
However, synthesis of a truncated PrP lacking the GPI anchor attachment signal still results in the production of PrPSc, thus eliminating this component as a necessary factor for PrPSc production (Rogers et al., 1993).
A signal sequence present near the C-terminus targets prion protein synthesis to the endoplasmic reticulum (ER), where the sequence is cleaved leaving ser-231 as a site for attachment of a glycosyl-phosphatidylinositol (GPI) group.
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The Raghavan Synthesis of Brefeldin A - Organic …
It is unclear whether ARF acts as a direct player in RNA replication. However, the role of ARF may be indirect since inhibition of ARF by BFA is itself indirect. It is more likely that the specific process in which ARF is normally involved, perhaps the activation of a step in the transport pathway, is relevant for replication in vivo. We hypothesize that infection somehow deregulates the transport pathway such that overall secretion is blocked, but the step inhibited by BFA, the ARF-dependent synthesis of secretory vesicles, remains active and is perhaps even stimulated by virus-encoded proteins such as 2BC, 2C, and/or 2B. As has been proposed by Maynell et al. () and Doedens et al. (), a block in the fusion of secretory vesicles with target membranes would result in their accumulation (, ), leading to the progressive disassembly of the donor organelle. These coated vesicles would eventually be uncoated through GTP hydrolysis, and the naked membranes may fuse nonspecifically, giving rise to the larger vacuolar structures seen during poliovirus infection of target cells (Fig. ). Viral replication complexes assemble at these membrane structures, forming the rosette structures described by Bienz et al. (). The recent observation that the replication-associated vesicles may originate primarily from the ER, Golgi, and lysosomes is consistent with a requirement for ARF activity in their generation, since evidence exists for possible ARF involvement in anterograde and retrograde transport from the ER to the Golgi (), intra-Golgi transport (), anterograde transport from the trans-Golgi network (), and endosomal function (, ). These transport steps are also affected by BFA (, ).
The Raghavan Synthesis of Brefeldin A
As yet, there is no direct evidence that virus-induced vesicular structures form in the cell-free replication system described here. However, the sensitivity of replication to BFA, coupled with an apparent requirement for a soluble cellular GTP-binding and -hydrolyzing activity and the inhibition of RNA replication by competitive inhibitors of ARF, suggests that the cellular pathway that produces transport vesicles in the normal cell, or a specific step thereof, is involved in RNA replication in vitro. We suggest that this may also be the case in vivo. It is unlikely that there is a functional secretory apparatus in the cell-free system, because we do not observe glycosylation and processing of a reporter protein (). However, our model requires only that the BFA-sensitive mechanism for generating transport vesicles be active, as it is in cell-free systems that reproduce the process (, ). These systems are similar in many respects to the cell-free replication system, making our interpretation of the results presented here, and their possible significance with regard to poliovirus RNA replication, a reasonable explanation of why BFA inhibits poliovirus RNA replication.
A synthesis of (±)-brefeldin A - ScienceDirect
We have observed that peptides which encode the N-terminal sequence of ARF proteins, and which are thought to act as competitive inhibitors for ARF interaction with binding partners during coated vesicle formation, are inhibitors of cell-free RNA replication. One possible explanation for this effect is that the peptides, which form amphipathic helices in aqueous solution, have a general detergent-like effect on membranes in the extract (), disrupting replication indirectly. Arguing against this possibility is the observation that P-13 and P-26 have unequal effects at equal concentrations, implying a specific mechanism of inhibition. Moreover, a truncated peptide (P-28) had little or no effect at equivalent concentrations. More importantly, inhibition by the P-13 peptide (consisting of the mARF1 N-terminal residues 2 to 17, identical to the corresponding human sequence) can be antagonized by the addition of increasing amounts of purified recombinant hARF1. This result is consistent with the inhibition occurring through competition and specifically suggests that the activity of ARF proteins is somehow required for viral RNA replication to occur efficiently in the cell-free system.
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