The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit.

Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses.

High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics.

The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed.

Label-free quantitative proteomic profiling identifies disruption of ubiquitin homeostasis as a key driver of Schwann cell defects in spinal muscular atrophy.

Low levels of survival of motor neuron (SMN) protein cause the neuromuscular disease spinal muscular atrophy (SMA), characterized by degeneration of lower motor neurons and atrophy of skeletal muscle. Recent work demonstrated that low levels of SMN also trigger pathological changes in Schwann cells, leading to abnormal axon myelination and disrupted deposition of extracellular matrix proteins in peripheral nerve. However, the molecular pathways linking SMN depletion to intrinsic defects in Schwann cells remained unclear. Label-free proteomics analysis of Schwann cells isolated from SMA mouse peripheral nerve revealed widespread changes to the Schwann cell proteome, including disruption to growth/proliferation, cell death/survival, and molecular transport pathways. Functional clustering analyses revealed significant disruption to a number of proteins contributing to ubiquitination pathways, including reduced levels of ubiquitin-like modifier activating enzyme 1 (Uba1). Pharmacological suppression of Uba1 in Schwann cells was sufficient to reproduce the defective myelination phenotype seen in SMA. These findings demonstrate an important role for SMN protein and ubiquitin-dependent pathways in maintaining Schwann cell homeostasis and provide significant additional experimental evidence supporting a key role for ubiquitin pathways and, Uba1 in particular, in driving SMA pathogenesis across a broad range of cells and tissues.

Novel blood collection method allows plasma proteome analysis from single zebrafish.

Zebrafish is an important model organism in biological research. One of the least explored tissues of zebrafish is blood, because the existing methods for isolating blood from this organism are tedious and irreproducible. The small volume of blood collected by these methods also prohibits many biochemical and cytological analyses. This technical obstacle limits the utilization of zebrafish in many applications, particularly in hematological research and plasma biomarker discovery. To overcome this limitation, we have established a novel method of extracting blood from zebrafish, based on the use of low centrifugal force to collect blood from a wound. This method consistently recovers more blood than traditional methods. Gel electrophoresis and flow cytometry showed that composition of blood harvested by this method is indistinguishable from traditional methods. The increase in yield enables us to perform biochemical experiments on zebrafish blood. In particular, we have demonstrated that quantitative proteomics can be performed on plasma collected from single zebrafish. Here, we have compared, by using shotgun proteomic analysis, the plasma proteomes of adult male and female zebrafish. Twenty-seven gender-dependent plasma proteins are identified and their biochemical importance discussed. Taken together, this novel technique enables analyses that were previously difficult to perform on zebrafish blood.