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The Dynamic Interplay between HIV-1, SAMHD1, and the Innate Antiviral Response

The innate immune response constitutes the first cellular line of defense against initial HIV-1 infection. Immune cells sense invading virus and trigger signaling cascades that induce antiviral defenses to control or eliminate infection. Professional antigen-presenting cells located in mucosal tissues, including dendritic cells and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. These cells are less permissive to HIV-1 infection compared to activated CD4+ T-cells, which is mainly due to host restriction factors that serve an immediate role in controlling the establishment or spread of viral infection. However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system. Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is the mammalian deoxynucleoside triphosphate triphosphohydrolase responsible for regulating intracellular dNTP pools and restricting the replication of HIV-1 in non-dividing myeloid cells and quiescent CD4+ T-cells. Here, we review and analyze the latest literature on the antiviral function of SAMHD1, including the mechanism of HIV-1 restriction and the ability of SAMHD1 to regulate the innate immune response to viral infection. We also provide an overview of the dynamic interplay between HIV-1, SAMHD1, and the cell-intrinsic antiviral response to elucidate how SAMHD1 modulates HIV-1 infection in non-dividing immune cells. A more complete understanding of SAMHD1’s role in the innate immune response to HIV-1 infection may help develop stratagems to enhance its antiviral effects and to more efficiently block HIV-1 replication and avoid the pathogenic result of viral infection.

The innate immune response constitutes the first cellular line of defense against initial HIV-1 infection. Immune cells sense invading virus and trigger signaling cascades that induce antiviral defenses to control or eliminate infection. Professional antigenpresenting cells located in mucosal tissues, including dendritic cells and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. These cells are less permissive to HIV-1 infection compared to activated CD4 + T-cells, which is mainly due to host restriction factors that serve an immediate role in controlling the establishment or spread of viral infection. However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system. Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is the mammalian deoxynucleoside triphosphate triphosphohydrolase responsible for regulating intracellular dNTP pools and restricting the replication of HIV-1 in non-dividing myeloid cells and quiescent CD4 + T-cells. Here, we review and analyze the latest literature on the antiviral function of SAMHD1, including the mechanism of HIV-1 restriction and the ability of SAMHD1 to regulate the innate immune response to viral infection. We also provide an overview of the dynamic interplay between HIV-1, SAMHD1, and the cell-intrinsic antiviral response to elucidate how SAMHD1 modulates HIV-1 infection in non-dividing immune cells. A more complete understanding of SAMHD1's role in the innate immune response to HIV-1 infection may help develop stratagems to enhance its antiviral effects and to more efficiently block HIV-1 replication and avoid the pathogenic result of viral infection.
Keywords: Hiv-1, sterile alpha motif and HD-domain containing protein 1, infection, innate immunity, myeloid cells inTRODUCTiOn Innate immunity is the cell-intrinsic defense mechanism that senses incoming pathogens and is characterized by type-I interferon (IFN-I) induction and the release of inflammatory cytokines that upregulate antiviral IFN-stimulated genes (ISGs) (1, 2). The activation of the innate response to pathogens is dependent on cellular pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs), including viral structures or nucleic acids. Interferon-inducible protein IFI16 and cyclic GMP-AMP synthase (cGAS) are cytosolic sensors of HIV-1 that detect viral DNA (3, 4). Recognition of PAMPs results in induction of IFN-I and ISGs to control initial infection and spread, while the concomitant induction of the inflammatory response and cytokines can initiate adaptive immune responses (5,6). Modulation of IFN-I activation is essential for viral clearance. However, overstimulation of IFN pathways can lead to inflammatory autoimmune disease (7).
HIV-1 is sensitive to ISGs and the IFN-induced antiviral response; so it is not surprising that HIV-1 is a poor inducer of IFN (8). HIV-1 benefits from evading innate immune activation and utilizes a variety of tactics to escape detection (9,10). Professional antigen-presenting cells located in mucosal tissues, including dendritic cells (DCs) and macrophages, are critical for recognizing HIV-1 at the site of initial exposure. However, these cells are less permissive to HIV-1 infection compared to activated CD4 + T-cells, mainly due to host restriction factors that control the establishment or spread of viral infection. Several host proteins can restrict HIV-1 at various points in the viral lifecycle, including APOBEC proteins, TRIM5α, and tetherin (11)(12)(13). However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system (13). SAMHD1 is host protein capable of blocking replication of retroviruses and several DNA viruses in cells (14)(15)(16)(17)(18). SAMHD1 is constitutively expressed at various levels in all cell types and highly expressed in myeloid lineage and resting CD4 + T-cells (14,15,19). IFN-I treatment increases SAMHD1 expression in certain cell types with low endogenous SAMHD1 levels (20,21). SAMHD1 has been implicated as a negative regulator of the IFN-I inflammatory response (22)(23)(24), however, the underlying mechanism is not fully understood. While HIV-2 encodes the SAMHD1 antagonist Vpx, the more pathogenic HIV-1 does not. It was hypothesized that HIV-1 lacks a countermeasure against SAMHD1 because it is beneficial for infection. In this review, we will discuss the contributions of SAMHD1 to both the direct restriction of HIV-1 and to the modulation of the antiviral innate response and to analyze the hypothesis that HIV-1 restriction by SAMHD1 leads to a diminished induction of innate immunity.
innATe iMMUne SenSinG OF Hiv-1 Although HIV-1 can be sensed by the innate immune system, the prevailing theory is that HIV-1 avoids immune surveillance through poor replication in immune cells causing ineffective triggering of innate cytosolic sensors (25). Several studies have identified the molecular basis of cytosolic sensors important for targeting viral pathogens. Here, we focus on HIV-1 DNA as a trigger of the innate antiviral response. After sensing viral DNA, cGAS generates the second messenger, cyclic guanosine monophosphate-adenosine monophosphate (26)(27)(28), that activates the stimulator of IFN genes (STING) (6). STING activation leads to phosphorylation of TANK-binding kinase 1 (TBK1) and the subsequent phosphorylation and dimerization of IFNregulatory transcription factors IRF3 and IRF7. Nuclear translocation of the IRF3/IRF7 homo-or-hetero dimers will activate IFN-I gene expression (Figure 1). This signaling cascade results in an upregulation of IFN-I and ISGs as a defense against viral infection (29,30). Reverse transcribed HIV-1 DNA was identified as the trigger to the cGAS-STING pathway (3). Although cGAS is the primary sensor of cytosolic viral DNA, IFI16 can also act as a sensor of HIV-1 single-stranded DNA that induces an IFN-β response in macrophages by a cGAS-STING-dependent pathway (4).

inTRODUCTiOn TO SAMHD1
Human SAMHD1 is a 626-amino acid protein containing an N-terminal nuclear localization signal followed by a sterile-alpha motif and histidine/aspartic acid (HD) domain. SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) (33,34) that converts dNTPs into the constituent deoxynucleoside and inorganic triphosphate upon stimulation by dGTP or GTP (33)(34)(35). SAMHD1 and ribonuclease reductase, the enzyme responsible for de novo dNTP synthesis through the conversion of ribonucleotide diphosphates to deoxyribonucleotides (36), are allosterically regulated to achieve balanced intracellular dNTP levels in a cell-cycle-dependent manner (37). During G1 to S-phase transition in actively proliferating cells, ribonuclease reductase expression increases, leading to expansion of the dNTP pool to facilitate DNA synthesis (38,39). The activity of SAMHD1 is activated by high dNTP levels, and degradation of nucleic acids in the absence of DNA replication protects the cell from innate immune activation and cancer development (40,41). Mutations in SAMHD1 that affect its enzyme activity are associated with Aicardi-Goutières syndrome (AGS), an encephalopathic autoimmune disease characterized by symptoms mimicking chronic viral infection (22). The accumulation of intracellular dNTPs caused by mutations in the genes encoding proteins involved in nucleic acid metabolism, including SAMHD1 and TREX1 (42), are sensed by PRRs, resulting in aberrant production of IFN-I (43). AGS patients present with increased production of IFN-α, the chemokine most characteristic of congenital virus infection. AGS patients with SAMHD1 mutations can present with signs of lupus erythematosus, with many symptoms mimicking those of HIV-1 infection (22,44). Furthermore, cells isolated from AGS patients with homozygous SAMHD1 mutation revealed that SAMHD1-deficient monocytes supported productive infection by HIV-1 (20), suggesting a link between SAMHD1 function in both autoimmunity and HIV-1 restriction.
Long interspersed element 1 (LINE-1) is the only autonomous and active human retroelement capable of producing new genomic insertions through its endogenous endonuclease and reverse transcriptase activities (45,46). A study on AGS-related SAMHD1 mutations indicate that all disease-related mutations reduced LINE-1 inhibition in dividing cells (47). Recent work suggests that SAMHD1 potently blocks LINE-1 transposition in cycling cells by triggering the sequestration of LINE-1 ORF1p into stress granules (48). Impaired inhibition of LINE-1 retrotransposition may lead to triggering of the autoimmune response by stimulating toll-like receptors (TLRs) (49), although this has not been confirmed. Impaired dNTPase activity and LINE-1 suppression by mutant SAMHD1 could explain the chronic inflammatory response characteristics of AGS disease. These studies outlining the pathogenic effect of SAMHD1 deficiency on autoimmune disease implicate SAMHD1 as a negative regulator of the innate immune system.

