Wielding a double-edged sword: viruses exploit host DNA repair systems to facilitate replication while bypassing immune activation

Viruses are obligate intracellular pathogens that hijack a myriad of host cell processes to facilitate replication and suppress host antiviral defenses. In its essence, a virus is a segment of foreign nucleic acid that engages host cell machinery to drive viral genome replication, gene transcription, and protein synthesis to generate progeny virions. Because of this, host organisms have developed sophisticated detection systems that activate antiviral defenses following recognition of aberrant nucleic acids. For example, recognition of viral nucleic acids by host DNA repair proteins results in compromised viral genome integrity, induction of antiviral inflammatory programs, cell cycle arrest, and apoptosis. Unsurprisingly, diverse viral families have evolved multiple strategies that fine-tune host DNA repair responses to suppress activation of antiviral defenses while simultaneously hijacking DNA repair proteins to facilitate virus replication. This review summarizes common molecular strategies viruses deploy to exploit host DNA repair mechanisms

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

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. UNG knockout human 293 and MCF10A cells elicit an A3B-dependent death. This synthetic lethal phenotype is dependent on A3B catalytic activity and reversible by UNG complementation. A3B expression in UNG-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.</p

Evolutionary Conservation of PP2A Antagonism and G2/M Cell Cycle Arrest in Maedi-Visna Virus Vif

The canonical function of lentiviral Vif proteins is to counteract the mutagenic potential of APOBEC3 antiviral restriction factors. However, recent studies have discovered that Vif proteins from diverse HIV-1 and simian immunodeficiency virus (SIV) isolates degrade cellular B56 phosphoregulators to remodel the host phosphoproteome and induce G2/M cell cycle arrest. Here, we evaluate the conservation of this activity among non-primate lentiviral Vif proteins using fluorescence-based degradation assays and demonstrate that maedi-visna virus (MVV) Vif efficiently degrades all five B56 family members. Testing an extensive panel of single amino acid substitution mutants revealed that MVV Vif recognizes B56 proteins through a conserved network of electrostatic interactions. Furthermore, experiments using genetic and pharmacologic approaches demonstrate that degradation of B56 proteins requires the cellular cofactor cyclophilin A. Lastly, MVV Vif-mediated depletion of B56 proteins induces a potent G2/M cell cycle arrest phenotype. Therefore, remodeling of the cellular phosphoproteome and induction of G2/M cell cycle arrest are ancient and conserved functions of lentiviral Vif proteins, which suggests that they are advantageous for lentiviral pathogenesis

Inhibition of ATM-directed antiviral responses by HIV-1 Vif.

Emerging evidence indicates that HIV-1 hijacks host DNA damage repair (DDR) pathways to facilitate multiple facets of virus replication. Canonically, HIV-1 engages proviral DDR responses through the accessory protein Vpr, which induces constitutive activation of DDR kinases ATM and ATR. However, in response to prolonged DDR signaling, ATM directly induces pro-inflammatory NF-κB signaling and activates multiple members of the TRIM family of antiviral restriction factors, several of which have been previously implicated in antagonizing retroviral and lentiviral replication. Here, we demonstrate that the HIV-1 accessory protein Vif blocks ATM-directed DNA repair processes, activation of NF-κB signaling responses, and TRIM protein phosphorylation. Vif function in ATM antagonism occurs in clinical isolates and in common HIV-1 Group M subtypes/clades circulating globally. Pharmacologic and functional studies combine to suggest that Vif blocks Vpr-directed activation of ATM but not ATR, signifying that HIV-1 utilizes discrete strategies to fine-tune DDR responses that promote virus replication while simultaneously inhibiting immune activation

CUL5 protein localization in HUVEC treated with increasing doses of thalidomide.

<p><b>A.</b> The control and thalidomide-treated HUVEC (20 and 50 μg/ml). <b>B.</b> Examples of HUVEC treated with 100 μg/ml thalidomide. Cells were immunostained using anti- CUL5 specific antibody as described in the Methods. Magnification is 100X.</p

Effect of thalidomide on CUL5 and NEDD8 localization in HUVEC after 24 hrs of treatment.

