Publications by authors named "Reuben S Harris"

176 Publications

Induction of APOBEC3 exacerbates DNA replication stress and chromosomal instability in early breast and lung cancer evolution.

Cancer Discov 2021 May 4. Epub 2021 May 4.

The Francis Crick Institute.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast DCIS, and in pre-invasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx pre-invasive to invasive NSCLC lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models, revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in pre-invasive disease, providing fuel for selection early in cancer evolution.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0725DOI Listing
May 2021

Structural Characterization of a Minimal Antibody against Human APOBEC3B.

Viruses 2021 04 12;13(4). Epub 2021 Apr 12.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.
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http://dx.doi.org/10.3390/v13040663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070380PMC
April 2021

Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor.

Viruses 2021 02 12;13(2). Epub 2021 Feb 12.

School of Fundamental Sciences, Massey University, Private Bag 11 222, New Zealand.

In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.
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http://dx.doi.org/10.3390/v13020290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918907PMC
February 2021

APOBECs and Herpesviruses.

Viruses 2021 02 28;13(3). Epub 2021 Feb 28.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

The APOBEC family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes - APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new host-pathogen conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation.
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http://dx.doi.org/10.3390/v13030390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998176PMC
February 2021

Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455;

Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.
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http://dx.doi.org/10.1073/pnas.2021120118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958190PMC
March 2021

Dual Functionality of HIV-1 Vif in APOBEC3 Counteraction and Cell Cycle Arrest.

Front Microbiol 2020 12;11:622012. Epub 2021 Jan 12.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, United States.

Accessory proteins are a key feature that distinguishes primate immunodeficiency viruses such as human immunodeficiency virus type I (HIV-1) from other retroviruses. A prime example is the virion infectivity factor, Vif, which hijacks a cellular co-transcription factor (CBF-β) to recruit a ubiquitin ligase complex (CRL5) to bind and degrade antiviral APOBEC3 enzymes including APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H). Although APOBEC3 antagonism is essential for viral pathogenesis, and a more than sufficient functional justification for Vif's evolution, most viral proteins have evolved multiple functions. Indeed, Vif has long been known to trigger cell cycle arrest and recent studies have shed light on the underlying molecular mechanism. Vif accomplishes this function using the same CBF-β/CRL5 ubiquitin ligase complex to degrade a family of PPP2R5 phospho-regulatory proteins. These advances have helped usher in a new era of accessory protein research and fresh opportunities for drug development.
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http://dx.doi.org/10.3389/fmicb.2020.622012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835321PMC
January 2021

Demystifying Cell Cycle Arrest by HIV-1 Vif.

Trends Microbiol 2021 May 19;29(5):381-384. Epub 2021 Jan 19.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

Although APOBEC3 degradation is the canonical function of HIV-1 Vif, this viral protein also induces potent cell cycle arrest through a newly defined mechanism. Here, we review recent advances in this area and propose that the scope of this activity may go beyond subversion of the host cell cycle.
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http://dx.doi.org/10.1016/j.tim.2021.01.001DOI Listing
May 2021

R-Spondins 2 and 3 Are Overexpressed in a Subset of Human Colon and Breast Cancers.

DNA Cell Biol 2021 Jan 15;40(1):70-79. Epub 2020 Dec 15.

Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

Wnt signaling is activated in many cancer types, yet targeting the canonical Wnt pathway has been challenging for cancer therapy. The pathway might be effectively targeted at many levels depending on the mechanism by which it has become hyperactive. Recently, mouse genetic screens have found that R-spondins (RSPOs) act as oncogenes. Evidence includes recurrent genomic rearrangements that led to increased or expression in human colorectal adenocarcinomas, exclusive of mutations. RSPOs modulate Wnt signaling to promote epithelial cell proliferation and survival. These secreted proteins modulate Wnt signaling by binding to G-coupled receptors LGR4/5/6, ultimately inhibiting frizzled membrane clearance by RNF43 and ZNRF3. They also exert their function independent of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) by binding to ZNRF3 and RNF43. This results in increased β-catenin concentration that, after translocation to the nucleus, acts as a transcriptional coactivator of genes necessary for proliferation and cell survival. In this article, we aimed to identify the role of in colon and breast cancers by using and studies. We found that expression of and at high levels characterized a subset of colorectal cancers (CRCs). expression was found to characterize a subset of triple-negative breast cancers. In both instances, increased expression of was associated with an activated Wnt signaling gene expression profile. Furthermore, knockdown of decreased Wnt signaling and proliferation in human breast cancer cells. Our findings show and confirm that and expression is upregulated in a subset of colorectal adenocarcinomas and breast cancers and that both are attractive druggable oncoprotein targets against such cancers. We also describe novel fusion transcripts that occur in CRC.
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http://dx.doi.org/10.1089/dna.2020.5585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821429PMC
January 2021

Gain-of-function assay for SARS-CoV-2 M inhibition in living cells.

bioRxiv 2020 Nov 9. Epub 2020 Nov 9.