SAMHD1-MeDiATeD Hiv-1 ReSTRiCTiOn
HIV-1 replicates inefficiently in non-diving cells, such as quiescent CD4 + T-cells, DCs, and monocytes. HIV-1 infection can be enhanced in these cells by Vpx, an accessory protein encoded by HIV-2 and certain lineages of simian immunodeficiency viruses (SIVs) (50,51). This hinted at the existence of a cellular restriction factor counteracted by Vpx (50). SAMHD1 was identified as the mystery HIV-1 restriction factor by a mass spectrometry analysis of cellular proteins immunoprecipitated from cells expressing Vpx (14,15). Vpx interacts with the C-terminal domain of SAMHD1, thereby initiating proteasomal degradation by an E3 ubiquitin ligase complex, and relieving SAMHD1-mediated lentiviral restriction (14,15,52,53).
The mechanism and modulation of SAMHD1-mediated HIV-1 restriction is an area of intense scrutiny (Figure 2). Overexpression of SAMHD1 in PMA-treated monocytic U937 cells results in a depletion of dNTP levels (54). It was later confirmed that SAMHD1 restricts the replication of retroviruses and several DNA viruses by depleting the concentration of intracellular dNTPs to levels insufficient to support viral DNA synthesis (14-18, 54, 55). Structural studies strengthened a model of nucleotide-dependent tetramer assembly of SAMHD1 (56)(57)(58), where GTP binds to guanine-specific allosteric sites and dNTP binds to non-specific activator sites, initiating the formation of enzymatically active tetramers with the catalytic core of the HD domain (33,34,37,59). Moreover, binding of single-stranded nucleic acids (ssNAs) to the dimer-dimer interface of SAMHD1 inhibits the formation of the catalytically active tetramer (60).
As SAMHD1 is also highly expressed in activated CD4 + T-cells that support productive infection, several studies demonstrated posttranslational modification as a means of mechanistic regulation of SAMHD1 function in restricting HIV-1. SAMHD1 is phosphorylated at several residues; however, phosphorylation of threonine 592 was identified as essential for the negative FiGURe 2 | SAMHD1 negatively regulates the innate immune sensing of HIV-1 DNA. SAMHD1 blocks HIV-1 infection through intracellular dNTP depletion, thus preventing the accumulation of viral DNA accessible to sensing by IFI16 and cyclic GMP-AMP synthase (cGAS) and the activation of the type-I interferon (IFN-I) response. The dNTPase activity of SAMHD1 is structurally regulated. Consecutive binding of dGTP/GTP and any dNTP to two allosteric sites provokes formation of the catalytically active tetramer, which can be destabilized by phosphorylation, oxidation, or the binding of single-stranded nucleic acids (ssNAs). dN, deoxynucleosides; PPPs, triphosphate; two linked letters S indicate the disulfide bond. modulation of its HIV-1 restriction activity (61-65) and tetramer formation (66,67). SAMHD1 is phosphorylated by cyclindependent kinase 1 (CDK1) and CDK2 in complex with cell cycle regulatory protein cyclin A. This regulation of SAMHD1 function is associated with the cell cycle, as CDK1 and cyclin A are highly expressed in dividing cells. Furthermore, S-phase requires elevated dNTP levels, indicating modulation of the dNT-Pase activity of SAMHD1 during the cell cycle (64). SAMHD1 protein levels may be altered during various stages of the cell cycle depending on different cell types (68,69). Interestingly, proliferation-induced oxidation of SAMHD1 by hydrogen peroxide reversibly inhibits its dNTPase activity through the formation of tetramer-inhibiting disulfide bonds (70), suggesting a dynamic structure-based regulatory mechanism of SAMHD1's dNTPase activity that is influenced by the cell cycle (Figure 2).
Although the accepted consensus is that SAMHD1 restricts HIV-1 infection through the depletion of intracellular dNTPs, several studies suggested the existence of an additional yet-undiscovered mechanism of SAMHD1-mediated retroviral restriction. This undefined antiviral activity appears to be dependent on phosphorylation (61,63,65) and is not fully dependent on low dNTP levels (71). SAMHD1 acts as a ssNA binding protein that degrades single-stranded DNA and RNA via a metal-dependent 3′-5′ exonuclease activity in vitro (72)(73)(74). It has been suggested that SAMHD1 utilizes its nucleic acid binding potential to exert a ribonuclease activity against incoming HIV-1 genomic RNA in a phosphorylation-dependent manner (75). SAMHD1 was shown to restrict retroviruses though degradation of HIV-1 RNA in human monocyte-derived macrophages (MDMs), monocytes, and CD4 + T-cells (75,76). It was proposed that SAMHD1 degrades incoming HIV-1 gRNA, thereby restricting infection and preventing innate immune sensing of viral nucleic acids. However, recent studies have been unable to confirm the controversial findings (55,(77)(78)(79). As a nuclear-localized protein (80), incoming viral genomic RNA would be inaccessible by SAMHD1 for hydrolysis. Additional studies showed that dNTPase inactive SAMHD1 mutant retained exonuclease activities in vitro, indicating the exonuclease activity could not be attributed to the known dNTP-binding active site (77). Seamon et al. (77) suggested that the nuclease activity attributed to SAMHD1 was due to contamination during purification. Cell-based assays also failed to recapitulate the findings, thereby confirming the lack of SAMHD1 RNase activity to restrict HIV-1 in infected cells (55,78). Ryoo et al. suggested that the differences in experimental conditions are responsible for the conflicting results, including a shorter infection time and the use of RNaseH-defective reverse transcriptase (81). They further identified SAMHD1 as a phosphorolytic not hydrolytic ribonuclease (82).
THe inTeRSeCTiOn OF Hiv-1, SAMHD1, AnD THe innATe AnTiviRAL ReSPOnSe SAMHD1 cDNA was originally identified as a ortholog of the mouse IFN-γ-induced gene Mg11 in human DCs (83). A link to the innate immune response was strengthened by the discovery that cytokines, including toll-like agonists and IFNs, can induce SAMHD1 expression (84,85). Cell lines treated with IFN-I (21,86) and human primary monocytes treated with IFN-α and IFN-γ (20,84,87) show enhanced expression of SAMHD1. While SAMHD1 is highly expressed in MDMs, monocytederived dendritic cells (MDDCs), and primary CD4 + T-cells, IFN treatment does not increase SAMHD1 protein levels further (21,(88)(89)(90). However, treatment of MDMs and MDDCs with IFN-I results in reduced phosphorylation of SAMHD1 at residue T592 (61), indicating a shift from catalytically inactive to active SAMHD1. Interestingly, the SAMHD1 promoter is a direct target of IRF3. The overexpression and activation of IRF3 enhances SAMHD1 promoter activity in HeLa cells (86).
HIV-1 does not trigger a sterilizing immune response (91) and is a poor activator of inflammatory pathways (8), resulting in an impaired response to HIV-1 and the development of persistent infection. The DC response to HIV-1 infection contributes to this dysfunctional immune response (92). Myeloid cells constantly sample the cellular environment to identify pathogens and send out danger signals in the form of IFN-I. DCs are essential for activating the adaptive immune response to infection, as maturation leads to T-cell responses through antigen priming (91,93). Interestingly, HIV-1 infects DCs without activating an effective antiviral response. As SAMHD1 limits HIV-1 cDNA synthesis in myeloid cells (14,54), it was hypothesized that degradation of SAMHD1 by Vpx in DCs would result in productive HIV-1 infection and the synthesis of viral proteins that would directly enter antigen presentation, thereby strengthening the T-cell response to infection (94). This could be why the vpx gene was lost from the ancestor of HIV-1 during the coevolution of primate SAMHD1 and lentiviruses (95).
Vpx-mediated degradation of SAMHD1 in DCs leads to enhanced HIV-1 infection, and studies in primary MDMs and MDDCs indicate that Vpx-mediated SAMHD1 degradation results in cGAS stimulation and IRF3 activation (3). Early work suggested that enhanced infection by SAMHD1 depletion leads to DC maturation (94). A study utilizing coculture of autologous activated CD4 + T lymphocytes with SAMHD1-deficient MDDCs infected with primary clinical HIV-1 isolates indicated enhancement of both infection and IFN response (96). Interestingly, cocultured primary T-lymphocytes, but not HIV-1, trigger a decrease in SAMHD1 expression in MDDCs independent of dNTP levels (96). This study suggests that crosstalk between lymphocytes and DCs induces downregulation of SAMHD1 expression, a requirement for stimulation of HIV-1 production in DCs, thereby inducing the innate sensing of HIV-1 and DC maturation (96).
Conversely, recent work indicates that DC maturation, measured by CD83 and CD86 expression, does not occur in SAMHD1-deficient cells due to additional manipulation of the innate immune system by HIV-1 (97). HIV-1 suppresses TLRinduced maturation of DCs independent of SAMHD1 expression, although Vpx-mediated depletion of SAMHD1 enhanced the effect of HIV-1 infection on lipopolysaccharide-induced DC maturation (97). Vesicular stomatitis virus G-proteinpseudotyped HIV-1 suppressed maturation similar to strains containing HIV-1 envelope protein, suggesting that viral replication, not envelope-receptor interactions, is required for suppression of maturation (97). Removing the SAMHD1-mediated block of reverse transcription resulted in a stronger suppression of maturation. Although infection and subsequent innate immune sensing in DCs is blocked by SAMHD1, HIV-1 maintains an additional SAMHD1-independent mechanism of suppressing DC maturation through downregulation of TLRs (97).
Two additional models suggest that, in MDDCs, HIV-1 attempts to hide its genomic RNA and newly synthesized cDNA from cytosolic sensors by obstructing the nucleic acids using viral capsid. The models differ with respect to the effect of recruitment of cellular cyclophillins and cleavage and polyadenylationspecific factor 6 (CPSF6) by capsid. One model suggests increased cyclophillin A (CypA) binding to the capsid increases sensitivity to innate sensing (94), while another proposes CypA binding coordinates uncoating, reverse transcription, and nuclear import of the preintegration complex (98), all to minimize the exposure of viral nucleic acids to cytosolic sensors. Future work is needed to clarify the contribution of CypA and SAMHD1 to the negative regulation of the innate immune response in myeloid cells to provide insight into HIV-1 mechanisms of evasion.
Non-cycling CD4 + T-cells and macrophages are less permissive to HIV-1 because of SAMHD1. However, during HIV-1 infection in vivo, activated CD4 + T-cells and macrophages are infected due to phosphorylation of SAMHD1. Although cytosolic HIV-1 DNA is abundant in these permissive cells, a cell-autonomous IFN response is not triggered (99). This is due at least in part to host protein TREX1. As a single-stranded DNA exonuclease, TREX-1 digests cytoplasmic DNA from retroviral DNA intermediates, thereby preventing the activation of mislocalized DNA by an innate immune sensor (99). Cytosolic HIV-1 DNA is accumulated in HIV-1 infected TREX1-deficient CD4 + T-cells and macrophages, which leads to inhibition of TBK1-dependent IFN-I response (99). This suggests a competition between two DNA sensors: cGAS leading to antiviral effects, and TREX1 leading to enhanced viral replication (100).