<p><b>A.</b> Expression of CUL5 and NEDD8 in Control, Thalidomide (20 μg/ml) and PMA (10 nM) treated HUVEC for 24 hours. Immunostaining with anti-VACM-1/CUL5 and anti NEDD8 antibodies was performed as described in the Methods. Images are from CUL5 staining (FITC-green) and NEDD8 staining (Texas red) and merged pictures. Magnification is 40X. <b>B.</b> Control and thalidomide treated cells expressing nuclear CUL5 and NEDD8 were quantitated (n = 10 and n = 3, respectively; *, p <0.05).</p

Thalidomide treatment inhibits growth in RAMEC but not in RAMEC transfected with a dominant negative mutant of CUL5 (^S730ACUL5) cDNA.

<p><b>A.</b> A representative growth assay results from CMV vector transfected RAMEC control cells treated with increasing doses of thalidomide. VEGF (50 nM) was used as a control. <b>B.</b> Western blot analysis of cell lysates from CMV vector transfected RAMEC treated with increasing doses of thalidomide for 24 hours. To ascertain equal protein loading blots were stripped and re-probed with anti-GAPDH specific antibody as described in <i>Methods</i>. <b>C.</b> The signal intensities shown in B above, were quantitated. <b>D.</b> A representative wound assay in CUL5 cDNA transfected cells treated with thalidomide. Arrows indicate space in the wound assay at time 0 and 18 hrs. <b>E.</b> Growth data shown in (D) was quantitated and expressed as a percent (%) regrowth from time 0. The effects of 0 μg/mL (black bars), 10 μg/ml (gray bars), and 50 μg/ml (white bars) of thalidomide were examined (*, p<0.05). <b>F.</b> A representative growth assay results from ^S730ACUL5 cDNA transfected RAMEC cells treated with increasing doses of thalidomide. VEGF (50 nM) was used as a control. <b>G.</b> Dose dependent effect of thalidomide on CUL5 in ^S730ACUL5 cDNA transfected RAMEC as detected with anti CUL5 protein specific antibody. <b>H</b>. Data shown in G were quantitated and expressed as mean ± standard error. (RAMEC CMV n = 2 and RAMEC−^S730AVACM-1 (<i>n</i> = 3, * = <i>p</i> < 0.05). <b>I.</b> A representative light microscopy experiment using the wound assay in ^S730ACUL5 cDNA transfected cells. Arrows indicate space in the wound assay. <b>J.</b> Growth data shown in (I) were quantitated and expressed as a percent regrowth from time 0. The effects of 0 (black bars), 10 μg/ml (gray bars), and 50 μg/ml (white bars) of thalidomide were examined (* = <i>p</i> < 0.05).</p

Effect of thalidomide on CUL5 and NEDD8 localization in HUVEC.

<p>CUL5 and NEDD8 colocalization in control cells treated with thalidomide (50 μg/mL) for 15 and 45 min, respectively. Immunostaining with anti- CUL5 and anti NEDD8 antibodies and nuclear DAPI staining was performed as described in the <i>Methods</i>. Images are of merged pictures from CUL5 staining (FITC-green) and NEDD8 staining (Texas red). Magnification is 40X.</p

Effect of thalidomide on CUL5 and NEDD8 localization in control and si-transfected HUVEC at 24 hours after treatment.

<p><b>A.</b> CUL5 (green) and NEDD8 (red) localization in control and si-CUL5 transfected HUVEC and treated with thalidomide (50 μg/mL) for 24 hrs. Magnification is 100X. <b>B.</b> HUVEC treated with thalidomide (50 μg/mL) immunostained with anti NEDD8 Ab. Magnification is 40X. <b>C.</b> NEDD8 signal in Control and Thalidomide treated cells shown in B was quantitated (<i>n</i> = 3; error bars are S.E.M., *, <i>p</i> < 0.05). D. Western blot analysis of neddylated CUL5 (upper band) and free NEDD8 (lower band) in control, thalidomide and PMA-treated HUVEC (24 hours).</p