Drosophila S2 Cells Are Non-Permissive for Vaccinia Virus DNA Replication Following Entry via Low pH-Dependent Endocytosis and Early Transcription

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis

The Foundations of Agency - and Ethics?

In this article, I take off from some central issues in Paul Katsafanas' recent book Agency and the Foundations of Ethics. I argue that Katsafanas' alleged aims of action fail to do the work he requires them to do. First, his approach to activity or control is deeply problematic in the light of counterexamples, but as the related issues are substantially under-theorized, we do not at present know what agential activity or control may imply. More importantly, the view of activity or control he needs to get his argument going is most likely false, as it requires our values to do work that they are too fickle to do. Second, I take issue with the Nietzschean drive psychology underlying the second agential aim, viz. power. I argue that ordinary desires better describe a number of phenomena that Katsafanas uses drives to explain, and that some actions can aim in the opposite direction. As only drive-motivated actions aim at power, action does not, therefore, constitutively aim at power. Finally, I sketch a Humean approach to constitutivism, and argue that it both explains the desiderata that Katsafanas posits as well as solves the problems for his view. Constitutivists should prefer it to his view and develop it further

Dual Mechanism for the Translation of Subgenomic mRNA from Sindbis Virus in Infected and Uninfected Cells

Infection of BHK cells by Sindbis virus (SV) gives rise to a profound inhibition of cellular protein synthesis, whereas translation of viral subgenomic mRNA that encodes viral structural proteins, continues for hours. To gain further knowledge on the mechanism by which this subgenomic mRNA is translated, the requirements for some initiation factors (eIFs) and for the presence of the initiator AUG were examined both in infected and in uninfected cells. To this end, BHK cells were transfected with different SV replicons or with in vitro made SV subgenomic mRNAs after inactivation of some eIFs. Specifically, eIF4G was cleaved by expression of the poliovirus 2A protease (2Apro) and the alpha subunit of eIF2 was inactivated by phosphorylation induced by arsenite treatment. Moreover, cellular location of these and other translation components was analyzed in BHK infected cells by confocal microscopy. Cleavage of eIF4G by poliovirus 2Apro does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems. SV infection induces phosphorylation of eIF2α, a process that is increased by arsenite treatment. Under these conditions, translation of subgenomic mRNA occurs to almost the same extent as controls in the infected cells but is drastically inhibited in uninfected cells. Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells. Finally, immunolocalization of different eIFs reveals that eIF2 α and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions. These findings support the notion that canonical initiation takes place when the subgenomic mRNA is translated out of the infection context, while initiation can occur without some eIFs and even at non-AUG codons in infected cells

Constitutivism, Moral

Moral constitutivism purports to explain moral normativity by appeal to the nature of either agency or rational powers. Ambitious constitutivism aspires to ground the categorical authority of morality and to derive the content of the basic moral norms while avoiding the problems of moral realism. As a general strategy, moral constitutivism faces three serious challenges. First, the shmagency challenge. The worry is that the authority of the norms derived from the nature of agency is only conditional on having a reason to be an agent rather than some other kind of subject, a shmagent or an alienated agent. Constitutivists usually reply by appealing to the inescapability of agency. Second, there is a worry that agency is too thin a basis for the derivation of the substantive content of moral norms. Finally, there is the worry that constitutivism might be unable to make room for bad actions. The entry considers possible responses by moral constitutivism to these concerns and whether, if these responses are unsatisfactory, moral constitutivism might still have some explanatory power but of a less ambitious sort

Appraising the apoptotic mimicry model and the role of phospholipids for poxvirus entry

Entry of vaccinia virus (VACV) into cells occurs by fusion with the plasma membrane and via a low pH-dependent endosomal pathway, presumably involving unidentified cellular receptors. In addition to ≈25 viral proteins, the membrane of VACV mature virions contains several phospholipids including phosphatidylserine (PS). A recent model posits that PS flags virions as apoptotic debris to activate a common cellular uptake pathway to gain cell entry, perhaps through an interaction with a PS-specific cell surface receptor. To evaluate the apoptotic mimicry model, we reconstituted the membrane of detergent-extracted virions with several different phospholipids. Although the ability of the L-stereoisomer of PS to reconstitute infectivity was confirmed, the nonbiologically relevant D-stereoisomer of PS, and phosphatidylglycerol, which are not normally present in the virion membrane, functioned as well. Regardless of which phospholipid reconstituted infectivity, virus entry was inhibited by a neutralizing monoclonal antibody to a virion surface protein and by the drugs blebbistatin and bafilomycin A1, suggesting that in each case virus uptake was specific and occurred by a similar mechanism involving macropinocytosis and a low-pH endocytic pathway. Lipid-reconstituted and nonreconstituted, membrane-extracted virions were equally capable of binding to cells. However, the physical association of phospholipids with virus particles during membrane reconstitution correlated directly with rescue of particle infectivity and cell entry capability. Our results support a role for PS in poxvirus entry, but demonstrate that other phospholipids, not known to signal uptake of apoptotic debris, can function similarly