The main protease, M , of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here we demonstrate a quantitative reporter for M function in living cells, in which protease inhibition by genetic or chemical methods results in strong eGFP fluorescence. This robust gain-of-function system readily distinguishes between inhibitor potencies and can be scaled-up to high-throughput platforms for drug testing.
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http://dx.doi.org/10.1101/2020.11.09.375139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668733PMC
November 2020

Characterization of the mechanism by which the RB/E2F pathway controls expression of the cancer genomic DNA deaminase APOBEC3B.

Elife 2020 09 28;9. Epub 2020 Sep 28.

Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Institute for Molecular Virology, Center for Genome Engineering, University of Minnesota, Minneapolis, United States.

APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in breast cancer. Molecular mechanisms responsible for upregulation in cancer are poorly understood. Here we show that a single E2F cis-element mediates repression in normal cells and that expression is activated by its mutational disruption in a reporter construct or the endogenous gene. The same E2F site is required for induction by polyomavirus T antigen indicating a shared molecular mechanism. Proteomic and biochemical experiments demonstrate the binding of wildtype but not mutant E2F promoters by repressive PRC1.6/E2F6 and DREAM/E2F4 complexes. Knockdown and overexpression studies confirm the involvement of these repressive complexes in regulating A3B expression. Altogether, these studies demonstrate that expression is suppressed in normal cells by repressive E2F complexes and that viral or mutational disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution.
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http://dx.doi.org/10.7554/eLife.61287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553775PMC
September 2020

A role for gorilla APOBEC3G in shaping lentivirus evolution including transmission to humans.

PLoS Pathog 2020 09 10;16(9):e1008812. Epub 2020 Sep 10.

Laboratory of Systems Virology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.

The APOBEC3 deaminases are potent inhibitors of virus replication and barriers to cross-species transmission. For simian immunodeficiency virus (SIV) to transmit to a new primate host, as happened multiple times to seed the ongoing HIV-1 epidemic, the viral infectivity factor (Vif) must be capable of neutralizing the APOBEC3 enzymes of the new host. Although much is known about current interactions of HIV-1 Vif and human APOBEC3s, the evolutionary changes in SIV Vif required for transmission from chimpanzees to gorillas and ultimately to humans are poorly understood. Here, we demonstrate that gorilla APOBEC3G is a factor with the potential to hamper SIV transmission from chimpanzees to gorillas. Gain-of-function experiments using SIVcpzPtt Vif revealed that this barrier could be overcome by a single Vif acidic amino acid substitution (M16E). Moreover, degradation of gorilla APOBEC3F is induced by Vif through a mechanism that is distinct from that of human APOBEC3F. Thus, our findings identify virus adaptations in gorillas that preceded and may have facilitated transmission to humans.
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http://dx.doi.org/10.1371/journal.ppat.1008812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482973PMC
September 2020

APOBEC3A catalyzes mutation and drives carcinogenesis in vivo.

J Exp Med 2020 12;217(12)

Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN.

The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members, including APOBEC3B, fail to promote liver tumor formation. Tumor DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor datasets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.
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http://dx.doi.org/10.1084/jem.20200261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953736PMC
December 2020

Functional and Structural Insights into a Vif/PPP2R5 Complex Elucidated Using Patient HIV-1 Isolates and Computational Modeling.

J Virol 2020 10 14;94(21). Epub 2020 Oct 14.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G/M cell cycle arrest. Vif initiates G/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-β/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis. A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.
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http://dx.doi.org/10.1128/JVI.00631-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565612PMC
October 2020

Endogenous APOBEC3B overexpression characterizes HPV-positive and HPV-negative oral epithelial dysplasias and head and neck cancers.

Mod Pathol 2021 02 6;34(2):280-290. Epub 2020 Jul 6.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.