ReMAininG QUeSTiOnS
Although it is clear that HIV-1 utilizes a variety of mechanisms to evade myeloid cell activation, controversial questions still exist.
Conflicting reports could be due to the use of different cell types, and the differential use of clinical HIV-1 isolates, replicationcompetent lab strains, or pseudotyped virus. It is essential to confirm experimental findings with primary cells that accurately recapitulate in vivo mucosal infection sites. Further understanding of the strategies HIV-1 utilizes to evade the innate response will allow for better ideas on how to increase the innate immune response to HIV-1.
The existence of a yet-undiscovered mechanism of HIV-1 restriction that is dependent on phosphorylation cannot be overlooked (63). Pretreatment MDMs with Vpx enhances the rate of HIV-1 cDNA synthesis (101), suggesting that the decrease in reverse transcription kinetics conferred by SAMHD1-mediated modulation of dNTP levels negatively regulates the rate of proviral DNA synthesis in non-dividing cells. When transcription is silenced, integrated proviral DNA can lead to latency (102). Although SAMHD1 is highly expressed in cells purported to harbor latent provirus (19,103) and the HIV-1 proviral promoter is activated by transcription factors (104), the effect of SAMHD1 expression on latency development or reversal has not been explored. It is possible that SAMHD1 utilizes its nucleic acid binding ability to restrict HIV-1 infection postintegration, although a recent study confirmed SAMHD1 exerts no effect on HIV-1 Gag synthesis, viral particle release, and virus infectivity in 293T cells transfected with a proviral DNA construct (55). SAMHD1 may exert a direct effect on proviral DNA through binding, as purified recombinant SAMHD1 was shown to bind in vitro transcribed fragments of gag and tat cDNA (72), or indirect effects may occur due to SAMHD1 modulation of inflammatory pathways. It is plausible that suppression of latency reactivation by SAMHD1 would further prevent activation of the innate antiviral response. Although viral nucleic acids can be sensed by IFI16 or cGAS in the absence of SAMHD1 (24,105), whether other pro-inflammatory pathways are affected by SAMHD1 expression remains unknown.
Discovering the mechanisms used by HIV-1 to avoid innate immune sensors is critical for the design of new therapies to eradicate HIV-1 infection. Therapeutic strategies aiming to inhi bit host factors that promote HIV-1 replication and to stimulate the immune response could diminish viral infection and transmission. Current work aims to determine whether a role exists for drugs targeting SAMHD1. Expression of SAMHD1 can increase the susceptibility of HIV-1 to nucleoside reverse transcriptase inhibitors by reducing the levels of competitive dNTPs (106)(107)(108)(109), suggesting modulation of SAMHD1 function may be a means to enhance drug effectiveness. Conversely, as SAMHD1 expression enables immune evasion by HIV-1 (13), it is tempting to hypothesize that SAMHD1 could be used as a drug target to enhance the innate immune response to viral infection. However, research is just beginning to uncover mechanisms to modify the dNTPase activity of SAMHD1 (110,111). Importantly, as an ISG and a negative regulator of the innate immune system, SAMHD1 may be involved in an unknown negative feedback loop aimed at modulating the complex and delicate system of inflammatory pathways.
The effect of SAMHD1 on IFN-I induction during viral infection should be further studied in vivo. Although initial robust IFN-I responses can lead to an upregulation of antiviral genes and a block in infection, chronic immune hyperactivity could lead to desensitization of IFN-I and an eventual suppression of antiviral gene expression. This phenotype was observed when Sandler et al. manipulated the IFN-α2a response to SIV infection in rhesus macaques (112). The dismantling of the antiviral state after long-term IFN-α2a treatment led to an increase in SIV reservoir size and an accelerated CD4 + T-cell loss (112). Studies are necessary to determine whether stimulation of the IFN-I response through inhibition of SAMHD1 function leads to chronic inflammation and progression to AIDS in vivo.

COnCLUSiOn
The identification of SAMHD1 as a regulator of the innate immune response to viral infection has led to the development of an exciting field of research. The structural and functional studies of SAMHD1 connect the physiology of HIV-1 infection to the innate antiviral response and the dynamic regulatory mechanisms in cells. Further work will aid in the development of stratagems to enhance the antiviral effects of the intrinsic immune system.

AUTHOR COnTRiBUTiOnS
JA wrote the manuscript with input and edits from CG and LW.

ACKnOwLeDGMenTS
We thank the Wu lab members for valuable discussions.

FUnDinG
This work was supported by a grant (AI104483) from the National Institutes of Health (NIH) to LW. LW is also supported in part by NIH grants (AI120209 and GM128212). JA was supported by C. Glenn Barber funds from the College of Veterinary Medicine at The Ohio State University.


extfieldsofstudy: Biology Medicine

text:
SAMHD1 protects cancer cells from various nucleoside-based antimetabolites

ABSTRACT Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.

