Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

SummaryCritical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism.

Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.DOI: http://dx.doi.org/10.7554/eLife.18296.001

Graphical AbstractHighlights d Tumor-derived extracellular vesicles (TEV) downregulate IFNAR1 and CH25H d CH25H acts to restrict TEV uptake and limit the education of healthy cells d Downregulation of CH25H in normal cells promotes melanoma metastasis d Disruption of TEV uptake and education by reserpine elicits anti-metastatic effects SUMMARY Tumor-derived extracellular vesicles (TEV) ''educate'' healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy.

Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase‐catalyzed glycosylation of the best‐selling biotherapeutic Herceptin, an anti‐HER2 antibody. Precise MS analysis of the intact four‐chain Ab heteromultimer reveals nonspecific, non‐enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non‐natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or “glycorandomization”) were readily generated.