The DNA cytosine deaminase APOBEC3B (A3B) is a newly recognized endogenous source of mutations in a range of human tumors, including head/neck cancer. A3B inflicts C-to-T and C-to-G base substitutions in 5'-TCA/T trinucleotide motifs, contributes to accelerated rates of tumor development, and affects clinical outcomes in a variety of cancer types. High-risk human papillomavirus (HPV) infection causes A3B overexpression, and HPV-positive cervical and head/neck cancers are among tumor types with the highest degree of APOBEC signature mutations. A3B overexpression in HPV-positive tumor types is caused by the viral E6/E7 oncoproteins and may be an early off-to-on switch in tumorigenesis. In comparison, less is known about the molecular mechanisms responsible for A3B overexpression in HPV-negative head/neck cancers. Here, we utilize an immunohistochemical approach to determine whether A3B is turned from off-to-on or if it undergoes a more gradual transition to overexpression in HPV-negative head/neck cancers. As positive controls, almost all HPV-positive oral epithelial dysplasias and oropharyngeal cancers showed high levels of nuclear A3B staining regardless of diagnosis. As negative controls, A3B levels were low in phenotypically normal epithelium adjacent to cancer and oral epithelial hyperplasias. Interestingly, HPV-negative and low-grade oral epithelial dysplasias showed intermediate A3B levels, while high-grade oral dysplasias showed high A3B levels similar to oral squamous cell carcinomas. A3B levels were highest in grade 2 and grade 3 oral squamous cell carcinomas. In addition, a strong positive association was found between nuclear A3B and Ki67 scores suggesting a linkage to the cell cycle. Overall, these results support a model in which gradual activation of A3B expression occurs during HPV-negative tumor development and suggest that A3B overexpression may provide a marker for advanced grade oral dysplasia and cancer.
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http://dx.doi.org/10.1038/s41379-020-0617-xDOI Listing
February 2021

Structural basis of host protein hijacking in human T-cell leukemia virus integration.

Nat Commun 2020 06 19;11(1):3121. Epub 2020 Jun 19.

Masonic Cancer Center, University of Minnesota, 2231 6th Street S.E., Minneapolis, MN, 55455, USA.

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Å resolution. The structure together with functional analyses reveal that the B56γ (B'γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses.
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http://dx.doi.org/10.1038/s41467-020-16963-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305164PMC
June 2020

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B.

FASEB Bioadv 2020 Jan 24;2(1):49-58. Epub 2019 Dec 24.

Department of Biochemistry, Molecular Biology and Biophysics University of Minnesota Minneapolis MN USA.

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.
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http://dx.doi.org/10.1096/fba.2019-00068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996314PMC
January 2020

MagnEdit-interacting factors that recruit DNA-editing enzymes to single base targets.

Life Sci Alliance 2020 04 24;3(4). Epub 2020 Feb 24.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA

Although CRISPR/Cas9 technology has created a renaissance in genome engineering, particularly for gene knockout generation, methods to introduce precise single base changes are also highly desirable. The covalent fusion of a DNA-editing enzyme such as APOBEC to a Cas9 nickase complex has heightened hopes for such precision genome engineering. However, current cytosine base editors are prone to undesirable off-target mutations, including, most frequently, target-adjacent mutations. Here, we report a method to "attract" the DNA deaminase, APOBEC3B, to a target cytosine base for specific editing with minimal damage to adjacent cytosine bases. The key to this system is fusing an APOBEC-interacting protein (not APOBEC itself) to Cas9n, which attracts nuclear APOBEC3B transiently to the target site for editing. Several APOBEC3B interactors were tested and one, hnRNPUL1, demonstrated proof-of-concept with successful C-to-T editing of episomal and chromosomal substrates and lower frequencies of target-adjacent events.
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http://dx.doi.org/10.26508/lsa.201900606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7043409PMC
April 2020

The DNA Cytosine Deaminase APOBEC3B is a Molecular Determinant of Platinum Responsiveness in Clear Cell Ovarian Cancer.

Clin Cancer Res 2020 07 14;26(13):3397-3407. Epub 2020 Feb 14.

Department of Biochemistry, Molecular Biology and Biophysics, College of Biological Sciences, University of Minnesota, Minneapolis, Minnesota.

Purpose: Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention.

Experimental Designs: APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens ( = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates ( = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages.

Results: One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival ( = 0.026) and moderately with overall survival ( = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues.

Conclusions: These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334080PMC
July 2020

HIV-1 Vif Triggers Cell Cycle Arrest by Degrading Cellular PPP2R5 Phospho-regulators.