Introduction
Sterile a motif and histidine/aspartic acid domain containing protein 1 (SAMHD1) was identified in 2011 to restrict human immunodeficiency virus type 1 (HIV-1) in myeloid cells, a breakthrough in unravelling the, up to then, enigmatic reasons underlying the low permissivity of macrophages or dendritic cells to HIV-1 infection. 1,2 Soon after this discovery, the restriction mechanism of SAMHD1 was suggested to involve its enzymatic triphosphohydrolase activity, which was proposed to deplete intracellular deoxynucleoside triphosphate (dNTP) substrates for HIV-1 reverse transcription. 3,4 Subsequent studies showed that this activity of SAMHD1 is subjected to allosteric regulation by nucleotides, with the first allosteric site (A1) requiring a guanine nucleotide (GTP or dGTP) and the second allosteric site (A2) requiring any dNTP, which together allow formation of the catalytically active SAMHD1 tetramer (Fig. 1). Owing to its activity toward all canonical dNTPs and the elegant allosteric regulatory mechanism, SAMHD1 has been proposed to oppose ribonucleotide reductase (RNR) as a major regulator of DNA precursor pools, and a cell-cycle dependent differential activation of RNR (S-phase) and SAMHD1 (G 1/0 -phase) has been suggested. 5 Underscoring a fundamental biologic role for SAMHD1, germ-line mutations in the gene encoding SAMHD1 are associated with human diseases, such as the neurodegenerative and hyperinflammatory Aicardi-Gouti eres syndrome (AGS). 6 In addition, somatic aberrations of SAMHD1 have been found in chronic lymphocytic leukemia, 7 lung cancer, 8 and colorectal cancer. 9 More recently, we and others have identified SAMHD1 as an obstacle toward antimetabolite-based cancer therapies, [10][11][12] which will be the focus of this Extra View.

