Interaction between the Rev1 C-Terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis

Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of “inserter” to “extender” DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the “inserter” Polη, Polι, or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit “extender” Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

An integrated map of HIV-human protein complexes that facilitate viral infection.

Recent proteomic and genetic studies have aimed to identify a complete network of interactions between HIV and human proteins and genes. This HIV-human interaction network provides invaluable information as to how HIV exploits the host machinery and can be used as a starting point for further functional analyses. We integrated this network with complementary datasets of protein function and interaction to nominate human protein complexes with likely roles in viral infection. Based on our approach we identified a global map of 40 HIV-human protein complexes with putative roles in HIV infection, some of which are involved in DNA replication and repair, transcription, translation, and cytoskeletal regulation. Targeted RNAi screens were used to validate several proteins and complexes for functional impact on viral infection. Thus, our HIV-human protein complex map provides a significant resource of potential HIV-host interactions for further study

Workflow Overview.

<p>HIV-interacting proteins and RNAi phenotypes are mapped to a network of human protein functional interactions (yellow and red nodes respectively). Network propagation is performed separately for each of these two mappings. Significant genes are selected based on the combination of both propagation results (blue nodes). Finally, enriched HIV-human protein complexes are identified within the list of significant genes (HIV proteins added as green nodes, protein complexes highlighted by circles).</p

Predictive power and statistical results.

<p>(A) Pearson correlation of RNAi and APMS network propagation scores (green dot). The green line shows the density plot of random correlation coefficients based on permuting the relationship between network nodes and protein names. Note that random correlation is not zero due to the network structure of HumanNet which is not randomized. (B) The RNAi and APMS network propagation scores for each protein. Blue dots are proteins significant in both propagations. (C) ROC curve showing the predictive power of RNAi-propagation (blue) and APMS-propagation (red).</p

Selected complexes and RNAi screening results.

<p>(A) Profilin-1 complex interacting with GP160. (B) DNA-PK-Ku-eIF2-NF90-NF20 complex interacting with NC. (C) LARC complex interacting with Gag. Interactions within the complex represent functional interactions from HumanNet (green), manually curated interactions from the Metabase resource (gray) or from both sources (red). Pink vs. turquoise stars correspond to proteins that were confirmed in our RNAi validation screen vs. previous screens, respectively. Orange nodes are kinases, red transcription factors, blue are binding proteins as classified in Metabase. The bar plots show the HIV luciferase activity of the sample normalized by the HIV luciferase activity of control siRNAs. (D) HIV luciferase activity for three non-targeting siRNAs (positive controls) and luciferase-targeting siGL3 (negative control) performed simultaneously with siRNA transfections shown in A, B, and C.</p

Comparison of results from different studies.

<p>The table lists all protein complexes identified by our method, as well as the complexes identified in three previous analyses from Jaeger et al, Murali et al, and Bushman et al. Bold complexes correspond to those uniquely identified in our study, italic to those identified by us and by at least one previous study. The remainder corresponds to protein complexes identified in previous analyses only.</p

Map of HIV-human protein complexes.

<p>40 identified human protein complexes are shown together with the HIV protein targeting the complex. Green rectangles correspond to HIV proteins. Human complexes are shown as ellipses. A color gradient from red (high) to yellow (low) indicates the average rank of the complex in the APMS- and RNAi-propagations. Node size corresponds to number of subunits in the complex. Gray edges represent functional interactions between the human complexes; green edges are HIV-human interactions. Purple boxes indicate protein complexes that were selected for follow-up RNAi screens.</p

Validation of mRNA knock-down by siRNAs found to alter HIV infection.

<p>293T cells were transfected with siRNA against the identified genes and two non-targeting scramble siRNAs. 72 h post-transfection, total RNA was harvested and used to make a cDNA library. The presence of the target gene and a housekeeping gene, TBP, was measured using QPCR. Target gene levels were normalized to TBP within in each sample. Values reported are normalized target gene levels compared to values observed in transfections with non-targeting scramble siRNAs.</p