Cell Rep 2019 10;29(5):1057-1065.e4

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

HIV-1 Vif hijacks a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes and PP2A phosphatase regulators (PPP2R5A-E). APOBEC3 counteraction is essential for viral pathogenesis. However, Vif also functions through an unknown mechanism to induce G2 cell cycle arrest. Here, deep mutagenesis is used to define the Vif surface required for PPP2R5 degradation and isolate a panel of separation-of-function mutants (PPP2R5 degradation-deficient and APOBEC3G degradation-proficient). Functional studies with Vif and PPP2R5 mutants were combined to demonstrate that PPP2R5 is, in fact, the target Vif degrades to induce G2 arrest. Pharmacologic and genetic approaches show that direct modulation of PP2A function or depletion of specific PPP2R5 proteins causes an indistinguishable arrest phenotype. Vif function in the cell cycle checkpoint is present in common HIV-1 subtypes worldwide and likely advantageous for viral pathogenesis.
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http://dx.doi.org/10.1016/j.celrep.2019.09.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903395PMC
October 2019

The Role of RNA in HIV-1 Vif-Mediated Degradation of APOBEC3H.

J Mol Biol 2019 12 16;431(24):5019-5031. Epub 2019 Oct 16.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

As many as five members of the APOBEC3 family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which forms a hybrid ubiquitin ligase complex that directly binds APOBEC3 enzymes and targets them for proteasomal degradation. APOBEC3H (A3H) is unique among family members by dimerization through cellular and viral duplex RNA species. RNA binding is required for localization of A3H to the cytoplasmic compartment, for efficient packaging into nascent HIV-1 particles and ultimately for effective virus restriction activity. Here we compared wild-type human A3H and RNA binding-defective mutants to ask whether RNA may be a factor in the functional interaction with HIV-1 Vif. We used structural modeling, immunoblotting, live cell imaging, and split green fluorescence protein (GFP) reconstitution approaches to assess the capability of HIV-1 Vif to promote the degradation of wild-type A3H in comparison to RNA binding-defective mutants. The results combined to show that RNA is not strictly required for Vif-mediated degradation of A3H, and that RNA and Vif are likely to bind this single-domain DNA cytosine deaminase on physically distinct surfaces. However, a subset of the results also indicated that the A3H degradation process may be affected by A3H protein structure, subcellular localization, and differences in the constellation of A3H interaction partners, suggesting additional factors may also influence the fate and functionality of this host-pathogen interaction.
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http://dx.doi.org/10.1016/j.jmb.2019.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948013PMC
December 2019

The deaminase APOBEC3B triggers the death of cells lacking uracil DNA glycosylase.

Proc Natl Acad Sci U S A 2019 10 14;116(44):22158-22163. Epub 2019 Oct 14.

Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455;

Human cells express up to 9 active DNA cytosine deaminases with functions in adaptive and innate immunity. Many cancers manifest an APOBEC mutation signature and APOBEC3B (A3B) is likely the main enzyme responsible. Although significant numbers of APOBEC signature mutations accumulate in tumor genomes, the majority of APOBEC-catalyzed uracil lesions are probably counteracted in an error-free manner by the uracil base excision repair pathway. Here, we show that A3B-expressing cells can be selectively killed by inhibiting uracil DNA glycosylase 2 (UNG) and that this synthetic lethal phenotype requires functional mismatch repair (MMR) proteins and p53. knockout human 293 and MCF10A cells elicit an A3B-dependent death. This synthetic lethal phenotype is dependent on A3B catalytic activity and reversible by complementation. A3B expression in -null cells causes a buildup of genomic uracil, and the ensuing lethality requires processing of uracil lesions (likely U/G mispairs) by MSH2 and MLH1 (likely noncanonical MMR). Cancer cells expressing high levels of endogenous A3B and functional p53 can also be killed by expressing an UNG inhibitor. Taken together, UNG-initiated base excision repair is a major mechanism counteracting genomic mutagenesis by A3B, and blocking UNG is a potential strategy for inducing the selective death of tumors.
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http://dx.doi.org/10.1073/pnas.1904024116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825264PMC
October 2019

A Rabbit Monoclonal Antibody against the Antiviral and Cancer Genomic DNA Mutating Enzyme APOBEC3B.

Antibodies (Basel) 2019 Sep 10;8(3). Epub 2019 Sep 10.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.
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http://dx.doi.org/10.3390/antib8030047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783943PMC
September 2019

A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits.