SAMHD1 is a mediator of ara-C toxicity in AML cell models
The global annual incidence of leukemia is about 350,000 cases, predominantly consisting of acute myelogenous leukemia (AML). 13 With the exception of pediatric patients, 14 only about one fifth of AML patients survive a 5-year period after diagnosis, and survival rates decrease substantially with the age at diagnosis. 15 Standard treatment of AML involves combination therapy of an anthracycline (most commonly doxorubicin or daunorubicin) and cytarabine (ara-C). 15 The latter is a nucleoside analog that was first used for AML-induction therapy in 1968 16 and was later shown to also be very effective in postremission therapy. 17 However, most of patients will eventually succumb to resistant diseases and relapses, [15][16][17] and while it has been accepted that a worse clinical outcome directly correlates with a decrease in intracellular levels of the active metabolite ara-CTP, 18-26 the underlying molecular reasons for this remained enigmatic. Shedding light on this, we and others have recently shown that SAMHD1 is a major determinant in dictating sensitivity of AML blasts to ara-C treatment, as the cell-active triphosphate metabolite of ara-C, ara-CTP, is a SAMHD1 substrate. [10][11][12] In particular, RNA interference (RNAi)-mediated SAMHD1 knockdown, CRISPR/Cas9induced SAMHD1 knockout or SAMHD1 degradation with Vpx-containing virus-like particles (VLPs) from simian immunodeficiency virus (SIV) increased sensitivity of AML cells to ara-C cytotoxicity. [10][11][12] Confirming the importance of the catalytic activity of SAMHD1, overexpression of wild type SAMHD1, but not allosteric site mutant D137N or the catalytic site mutants D311A or H233A, significantly reduced ara-C cytotoxicity. 10,11 Interestingly, despite a proposed role of SAMHD1 phosphorylation in the restriction of retroviruses, 27 we did not find any evidence for a role of the SAMHD1 phosho-site T592 in ara-CTP turn over, 11 which is consistent with experimental evidence that dNTPase activity might be dispensable for HIV-1 restriction. 28 SAMHD1 phosphorylation ablates tetramer-formation as well as HIV-1 restriction, however the dNTPase activity of phospho-SAMHD1 is only affected in conditions of low nucleotide levels. 29 Our study also investigated the mechanism underlying ara-C cytotoxicity and showed that activation of the intra-S-phase and DNA-damage response pathways are substantially elevated in ara-C treated leukemic cells lacking SAMHD1 11 , consistent with the established mechanism of action of ara-C. [30][31][32][33] While Schneider et al. specifically looked at AML tumor cell lines, our study provides evidence that other haematological malignancies may involve SAMHD1 as a barrier to treatment efficacy and could possibly be antagonised to improve therapy. 11 Similar to monocytic THP-1 cells, the cutaneous T-cell lymphoma line Hut-78, derived from a patient with S ezary syndrome, 34 was also sensitized to ara-C treatment when SAMHD1 was depleted, and reconstitution of dNTPase-proficient SAMHD1 reduced ara-C cytotoxicity. 11 This is also in support of a negative correlation of SAMHD1 mRNA expression and ara-C cytotoxicity in a panel of cell lines containing both myeloid and lymphoid neoplasms; 11 hence SAMHD1s role as a modifier of ara-C toxicity is not restricted to myeloid neoplasms. Figure 1. Intracellular conversion of cytarabine (ara-C) to ara-CTP and detoxification by SAMHD1. The schematic depicts canonical pathways for the intracellular synthesis of ara-CTP, the active metabolite of ara-C, which exerts DNA-damage and antiproliferative downstream effects by interfering with DNA synthesis. SAMHD1 is activated by binding of GTP or dGTP to allosteric site 1 (AS1), binding of a dNTP to allosteric site 2 (AS2) and binding of its substrate in the catalytic site. Ara-CTP is a substrate for SAMHD1 but not an allosteric activator. Abbreviations: CDA, cytidine deaminase; DCK, deoxyctidine kinase; NT5C2, cytosolic nucleotidase-II; DCTD, deoxycytidylate deaminase.
Mouse models confirm in vivo role of SAMHD1 dNTPase activity in reducing ara-C treatment efficacy To address whether human AML tumor cells with differential SAMHD1 expression would respond differently to ara-C treatment, we used both a heterotopic as well as an orthotopic AML mouse model. Firstly, nude mice were subcutaneously transplanted with CRISPR/Cas9 THP-1 cell clones expressing SAMHD1 or not. 11 Secondly, we injected CRISPR/Cas9 HL-60/ iva cell clones containing or lacking a functional SAMHD1 gene, respectively, into the tail-vein of NOD/SCID mice. 11 Lack of SAMHD1 expression dramatically increased the sensitivity of AML xenotransplants to ara-C induced toxicity, resulting in pronounced survival improvements. 11 As mentioned above, it has been reported that restriction of retroviral infection by SAMHD1 can be uncoupled from its dNTPase activity, 28 and thus we wanted to confirm that modulation of ara-C efficacy in vivo is dependent on the enzymatic activity of SAMHD1 and not mediated by other functions of SAMHD1. To perform in vivo structure-function analyses, we reconstituted SAMHD1 expression by lentiviral transduction and ectopically expressed either wild type or the catalytically inactive H233A mutant of SAMHD1 in HL-60/iva CRISPR/ Cas9 SAMHD1 ¡/¡ cells. We confirmed equal expression of the SAMHD1 variants by immunoblotting (Fig. 2a) and then xenotransplanted NOD/SCID mice i.v. Subsequently, these mice were treated with 50 mgkg ¡1 ara-C for 5 consecutive days from day 6 post xenotransplantation, and signs of disease of these mice were monitored by veterinarian examination as described previously. 11 Mice transplanted with cells expressing wild type SAMHD1 were substantially more resistant to ara-C treatment and developed signs of disease after a median time of 23 days, while 5 out of 6 mice in which cells were engrafted expressing the H233A mutant of SAMHD1 were still without signs of disease at the time of sacrifice ( Fig. 2b and c). This demonstrates that in vivo detoxification of ara-C requires catalytically competent SAMHD1. SAMHD1 controls the therapeutic response of AML to ara-C Targeting SAMHD1 with RNAi or Vpx-VLP treatment in patient-derived AML blasts sensitized those to ara-C-induced toxicity, although there was some donor-to-donor variability in the magnitude of sensitization. 10,11 A retrospective analysis of the adult AML cohort from The Cancer Genome Atlas (TCGA), 10,11 as well as the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) cohort of children with AML, 11 demonstrated that patients with lower SAMHD1 expression levels in AML blasts had better clinical outcome. Interestingly, our analyses did not show a significant difference in SAMHD1 expression in patients that achieved complete remission as compared with patients that did not. 11 The survival advantage only became apparent later, i.e. during consolidation courses. This seemingly is a contradiction to the data presented by Schneider et al. that based their conclusions for the clinical significance of SAMHD1 upon immunohistoand immunocytochemistry. As the therapy regimens did not differ substantially between the patient cohorts analyzed in both studies, this discrepancy might be explained by methodological differences. We assessed mRNA expression of SAMHD1 as a continuous measure, whereas Schneider et al. scored the staining intensity by eye categorically. It can be reasoned that protein levels do not necessarily correlate with mRNA abundance, while another possible confounder may be staining of patient-AML specific non-SAMHD1 epitopes due to the polyclonal nature of the antibody used by Schneider et al., however there is no reason to assume that unspecific staining would be more prevalent in AML patients that fail induction therapy. Ultimately, only the analysis of an independent patient cohort by immunohistochemistry, preferably using a more specific antibody, may resolve this apparent contradiction. This is not merely an academic discussion, as this will have large implications when considering SAMHD1 as a possible biomarker for treatment stratification. If the mRNA levels analyzed in 2 independent patient cohorts in our study are representative, dose modification (i.e., dose reduction for low SAMHD1 expressers and/or dose escalation for high SAMHD1 expressers) would only be justified in the post-remission phase. We feel that this is in line with the notion that anthracyclines contribute most to remission induction, 35 and in line with the study by Mayer et al., which showed improved survival in AML with escalating doses of ara-C during the post-remission phase. 17 In any case, reliable cut-off values of SAMHD1 expression applicable for Figure 2. Overexpression of wild type but not catalytic-inactive SAMHD1 confers resistance to ara-C treatment in vivo. HL-60/iva CRISPR/Cas9 cells lacking endogenous SAMHD1 expression were transduced with a lentiviral vector encoding for HA-tagged wild type (black) or the catalytically-inactive H233A mutant (red) SAMHD1. Equal expression levels of ectopic SAMHD1 were confirmed by western blotting (a). Cells were xenotransplanted into NOD/SCID IL2R ¡/¡ female mice; (n D 12 for each cell line), which were subsequently treated with either PBS or ara-C. Clinical signs of disease (b) and percentage of survival (c) were determined over time. For details see Methods.
dose-adjustments would have to be determined by prospective studies specifically designed for this purpose and cannot simply be inferred from retrospective analyses. Furthermore, the therapeutic phase with most negative impact of SAMHD1 will also inform the study design of implementing putative SAMHD1 inhibitors into AML treatment protocols. In our manuscript, we suggested to explore Vpx as a biologic SAMHD1 inhibitor. 11 As this protein ultimately has to be delivered to the cytoplasm/ nucleoplasm, we made use of non-replicating VLPs as vehicle, whose pseudotyping with VSV-G guarantees delivery to the cytoplasmic compartment. 36 In fact, Vpx-VLPs have been shown to increase transduction efficiency as a direct effect of Vpx, 37,38 and Vpx has been suggested to improve the efficacy of lentiviral gene therapy. 39,40 Lentiviral vectors are being tested in clinical trials, mainly for ex vivo transduction of hematopoetic stem cells to correct hemoglobinopathies and primary immunodeficiencies, 41,42 for instance. To treat the systemic disease AML, in vivo delivery of Vpx would be required. Pre-clinical safety and toxicity data are available for lentiviral vectors, but studies for in vivo transduction to correct non-haematological genetic disorders are only in preparation. 43 One major obstacle for Vpx therapy in AML is the efficiency of bone marrow targeting, however in vivo mouse experiments show that this barrier can be overcome. 44,45 Alternative strategies to deliver Vpx to leukemic blasts are mRNA-based therapeutics, 46 the use of small DNA viruses, 47 liposomal packaging of Vpx protein 48,49 or coupling of Vpx to antibodies targeting AML cells. 50 SAMHD1 potentially bears some tumor suppressor functionality, 7-9,51-53 and thus transient inhibition of SAMHD1 only for the time of co-administration of ara-C or other antimetabolites would be preferable from a toxicity and safety perspective. Hence, viral transduction strategies with potential off-target effects 54 might conceptually be the least favorable approach, and small molecule inhibitors would be ideal for controllable, safe, transient and cost-effective SAMHD1 inhibition. As germline mutations of SAMHD1 are causing autoimmune-like symptoms in AGS patients, 6 it is also important to evaluate inflammatory reactions in putative SAMHD1 inhibitor trials. If existent, these might even enhance anti-tumor effects e.g. due to interferon secretion. Although to date no cell-active SAMHD1 inhibitors have been reported, methodologies have been developed that can be used to screen small molecule libraries in vitro. 55,56 From these and other studies, several nucleoside triphosphate-based small molecules have been reported to successfully inhibit SAMHD1 in vitro, 12,55-57 and although these compounds currently lack potency, they provide starting points for rational design of SAMHD1 inhibitors.
Following the line of discussion in our recent manuscript regarding targeting SAMHD1 to improve therapy, 11 one could speculate that AML patients with high SAMHD1 levels in leukemic blasts would eventually have a better prognosis than low SAMHD1 expressers due to the tumor suppressive activity of SAMHD1 in putative post-treatment residual AML cells. In any case, further studies on the role of SAMHD1 in maintaining genome stability and cell homeostasis are needed. In addition to general dNTP pool homeostasis, potential further explanations for tumor suppressor functions of SAMHD1 could be sanitation of endogenously occurring or stress-induced modified nucleotides, like dUTP 58 or oxidised dNTPs, [59][60][61] or perhaps the potential role of SAMHD1 in the DNA damage response, 7 where it could be involved in fine-tuning dNTP requirements for repair synthesis. SAMHD1 neutralizes the cytotoxic effects of diverse nucleoside-based antimetabolites SAMHD1 is a promiscuous dNTPase that is regulated allosterically by dNTPs, and thus it is likely that several dNTP analogs, whether endogenously occurring or as part of antimetabolitebased therapies, will be SAMHD1 substrates and/or activators. Among the first modified nucleotides to be tested as substrates were several nucleoside reverse transcriptase inhibitors (NRTIs), and overall these were restrictive to SAMHD1-induced hydrolysis. Interestingly, SAMHD1 was reported to increase activity of these nucleoside analogs in cells by depleting the competing endogenous nucleoside triphosphates. 59,62 Despite lack of activity toward NRTIs, several base-modified nucleotides were reported as SAMHD1 substrates, such as O6-methyl-dGTP and 5-methyl-2 0 -dCTP, 59 however the biological relevance of this has yet to be examined. The development of a continuous enzyme-coupled assay for measuring the triphosphohydrolase activity of SAMHD1 enabled the investigation of potential nucleotide substrates, activators and inhibitors. 55 It was in this report that activity of SAMHD1 toward the nucleoside triphosphate metabolite of clofarabine, an anti-cancer drug used to treat hematological malignancies, was demonstrated. 55 Clofarabine-triphosphate was also suggested to be an allosteric activator. 55 This was confirmed in our study, and we also demonstrated that SAMHD1 modulates clofarabine-induced toxicity in THP-1 and Hut-78 cells, 11 however to a lesser extent than ara-C (see Fig. 3a and e for comparison). Supportive of a wider role of SAMHD1 in controlling clofarabine cytotoxicity, a significant negative correlation was observed between SAMHD1 expression and clofarabine-induced cytotoxicity in a panel of 133 haematological and lymphoid tissue-derived cell lines (Fig. 4a).
A recent study used in silico predictions to identify additional substrates of SAMHD1, and verified several of these predictions using an in vitro HPLC-based assay. 12 To determine whether SAMHD1 modulates cytotoxicity of these and other compounds, we treated parental THP-1 or THP-1 CRISPR/Cas9 SAMHD1 knockout cells with a panel of these nucleoside analogs and measured cytotoxicity as described previously. 11 As anticipated, THP-1 cells lacking SAMHD1 were 2 orders of magnitude more sensitive to cytarabine treatment (Fig. 3a). Similarly, SAMHD1 ¡/¡ cells displayed increased sensitivity to vidarabine (Fig. 3b), nelarabine (Fig. 3c), fludarabine (Fig. 3d), decitabine (Fig. 3f) and trifluridine (Fig. 3g), indicating that triphosphate variants of these drugs could be substrates for SAMHD1. Although vidarabine is mainly used as an antiviral, it has been suggested as an antineoplastic agent, 63 and trifluridine is another example of an antiviral drug that recently has been repurposed for the use in cancer treatment. [64][65][66] In addition to a possible role for future cancer therapies, this may become interesting when considering SAMHD1 as a possible intrinsic resistance gene toward treatment of herpes viral infections. 67 In further support of a key role for SAMHD1 in controlling cytotoxicity of nelarabine and decitabine, significant negative correlations of SAMHD1 mRNA expression and toxicity of these drugs were observed in a panel of myeloid and lymphoid cells lines (Fig. 4b and c). We also reported that gemcitabine triphosphate and 6-thioguanine triphosphate are not substrates of SAMHD1, and accordingly, the absence of SAMHD1 did not affect toxicity of these drugs. 11 The data presented here, together with the recent reports, 10,12,55 suggests that SAMHD1 is a key player in reducing the efficacy of antimetabolite-based cancer therapies; further investigations will determine whether this is the case and to which extent this is relevant in clinical settings. The complex allosteric regulation of SAMHD1 in combination with the enzymatic activity creates a scenario in which the net effect of a SAMHD1-interacting nucleoside toward SAMHD1 activity is composed to varying degrees of (i) being a substrate and/or competitive inhibitor at the catalytic site as well as (ii) an allosteric activator or inhibitor at either one or both of the allosteric sites. 55 Eventually, a very simple strategy for choosing the right antimetabolite for a SAMHD1-positive cancer might be to select one that is not a SAMHD1-substrate.

