The APOBEC family of single-stranded (ss)DNA cytosine deaminases provides innate immunity against virus and transposon replication 1 – 4 . A well-studied mechanism is APOBEC3G restriction of HIV-1, which is counteracted by a virus-encoded degradation mechanism 1 – 4 . Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded (ds)DNA viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein-Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers 5 , utilizes a two-pronged approach to counteract restriction by APOBEC3B. The large subunit of the EBV ribonucleotide reductase, BORF2 6 , 7 , bound to APOBEC3B in proteomics studies and immunoprecipitation experiments. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. Upon lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titers, lower infectivity, and hypermutation. The Kaposi’s sarcoma herpesvirus (KSHV) homolog, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model in which the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B.

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

SummaryInitiation and resolution of interhomolog connections: crossover and non-crossover sites along mouse synaptonemal complexes

SUMMARY The RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) heptapeptide repeats (Y1-S2-P3-T4-S5-P6-S7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD interaction domains (CIDs) with CTD repeats phosphorylated at S2 and S7. Our high resolution crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an interesting example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N-and C-terminal tags and multiple promoters), is compatible with Gateway TM cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming. Molecular & Cellular Proteomics 9:811-823, 2010.The analysis of protein-protein interactions (PPIs) 1 and protein complexes is of central importance to biological research and facilitates our understanding of how molecular events drive phenotypic outcomes. Moreover, large scale protein interaction data can be used to generate protein interaction networks, which can then be used to predict disease genes and model biology in any living organism.A number of methods (e.g. yeast two-hybrid) have been developed to examine binary protein interactions in a systematic format and applied to model systems (1-8). However, affinity purification (AP) coupled with tandem MS has become the method of choice for the identification of protein complexes (9, 10). Large scale PPI studies using a high throughput and systematic AP-MS approach have been performed for Escherichia coli (11,12) and Saccharomyces cerevisiae (13-15). In fact, large scale efforts using AP-MS have connected an estimated 60% of the yeast proteome, demonstrating the power of coupling systematic biochemical purifications with mass spectrometry (13-16).AP-MS has also been used extensively for purification of mammalian protein complexes (17), but this has been mostly restricted to small scale studies and the use of either cell lines that are easy to transfect or highly validated antibodies against specific targets. For example, Glatter et al. (18) recently developed an integrated workflow where a high density interactome was developed for the protein phosphatase 2A complex. This workflow relies on "flip-in" technology to introduce transgenes into a common genomic site in HEK293 cells and, similar to work by other groups (19,20), utilizes an From the ‡Banting and Best

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is essential for viral replication, making it a promising target for antiviral drug and vaccine development. SARS-CoV-2 infected patients exhibit an uncoordinated immune response; however, the underlying mechanistic details of this imbalance remain obscure. Here, starting from a functional proteomics workflow, we catalogued the protein-protein interactions of SARS-CoV-2 proteins, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N localizes to stress granules and sequesters G3BPs away from their typical interaction partners, thus attenuating stress granule formation. We found that N binds directly to host mRNAs in cells, with a preference for 3´ UTRs, and modulates target mRNA stability. We show that the N protein rewires the G3BP1 mRNA-binding profile and suppresses the physiological stress response of host cells, which may explain the imbalanced immune response observed in SARS-CoV-2 infected patients.