J Virol 2019 12 13;93(23). Epub 2019 Nov 13.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes. The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.
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http://dx.doi.org/10.1128/JVI.01539-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854502PMC
December 2019

The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1.

J Biol Chem 2019 08 19;294(32):12099-12111. Epub 2019 Jun 19.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455; Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, Minnesota 55455. Electronic address:

Apolipoprotein B mRNA editing enzyme catalytic subunit-like protein 3B (APOBEC3B or A3B), as other APOBEC3 members, is a single-stranded (ss)DNA cytosine deaminase with antiviral activity. A3B is also overexpressed in multiple tumor types, such as carcinomas of the bladder, cervix, lung, head/neck, and breast. A3B generates both dispersed and clustered C-to-T and C-to-G mutations in intrinsically preferred trinucleotide motifs (TA/TG/TT). A3B-catalyzed mutations are likely to promote tumor evolution and cancer progression and, as such, are associated with poor clinical outcomes. However, little is known about cellular processes that regulate A3B. Here, we used a proteomics approach involving affinity purification coupled to MS with human 293T cells to identify cellular proteins that interact with A3B. This approach revealed a specific interaction with cyclin-dependent kinase 4 (CDK4). We validated and mapped this interaction by co-immunoprecipitation experiments. Functional studies and immunofluorescence microscopy experiments in multiple cell lines revealed that A3B is not a substrate for CDK4-Cyclin D1 phosphorylation nor is its deaminase activity modulated. Instead, we found that A3B is capable of disrupting the CDK4-dependent nuclear import of Cyclin D1. We propose that this interaction may favor a more potent antiviral response and simultaneously facilitate cancer mutagenesis.
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http://dx.doi.org/10.1074/jbc.RA119.008443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690700PMC
August 2019

HIV-1 restriction by endogenous APOBEC3G in the myeloid cell line THP-1.

J Gen Virol 2019 07 30;100(7):1140-1152. Epub 2019 May 30.

3 Center for Genome Engineering, Minneapolis, MN 55455, USA.

HIV-1 replication in CD4-positive T lymphocytes requires counteraction of multiple different innate antiviral mechanisms. Macrophage cells are also thought to provide a reservoir for HIV-1 replication but less is known in this cell type about virus restriction and counteraction mechanisms. Many studies have combined to demonstrate roles for APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H in HIV-1 restriction and mutation in CD4-positive T lymphocytes, whereas the APOBEC enzymes involved in HIV-1 restriction in macrophages have yet to be delineated fully. We show that multiple APOBEC3 genes including APOBEC3G are expressed in myeloid cell lines such as THP-1. Vif-deficient HIV-1 produced from THP-1 is less infectious than Vif-proficient virus, and proviral DNA resulting from such Vif-deficient infections shows strong G to A mutation biases in the dinucleotide motif preferred by APOBEC3G. Moreover, Vif mutant viruses with selective sensitivity to APOBEC3G show Vif null-like infectivity levels and similarly strong APOBEC3G-biased mutation spectra. Importantly, APOBEC3G-null THP-1 cells yield Vif-deficient particles with significantly improved infectivities and proviral DNA with background levels of G to A hypermutation. These studies combine to indicate that APOBEC3G is the main HIV-1 restricting APOBEC3 family member in THP-1 cells.
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http://dx.doi.org/10.1099/jgv.0.001276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176283PMC
July 2019

HIV-1 restriction by endogenous APOBEC3G in the myeloid cell line THP-1.

J Gen Virol 2019 07 30;100(7):1140-1152. Epub 2019 May 30.

3 Center for Genome Engineering, Minneapolis, MN 55455, USA.

HIV-1 replication in CD4-positive T lymphocytes requires counteraction of multiple different innate antiviral mechanisms. Macrophage cells are also thought to provide a reservoir for HIV-1 replication but less is known in this cell type about virus restriction and counteraction mechanisms. Many studies have combined to demonstrate roles for APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H in HIV-1 restriction and mutation in CD4-positive T lymphocytes, whereas the APOBEC enzymes involved in HIV-1 restriction in macrophages have yet to be delineated fully. We show that multiple APOBEC3 genes including APOBEC3G are expressed in myeloid cell lines such as THP-1. Vif-deficient HIV-1 produced from THP-1 is less infectious than Vif-proficient virus, and proviral DNA resulting from such Vif-deficient infections shows strong G to A mutation biases in the dinucleotide motif preferred by APOBEC3G. Moreover, Vif mutant viruses with selective sensitivity to APOBEC3G show Vif null-like infectivity levels and similarly strong APOBEC3G-biased mutation spectra. Importantly, APOBEC3G-null THP-1 cells yield Vif-deficient particles with significantly improved infectivities and proviral DNA with background levels of G to A hypermutation. These studies combine to indicate that APOBEC3G is the main HIV-1 restricting APOBEC3 family member in THP-1 cells.
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http://dx.doi.org/10.1099/jgv.0.001276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176283PMC
July 2019

Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1.

