Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation

Cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis, effectively inhibited the formation and release of virus particles from chicken embryo fibroblasts infected with Sindbis or vesicular stomatitis virus (VSV). When added for 1 h at 3 h postinfection, the antibiotic blocked VSV particle production by 80 to 90% and inhibited incorporation of [3H]palmitic acid into the VSV glycoprotein by an equivalent amount. The effect of this antibiotic on virus protein and RNA biosynthesis was significantly less than that on fatty acid acylation. Nonacylated virus glycoproteins accumulated inside and on the surface of cerulenin-treated cells. These data indicate that fatty acid acylation is not essential for intracellular transport of these membrane proteins, but it may have an important role in the interaction of glycoproteins with membranes during virus assembly and budding.     

Defective interfering particles of Sindbis virus do not interfere with the homologous virus obtained from persistently infected BHK cells but do interfere with Semliki Forest virus.

Defective interfering particles derived from wild-type Sindbis virus no longer interfere with the infectious virus cloned from BHK cells persistently infected with Sindbis virus for 16 months. These particles do interfere with the replication of Semliki Forest virus.     

Fluorescence photobleaching recovery measurements reveal differences in envelopment of sindbis and vesicular stomatitis viruses

Fluorescence photobleaching recovery (FPR) measurements of virus glycoproteins on the surfaces of cells infected with vesicular stomatitis virus (VSV) and Sindbis virus showed that the VSV glycoprotein (G) remained mobile throughout the infectious cycle, whereas Sindbis virus glycoproteins (E1, E2) were partially mobile early after infection and immobile at later times when greater amounts of these proteins were on the cell surface. A highly mobile fraction of Sindbis virus glycoproteins was detected throughout the replication cycle of a temperature-sensitive mutant unable to form virus particles. Thus immobilization of E1 and E2 was the result of increasing surface glycoprotein concentrations and virus budding. Together with other data, which included the detection of E1 and E2 in particles as soon as these proteins were transported to the cell surface, the FPR results suggest that Sindbis virus assembly initiates on intracellular vesicles, where glycoproteins aggregate and bind nucleocapsids. In contrast, our FPR data on VSV support a model previously suggested by others, in which a small fraction of cell-surface G is immobilized into budding sites formed by interactions with virus matrix and nucleoproteins. FPR measurements also provide direct evidence for strong interactions between E1 and E2, as well as between E1 and PE2, the precursor form of E2.     

Clearance of lysosomal hydrolases following intravenous infusion. Kinetic and competition experiments with beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Clearance experiments with highly purified lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum β-glucuronidase and epididymal N-acetyl-β-d-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland β-glucuronidase, liver lysosomal β-glucuronidase, and liver lysosomal N-acetyl-β-d-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of β-glucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with β-glucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding.     

Clearance of lysosomal hydrolases following intravenous infusion. The role of liver in the clearance of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Certain highly purified forms of rat lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, are rapidly cleared from the circulation following intravenous infusion. Several lines of evidence are presented which indicate that the primary site of enzyme uptake is the liver. Clearance of the two enzymes was unaffected by nephrectomy, whereas it was abolished by evisceration. Tissue distribution experiments with native and [¹²⁵I]β-glucuronidase indicate the liver as the major, if not exclusive, site of enzyme uptake. Experiments with the isolated perfused liver showed clearance of certain enzyme preparations but not others. Those enzymes cleared by the isolated perfused liver were likewise cleared in vivo. Liver fractionation studies following infusion of large doses of β-glucuronidase revealed a rapid, short-lived increase in microsomal β-glucuronidase and a slower but larger increase in lysosomal β-glucuronidase. The results indicate that β-glucuronidase, N-acetyl-β-d-glucosaminidase, and probably other glycosidases are rapidly incorporated into the lysosomal compartment of liver.     

Isolation of a Sindbis virus variant by passage on mouse plasmacytoma cells.

