BackgroundCellular heterogeneity is present in almost all gene expression profiles. However, transcriptome analysis of tissue specimens often ignores the cellular heterogeneity present in these samples. Standard deconvolution algorithms require prior knowledge of the cell type frequencies within a tissue or their in vitro expression profiles. Furthermore, these algorithms tend to report biased estimations.ResultsHere, we describe a Digital Sorting Algorithm (DSA) for extracting cell-type specific gene expression profiles from mixed tissue samples that is unbiased and does not require prior knowledge of cell type frequencies.ConclusionsThe results suggest that DSA is a specific and sensitivity algorithm in gene expression profile deconvolution and will be useful in studying individual cell types of complex tissues.

Interferon-γ (IFN-γ) primes macrophages for enhanced inflammatory activation by Toll-like receptors (TLRs) and microbial killing, but little is known about the regulation of cell metabolism or mRNA translation during priming. We found that IFN-γ regulates human macrophage metabolism and translation by targeting the kinases mTORC1 and MNK that both converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of mTORC1 by IFN-γ was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages revealed that IFN-γ selectively modulates the macrophage translatome to promote inflammation, further reprogram metabolic pathways, and modulate protein synthesis. These results add IFN-γ-mediated metabolic reprogramming and translational regulation as key components of classical inflammatory macrophage activation.

Mitophagy is a form of autophagy that selectively removes damaged mitochondria. Impaired mitochondria can be tagged by the kinase PINK1, which triggers recruitment of the E3-ubiquitin ligase Parkin and subsequent mitochondrial sequestration within autophagosomes. We previously found that human parainfluenza virus type 3 (HPIV3) infection induces autophagy, but the type and mechanisms of autophagy induction remain unknown. Here, we show that matrix protein (M) of HPIV3 translocates to mitochondria and interacts with Tu translation elongation factor mitochondrial (TUFM). M-mediated mitophagy does not require the Parkin-PINK1 pathway but rather an interaction between M and the LC3 protein that mediates autophagosome formation. These interactions with both TUFM and LC3 are required for the induction of mitophagy and lead to inhibition of the type I interferon response. These results reveal that a viral protein is sufficient to induce mitophagy by bridging autophagosomes and mitochondria.

Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the detailed molecular mechanisms and functions of picornavirus-mediated regulation of SG formation are not clear. Here, we found that the 2A protease of a picornavirus, EV71, induced atypical stress granule (aSG), but not typical stress granule (tSG), formation via cleavage of eIF4GI. Furthermore, 2A was required and sufficient to inhibit tSGs induced by EV71 infection, sodium arsenite, or heat shock. Infection of 2A protease activity-inactivated recombinant EV71 (EV71-2AC110S) failed to induce aSG formation and only induced tSG formation, which is PKR and eIF2α phosphorylation-dependent. By using a Renilla luciferase mRNA reporter system and RNA fluorescence in situ hybridization assay, we found that EV71-induced aSGs were beneficial to viral translation through sequestering only cellular mRNAs, but not viral mRNAs. In addition, we found that the 2A protease of other picornaviruses such as poliovirus and coxsackievirus also induced aSG formation and blocked tSG formation. Taken together, our results demonstrate that, on one hand, EV71 infection induces tSG formation via the PKR-eIF2α pathway, and on the other hand, 2A, but not 3C, blocks tSG formation. Instead, 2A induces aSG formation by cleaving eIF4GI to sequester cellular mRNA but release viral mRNA, thereby facilitating viral translation.