Future perspectives-SAMHD1 and beyond
Our study suggests that targeting SAMHD1 bears the potential to improve outcome in AML therapy when combined with high-dose ara-C treatment. 11 As shown by Hollenbaugh et al. and the present work, SAMHD1 may also have activity toward triphosphate metabolites of other drugs used against AML (in particular fludarabine and clofarabine) and drugs used against other types of cancers like nelarabine for T-lymphoblastic lymphoma and leukemia, decitabine for myelodysplastic syndrome (MDS), and trifluridine that in combination with the thymidine phosphorylase (TP) inhibitor tipiracil, is used against metastasised colorectal cancer and has been approved by the FDA and EMA. 68 Tipiracil is a prime example of how the understanding of both anabolic and catabolic antimetabolite metabolism is driving the development of combination therapies to improve the efficacy of anti-cancer drugs. TP is a glycosyltransferase that removes the deoxyribose monophosphate from thymidine leaving thymine as second reaction product. 69 It has been recognized that trifluridine monophosphate (tF-dTMP) is also a substrate for TP, counteracting the intracellular toxification of trifluridine. 70 Catabolic enzymes that reduce the effective concentration of antimetabolites have, however, been recognized long before. The deaminsases cytosine deaminase (CDA) and deoxycytidylate deaminase (DCTD) convert dC or dCMP, respectively, and its analogs ara-C or ara-CMP as well as 5-aza-dC or 5-aza-dCMP, [71][72][73] to their uracil derivatives that have no or much lower antineoplastic activity 74 (Fig. 1). Indeed, CDA inhibitors like tetrahydrouridine have been developed to increase both plasma (inhibiting plasma and liver CDA) and intracellular concentrations of ara-C. 72,75,76 Another example of augmenting the efficacy of antimetabolites is the use of small molecule inhibitors targeting dUTPase, which hydrolyses dUTP and 5-FdUTP, an active metabolite of 5-FU, to their monophosphate forms, thereby limiting their toxic incorporation into nascent DNA. 58,77,78 Hence, inhibitors of dUTPase can increase the efficacy of 5-FU. 79,80 A more recent target to improve cytotoxic therapies with thiopurines has emerged by discovering that the nudix enzyme family member NUDT15 can hydrolyse triphosphates of 6-thioguanine and mercaptopurine. 81,82 Hence, also in this case, combining these drugs with a NUDT15 inhibitor might improve therapy outcomes. It should, however, be added that these drugs are particularly suited for maintenance therapy strategies over longer periods of time. Hence, a careful preselection of patients that have a selective overexpression of NUDT15 in tumor cells should be aimed at when studying NUDT15 inhibitors clinically to omit drastic increases in toxicitythat even today are reasons to pause or cancel a treatment with thiopurines. 83 Based on substitutions of the stereoselective 2 0 sugar moiety, Hollenbaugh et al. predicted nelarabine triphosphate, fludarabine triphosphate, BV-ara-UTP, floxuridine triphosphate, trifluridine triphosphate, sorivudine triphosphate and cladribine triphosphate as SAMHD1 substrates. It will be of interest to simulate interactions of other nucleotide drugs with the SAMHD1 catalytic site, possibly in an effort for rational design of competitive SAMHD1 inhibitors.
Several SAMHD1 inactivating single nucleotide polymorphisms (SNPs) have been described in AGS, some of them located close to the active site. 6,84 Certain SAMHD1 SNPs were also reported for chronic lymphoblastic leukemia. 7 In addition, a recent study associated SAMHD1 SNPs with colon cancer. 9 While some of these SNPs have been shown to affect dNTPase function, possibly even in a dNTP-specific manner, there are currently hundreds of SNPs published in public databases for which the effects on SAMHD1 dNTPase activity and stability are unknown. Future studies will have to investigate their role in cancer, their antagonisation of antimetabolite-based chemotherapies and their potential to resist small molecule inhibitors of SAMHD1. Considering compounds that bind to the active site in a competitive manner, development of resistance to these inhibitors while retaining full ara-CTPase activity seems rather unlikely, but not impossible. Allosteric inhibitors, however, are conceptually more prone for resistance development as evidenced by allosteric inhibitors of HIV-1 reverse transcriptase. 85 The fact that trifluridine triphosphate is a potential substrate of SAMHD1 could have important implications for SAMHD1directed therapies that might be relevant for non-hematological solid tumors. 86 It is important to gain more insight into why certain nucleoside analogs such as 5-FU, gemcitabine, and trifluridine are clinically most useful against solid tumors, whereas other analogs like ara-C, clofarabine, nelarabine, fludarabine and decitabine are almost exclusively reserved for myeloid and lymphoid neoplasms. Our preliminary analyses indicate that this discrepancy cannot be solely explained by differential SAMHD1 expression (data not shown). Whether general differences in tumor biology, e.g., differences in proliferation rates, or tumor-type specific transporters, metabolic enzymes or differentially activated damage-response pathways constitute the main reason for a relative resistance of solid tumors to various antimetabolites, remains to be investigated. We are hopeful that these kinds of studies will ultimately increase the arsenal of drugs that can be used to more efficiently target solid tumors.

Concluding remarks
In conclusion, our study along with the reports by Schneider et al. and Hollenbaugh et al. demonstrate that SAMHD1 is a key barrier to ara-C efficacy during AML consolidation therapy and that SAMHD1 may be a suitable target to enhance chemotherapies against other malignancies. [10][11][12] The search for drugs to target and counteract SAMHD1 to improve chemotherapeutic treatments will hopefully lead to the discovery of compounds that, in combination with existing drugs, would reduce side effects and simultaneously make them more effective. A better understanding of their physiologic metabolism will hopefully pave the way for a less empirical and more rational design of future cancer treatments with antimetabolites. Personalised cytotoxic therapy of the future could select the best combinations from a plethora of antimetabolites in clinical use and tune their efficacy by selecting the right combination of accompanying inhibitors.

Plasmids and viral vectors
pCSxW encoding hemagglutinin-tagged wild type or catalytically-inactive H233A mutant SAMHD1, HIV-1 GagPol expression vector pCMV-DR8.91 and pMD.G (kind gift from D. Trono, School of Life Sciences, Lausanne, Switzerland) were described before. 11 To generate guideRNA-resistant versions of these SAMHD1 expression plasmids we PCR-amplified the SAMHD1 cDNAs using forward primer (EcoRI) OTS1478 5 0 -ATC gaa ttc ATG CAG CGA GCC GAT TCC GAG CAG CCG TCA GCA CGA CCC AGA TGT GAC GAT TCA CCA AGA ACC CCC TCA AAC AC-3 0 which contains non-coding nucleotide changes (underlined) and reverse primer (NotI) OTS1393 5 0 -GCA Tgc ggc cgc TCA CAT TGG GTC ATC TTT AAA AAG C-3 0 and cloned the amplicon back into pCSxW-HA using EcoRI/NotI. pLentiCRISPRv2 (Addgene plasmid #52961) encoding SAMHD1 specific gRNAs was described before. 11 For viral vector production 293T cells were transfected according to described previously protocols. 11 Generation of THP-1 SAMHD1 CRISPR/Cas9 cell lines and antibodies The construction of THP-1 and HL-60/iva CRISPR/Cas9 single cell clones with disrupted SAMHD1 gene has been described before. 11 We reconstituted expression of HA-tagged wild type or H233A mutant SAMHD1 by transduction with pCSxW derived lentiviral vectors encoding guide RNA-resistant SAMHD1 at a multiplicity of infection (MOI) of »100. Due to this high MOI there was no need to drug-select for ectopic expression. We used a monoclonal rat anti-HA antibody 3F10 (Roche) directly coupled to horse-reddish peroxidase to detect ectopically expressed SAMHD1, mouse monoclonal anti-SAMHD1 antibody 1F9 (Abcam) or rabbit polyclonal anti-Hsp90 antibody H-114 (Santa Cruz) as loading control.