ACS Infect Dis 2019 04 12;5(4):634-640. Epub 2019 Mar 12.

Department of Chemistry , Colorado State University , 200 W. Lake Street , Fort Collins , Colorado 80523 , United States.

Drugs that block HIV-1 entry are relatively limited. Enfuvirtide is a 36-residue synthetic peptide that targets gp41 and blocks viral fusion. However, Enfuvirtide-resistant HIV has been reported, and this peptide drug requires daily injection. Previously, we have reported helix-grafted display proteins, consisting of HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these biologics inhibit HIV-1 entry with relatively modest and varied potency (IC = 190 nM to >1 μM). Here, we report that gp41 C-peptide helix-grafted Sac7d (Sac7d-Cpep) potently suppresses HIV-1 entry in a live virus assay (IC = 1.9-12.4 nM). Yeast display sequence optimization of solvent exposed helix residues led to new biologics with improved expression in E. coli (a common biosimilar expression host), with no appreciable change in entry inhibition. Evolved proteins inhibit the entry of a clinically relevant mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide resistant. Fusion proteins designed for serum stability also potently suppress HIV-1 entry. Collectively, we report several evolved biologics that are functional against an Enfuvirtide-resistant strain and are designed for serum stability.
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http://dx.doi.org/10.1021/acsinfecdis.8b00362DOI Listing
April 2019

Polyomavirus T Antigen Induces Expression Using an LXCXE-Dependent and TP53-Independent Mechanism.

mBio 2019 02 5;10(1). Epub 2019 Feb 5.

Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA

APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering upregulation in virus-infected cells. The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5'-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.
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http://dx.doi.org/10.1128/mBio.02690-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428753PMC
February 2019

A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination.

Methods 2019 03 19;156:79-84. Epub 2018 Dec 19.

Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Institute for Molecular Virology, Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3'-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.
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http://dx.doi.org/10.1016/j.ymeth.2018.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397784PMC
March 2019

Natural APOBEC3C variants can elicit differential HIV-1 restriction activity.

Retrovirology 2018 12 17;15(1):78. Epub 2018 Dec 17.

Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Center for Genome Engineering, Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, USA.

Background: The APOBEC3 (A3) family of DNA cytosine deaminases provides an innate barrier to infection by retroviruses including HIV-1. A total of five enzymes, A3C, A3D, A3F, A3G and A3H, are degraded by the viral accessory protein Vif and expressed at high levels in CD4+ T cells, the primary reservoir for HIV-1 replication in vivo. Apart from A3C, all of these enzymes mediate restriction of Vif-deficient HIV-1. However, a rare variant of human A3C (Ile188) was shown recently to restrict Vif-deficient HIV-1 in a 293T-based single cycle infection system. The potential activity of this naturally occurring A3C variant has yet to be characterized in a T cell-based spreading infection system. Here we employ a combination of Cas9/gRNA disruption and transient and stable protein expression to assess the roles of major Ser188 and minor Ile188 A3C variants in HIV-1 restriction in T cell lines.

Results: Cas9-mediated mutation of endogenous A3C in the non-permissive CEM2n T cell line did not alter HIV-1 replication kinetics, and complementation with A3C-Ser188 or A3C-Ile188 was similarly aphenotypic. Stable expression of A3C-Ser188 in the permissive T cell line SupT11 also had little effect. However, stable expression of A3C-Ile188 in SupT11 cells inhibited Vif-deficient virus replication and inflicted G-to-A mutations.

Conclusions: A3C-Ile188 is capable of inhibiting Vif-deficient HIV-1 replication in T cells. Although A3C is eclipsed by the dominant anti-viral activities of other A3s in non-permissive T cell lines and primary T lymphocytes, this enzyme may still be able to contribute to HIV-1 diversification in vivo. Our results highlight the functional redundancy in the human A3 family with regards to HIV-1 restriction and the need to consider naturally occurring variants.
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http://dx.doi.org/10.1186/s12977-018-0459-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297987PMC
December 2018