A variant of Sindbis virus has been isolated by growing a stock of virus, previously passaged on chicken embryo cells, in mouse plasmacytoma (MOPC 315) cells in suspension culture. An indirect immunofluorescence test and infective center assay showed that only a small fraction of cells could be infected by the stock wild-type virus, but that the population of virus accumulating after a few passages on the mouse cells had host-range properties distinct from the stock virus. The mouse-passaged virus retained its virulence for the original host and back-passaging of this virus on chicken cells did not change its newly acquired properties. Thus, this variant appears to be a genetically distinct form of Sindbis that adsorbs to and grows much better than the stock virus on several types of mouse cells including cultures of mouse macrophages.     

Ranking buffel: Comparative risk and mitigation costs of key environmental and socio‐cultural threats in central Australia

Changed fire regimes and the introduction of rabbits, cats, foxes, and large exotic herbivores have driven widespread ecological catastrophe in Australian arid and semi‐arid zones, which encompass over two‐thirds of the continent. These threats have caused the highest global mammal extinction rates in the last 200 years, as well as significantly undermining social, economic, and cultural practices of Aboriginal peoples of this region. However, a new and potentially more serious threat is emerging. Buffel grass (Cenchrus ciliaris L.) is a globally significant invader now widespread across central Australia, but the threat this ecological transformer species poses to biodiversity, ecosystem function, and culture has received relatively little attention. Our analyses suggest threats from buffel grass in arid and semi‐arid areas of Australia are at least equivalent in magnitude to those posed by invasive animals and possibly higher, because unlike these more recognized threats, buffel has yet to occupy its potential distribution. Buffel infestation also increases the intensity and frequency of wildfires that affect biodiversity, cultural pursuits, and productivity. We compare the logistical and financial challenges of creating and maintaining areas free of buffel for the protection of biodiversity and cultural values, with the creation and maintenance of refuges from introduced mammals or from large‐scale fire in natural habitats. The scale and expense of projected buffel management costs highlight the urgent policy, research, and financing initiatives essential to safeguard threatened species, ecosystems, and cultural values of Aboriginal people in central Australia.     

Fine Tuning Inflammation at the Front Door: Macrophage Complement Receptor 3-mediates Phagocytosis and Immune Suppression for Francisella tularensis

Complement receptor 3 (CR3, CD11b/CD18) is a major macrophage phagocytic receptor. The biochemical pathways through which CR3 regulates immunologic responses have not been fully characterized. Francisella tularensis is a remarkably infectious, facultative intracellular pathogen of macrophages that causes tularemia. Early evasion of the host immune response contributes to the virulence of F. tularensis and CR3 is an important receptor for its phagocytosis. Here we confirm that efficient attachment and uptake of the highly virulent Type A F. tularensis spp. tularensis strain Schu S4 by human monocyte-derived macrophages (hMDMs) requires complement C3 opsonization and CR3. However, despite a>40-fold increase in uptake following C3 opsonization, Schu S4 induces limited pro-inflammatory cytokine production compared with non-opsonized Schu S4 and the low virulent F. novicida. This suggests that engagement of CR3 by opsonized Schu S4 contributes specifically to the immune suppression during and shortly following phagocytosis which we demonstrate by CD11b siRNA knockdown in hMDMs. This immune suppression is concomitant with early inhibition of ERK1/2, p38 MAPK and NF-κB activation. Furthermore, TLR2 siRNA knockdown shows that pro-inflammatory cytokine production and MAPK activation in response to non-opsonized Schu S4 depends on TLR2 signaling providing evidence that CR3-TLR2 crosstalk mediates immune suppression for opsonized Schu S4. Deletion of the CD11b cytoplasmic tail reverses the CR3-mediated decrease in ERK and p38 activation during opsonized Schu-S4 infection. The CR3-mediated signaling pathway involved in this immune suppression includes Lyn kinase and Akt activation, and increased MKP-1, which limits TLR2-mediated pro-inflammatory responses. These data indicate that while the highly virulent F. tularensis uses CR3 for efficient uptake, optimal engagement of this receptor down-regulates TLR2-dependent pro-inflammatory responses by inhibiting MAPK activation through outside-in signaling. CR3-linked immune suppression is an important mechanism involved in the pathogenesis of F. tularensis infection.