Mouse experiments
Ectopic SAMHD1 expression was achieved by lentiviral transduction of HL-60/iva CRISPR/Cas9 SAMHD1 ¡/¡ cells. Ectopically expressed wild type or the catalytically-inactive H233A mutant SAMHD1 contained synonymous nucleotide substitutions to escape the guide RNAs expressed in the cell. Transduced cell bulks were xenotransplanted intravenously into NOD/SCID mice (each n D 6). Mice were treated with 50 mgkg ¡1 ara-C for 5 consecutive days from day 6 post xenotransplantation. Signs of disease of these mice were monitored by veterinarian examination with endpoints defined as described previously. 11

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
to their AML databases and the Broad Institute for access to the Cancer Therapeutics Response Portal (CTRP).


s2fieldsofstudy: Biology Chemistry

text:
Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding

RNase P is a universal enzyme that removes 5′ leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP–tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP–tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

Transfer RNA maturation involves many post-transcriptional steps. Among them, 5Ј leader sequences are removed by an endonuclease activity called RNase P (1). This processing step was assumed to be universally performed by ribonucleoprotein (RNP) 5 particles containing a ribozyme and up to 10 protein subunits until the discovery of protein-only RNase P in eukaryotes (2).
Protein-only RNase P (PRORP) enzymes were first identified in human mitochondria and plants (3,4). Contrary to initial appreciations, PRORP enzymes are not restricted to organelles in a few species (5). They are actually widespread in eukaryotes, occurring in mitochondria and/or chloroplasts as well as in nuclei in four of five eukaryote supergroups (6). In some groups, i.e. in most Chloroplastida (Viridiplantae), Stramenopiles, and Trypanosomatida, PRORP enzymes have seemingly entirely replaced ribonucleoproteins for RNase P activity as experimentally shown for Arabidopsis thaliana (7), Chlamydomonas reinhardtii (8), and Trypanosoma brucei (9). PRORP enzymes were also characterized in the moss Physcomitrella patens (10). Intriguingly, in this species, a nuclear PRORP is not essential, although no recognizable ribonucleoprotein RNase P is present (6). Remarkably, the occurrence of PRORP and RNP RNase P appears mutually exclusive in compartments or in entire organisms (6). RNPs or PRORPs might have been retained in specific cell compartments or entire organisms because they have different substrate spectra in vivo. Alternatively, in specific clades, RNPs or PRORPs might have evolved additional functions that cannot be held by the other type of enzyme (6).
cro ARTICLE nuclease domain belonging to the N4BP1, YacP-like nuclease (NYN) family (15) and an N-terminal pentatricopeptide repeat (PPR) domain (16,17) believed to be responsible for RNA binding and substrate specificity. These two main domains are bridged by a central zinc-binding domain (2). A comparative kinetic analysis of RNase P activities by PRORP and RNPs has suggested that the two types of enzymes use different catalytic mechanisms (18). Remarkably, this comparative analysis also revealed that RNPs are better catalysts than PRORP enzymes at least in vitro. However, the identification of tRNA residues in contact with PRORP (11) and RNP RNase P (19) has suggested that RNPs and PRORP use a similar strategy to recognize their substrates (11). Still, how this is achieved at the protein level remained unknown, and the dynamics required for PRORP mode of action was unexplored (20).
To tackle these questions, we determined the crystal structure of Arabidopsis nuclear PRORP2 together with a SAXS model in solution and explored the role of its PPR domain for substrate binding. We found that two particular PPR motifs are required for specific tRNA recognition by PRORP2. Furthermore, we studied the PRORP2-tRNA complex in solution by SAXS to establish an interaction model. Taken together, our results identify structural features in PRORP2 important for the RNA binding process and suggest that the enzyme is flexible and undergoes conformational changes to perform its activity.

Crystal and solution structure of Arabidopsis nuclear PRORP2
Crystallization conditions were extensively screened for different PRORP2 constructs, including catalytically impaired and shortened forms. Crystals were only obtained for the full-length wild-type PRORP2 (21). Their analysis led to the determination of the enzyme structure at a resolution of 3.05 Å ( Fig. 1A and supplemental Table S1). As proposed based on sequence similarities with the organellar PRORP1 (2, 11), PRORP2 adopts the same characteristic ⌳-shape. The N-terminal arm consists of a PPR domain made of five PPR motifs, the C-terminal arm consists of the NYN metallonuclease domain, and a bipartite zincbinding domain (ZBD) bridges the two arms together. The first 27 residues, including the nuclear localization signal, as well as the last 20 amino acids, including the His 6 affinity tag, were probably floppy in the crystal packing and could not be visualized in the electron density map. The two copies of PRORP2 present in the triclinic unit cell are very similar (root mean square deviation, 0.85 Å; supplemental Fig. S1). They also superimpose well with PRORP2 monomers (average root mean square deviation, 1.32 Å) recently described by Karasik et al. (22). As in the latter crystal structure determined concomitantly to ours in the same triclinic environment and in fairly similar crystallization conditions, we do not observe any cation bound to conserved aspartate residues in the catalytic site, although we and Karasik et al. (22) show that, similar to PRORP1, Mg 2ϩ ions are required for catalysis (supplemental Fig. S2).
The full-length PRORP2 and a more compact form, PRORP2xs, were characterized in solution by SAXS as well (Fig. 1C). Their gyration radii (R g ) of respectively 35 and 32 Å derived from the Guinier analysis are in good agreement with the value calculated from the crystal structure (29.5 Å) deprived of N-and C-terminal extensions (supplemental Fig. S3). Possible conformations of PRORP2 were explored by a normal mode analysis (NMA), and the model fitting best to the SAXS data (supplemental Fig. S4) was extended to include missing N-and C-regions and refined under SAXS constraints (supplemental

Defining conditions for a stable PRORP2-tRNA complex
A prerequisite for the characterization of the PRORP2-tRNA complex was to define buffer conditions compatible with various biochemical and biophysical analyses and ensuring both protein and RNA stability. The effect of the salt concentration was tested on PRORP2 catalytic activity. Initial buffer conditions contained 250 mM NaCl to increase the solubility of the enzyme, but it appeared to be detrimental to both crystallization (21) and activity. As shown in supplemental Fig. S6, the cleavage of pre-tRNA sharply decreases beyond 150 mM NaCl. The latter concentration was chosen in combination with 5% glycerol (w/v) as a good compromise to maintain both PRORP2 monodispersity in solution and its activity.
Defining a compact but well processed RNA substrate was another prerequisite, especially for SAXS or crystallography to avoid long and flexible extensions. We therefore gradually trimmed the leader sequence of our Arabidopsis pre-tRNA Cys(GCA) constructs from 51 down to 5 nucleotides (supplemental Fig. S7). The best compromise was obtained with the L5T0 tRNA Cys (i.e. with a leader sequence of 5 nucleotides, no trailer sequence, and no added CCA), which proved to be fully cleaved and showed up as a single band on a native gel. This is in agreement with a previous study (22) showing that PRORP2 preferentially binds substrates with short 5Ј leaders and 3Ј trailers. The L5T0 construct was selected for all subsequent analyses. In the same line, the short PRORP2xs construct was preferred over the full-length catalytic mutant to avoid long and flexible regions and to facilitate SAXS modeling (supplemental Fig. S5).

PRORP2 affinity for pre-tRNA substrates
The experimental setup defined above was used to determine the affinity of Arabidopsis PRORP2 for pre-tRNA by several biophysical methods. Hence, a catalytically inactive mutant of PRORP2xs containing two alanines in place of two aspartates at positions 421 and 422 in the NYN domain (7) was used in combination with the L5T0 tRNA substrate. Isothermal titration calorimetry (ITC) revealed a first K D value of 1 M for the interaction of PRORP2 with tRNA with a ⌬H of Ϫ3.9 10 4 cal⅐mol Ϫ1 , a ⌬S of Ϫ107 cal⅐mol Ϫ1 ⅐degree Ϫ1 , and a stoichiometry of 0.4 ( Fig. 2A), thus suggesting that a proportion of either of the partners would not form a complex or that one of the partners (tRNA) partially oligomerizes as seen by size exclusion chromatography (SEC) in similar conditions (data not shown). Subsequent dynamic light scattering (DLS) and SEC measurements confirmed that the sample did not aggregate during the experiment. To confirm these results, the PRORP2-tRNA complex formation was also monitored by microscale thermophoresis (MST) using a tRNA Cys precursor 5Ј-labeled with a fluorescent dye (Cy5). This revealed a K D of 1 M with 95% confidence

Relative importance of PPR motifs for interaction with tRNA
PRORP proteins belong to the huge family of PPR proteins. These eukaryote-specific RNA-binding proteins are involved in Red, blue, and gray indicate three replicate experiments. In the AUC plot, different colors indicate the particular concentrations of PRORP2 and/or RNA in the respective AUC experiments. The star indicates signals for PRORP2 alone, the cross indicates signals for RNA alone, and the arrow indicates signals for PRORP2-pre-tRNA Cys complexes. K D values that could be derived from the three biophysical approaches are indicated in the respective panels.

Biophysical analysis of PRORP2-tRNA complex
a wide variety of post-transcriptional processes such as RNA editing, splicing, and the maturation of transcript ends (16,23). They are composed of ϳ35-amino acid tandem repeats of degenerate primary sequences although bearing a conserved helix-turn-helix structure. The investigation of PPR protein mode of action has revealed that each PPR motif specifically interacts with a defined ribonucleotide. Some residues are particularly important to achieve this specificity, i.e. two residues located toward the start and at the end of PPR motifs, termed here positions 5 and 35, according to Cheng et al. (23). The nature of amino acids at these positions defines a combinatorial code for RNA recognition by PPR motifs (24 -26). The PPR domain of Arabidopsis PRORP2 is composed of five such motifs (PPR1-PPR5) (supplemental Fig. S8). Positions 5 and 35 of motifs PPR2 and PPR3 are particularly well conserved in plants with the occurrences of a Gln or Asn at PPR2 position 5 and an Asn or Ser at PPR2 position 35 as well as a Thr at PPR3 position 5 and an Arg at PPR3 position 35 in an alignment of 100 plant PRORP sequences (6). Shorter alignments are exemplarily shown in supplemental Figs. S8 and S9. Although the "PPR code" is not yet fully understood (i.e. target RNAs cannot be predicted for many PPR proteins or motifs, and the exact involvement of other residues besides positions 5 and 35 for specific RNA binding is not entirely known), the latest resources and knowledge on PPR RNA recognition (23) enable the prediction that PRORP2 motif PPR2 would recognize a cytidine, whereas motif PPR3 would recognize a purine. In contrast, no clear prediction can be made for PPR motifs 1, 4, and 5.
Accordingly, to test the relative importance of individual PPR motifs for PRORP function, presumably for substrate recognition, PRORP2 was mutated at positions 5 and 35 of its five PPR motifs. The nature of the amino acids and mutations at these positions is indicated on supplemental Fig. S8. The five double mutants were expressed and purified to homogeneity. Solubility, stability, and structural integrity of mutants were verified by DLS and SAXS. All mutants behaved similarly to the wild-type enzyme and its catalytically inactive version in solution (supplemental Fig. S10) except PPR4 variant, which is unstable and prone to aggregation especially upon RNA removal. Therefore, the latter mutant could only be used for cleavage assays at low concentration.
Hence, the five mutant proteins were assayed for in vitro RNase P activity with two tRNA Cys precursors with leader sequences of either 5 or 51 nucleotides. The quantification of relative cleavages revealed that PPR mutants 1, 4, and 5 were as active as wild-type PRORP2. However, activity decreased by 45% (on average for the two tRNA substrates) in PPR2 mutant and by 91% in PPR3 mutant (Fig. 3A). This suggests that motifs PPR2 and -3 are the most important for substrate recognition by PRORP2 in accordance with comparable results obtained for the organellar PRORP1 (27).
Still, RNase P cleavage experiments represent indirect evidence for PRORP binding to tRNA. We therefore analyzed direct interaction between PPR mutants and RNA using sedimentation velocity AUC to determine which PPR motifs are indeed involved in tRNA binding. For this, the PRORP2 PPR double mutants were further mutated in the catalytic site as described above to obtain catalytically inactive proteins ena- Ϫ indicates reactions with RNA alone. PRORP cleavage products were separated by 10% denaturing PAGE and quantified with ImageJ. Numbers indicate percentages of cleavage as defined by Gobert et al. (11). Values were normalized so that 100 corresponds to cleavage observed for wild-type PRORP2. The molecular weights of markers (M) are given in ribonucleotides. B, analytical ultracentrifugation sedimentation plots of catalytically inactive WT PRORP2 and PPR mutants in complex with tRNA Cys precursors having 5-nucleotide-long leader sequences. Different colors indicate the particular concentrations of PRORP2 isoforms and/or RNA in the respective experiments. Red dotted lines indicate signals for PRORP2 alone, blue dotted lines indicate signals for RNA alone, and purple dotted lines indicate signals for PRORP2-tRNA Cys complexes. K D values that could be derived for the different complexes are indicated in the respective panels.

Biophysical analysis of PRORP2-tRNA complex
bling K D measurements. The observed broad distributions of sedimenting species (between the faster tRNA component and the complex species) are characteristic of rapidly reversible systems due to dynamically associating and dissociating events during sedimentation. The weight average s-values of the fast component of the reaction boundary (swfast) isotherms were generated and analyzed to get the binding constants. The K D of PPR5 mutant interaction with tRNA Cys was estimated to 0.9 M (95% confidence limits, 0.7, 1.1 M), similar to wild-type PRORP2 (95% confidence limits, 0.6, 1.9 M), whereas the K D values of PPR1 and PPR2 mutants were estimated to 2.2 M (95% confidence limits, 1.7, 3.0 M) and 2 M (95% confidence limits, 1.4, 2.7 M) respectively. For PPR3, no estimate of the K D was possible due to the absence of complex formation in the experimental conditions (Fig. 3B). The analysis of AUC data showed that K D variations were not due to the aggregation or misfolding of PPR mutants. No data could be obtained for PPR4 mutant because of its instability at the concentrations required for AUC (see above).
Altogether, results indicate that motif PPR3 is the most important to form the PRORP2-tRNA complex. Mutations of PPR2 motif affect both catalysis and RNA binding although to a lesser extent. PPR1 mutant is only moderately affected for RNA binding. This is probably because the mutation at position 35 introduces a negative charge that can interfere with RNA binding. Still, although PPR1 mutant does not display a loss of RNase P activity, our data suggest that the PPR1 motif is in the vicinity of the RNA in accordance with previous work showing contacts between the N-terminal extremity of PRORP and tRNA (28). In contrast, PPR motifs 4 and 5 do not seem to be involved in RNA interaction because their mutations do not alter RNA binding and cleavage.

Modeling of PRORP2-tRNA complex based on SAXS data
The conditions defined to favor a stable PRORP2-tRNA interaction were used to study the PRORP2xs-L5T0 pre-tRNA complex by SAXS. For this purpose, PRORP2xs, pre-tRNAs, and their complex were separated by analytical SEC just upstream of the SAXS cell. Because the three entities could hardly be resolved by SEC (Fig. 4A) and because of the M affinity of PRORP for tRNA, the SEC separation was carried out in the presence of 1.5 M free enzyme in the mobile phase to avoid complex dissociation. The complex was detected in the second part of the main scattering peak where the estimated R g along data collection makes a plateau at ϳ34 Å (Fig. 4B), i.e. a value 6% larger than that observed for PRORP2xs alone (supplemental Fig. S3). The SAXS profile and the P(r) distribution are also clearly distinct from those obtained with the isolated enzyme (supplemental Fig. S3).
To build a model of the PRORP2-tRNA complex, an ensemble of data previously available and determined here were considered.
1) Our previous footprinting analysis identified tRNA residues protected from RNase digestion and thus in contact with PRORP in the tRNA D and T loops (U16, G18, G19, and C56) (11).
2) These residues and others that are universally conserved among canonical tRNAs (29) were mutated. For some of them, mutations resulted in a complete loss of RNase P activity, i.e. G18, C56, and R57 (where R represents a purine) in the full- pre-tRNA (green chromatogram; injection, 0.2 nmol; peak elution time, 12.5 min), PRORP2xs (blue; injection, 0.9 nmol; peak elution time, 11.9 min), the complex formed during an ITC titration (orange; injection, 20 l of ITC mixture; peak elution time, 12.0 min), and a 2:1 PRORP2xs/L5T0 mixture (red; injection, 0.6:0.3 nmol; peak elution times, 11.9 and 12.3 min). This illustrates the difficulty to stabilize the complex and to separate it from individual partners. B, evolution of X-ray scattering intensity I(0) (blue plot) and R g (red plot) along the SEC elution of the complex in a mobile phase supplemented with PRORP2xs to minimize the dissociation of the complex. A data set of 240 SAXS images was collected in the 10 -18-min window symbolized by the blue arrow. SAXS images (frames 112-130) were averaged in the region indicated by the green bar where R g is ϳ34 Å, leading to the profile shown in C. C, best solution model of the complex obtained under distance (PPR2 and -3 interacting with C56 and G18, respectively) and SAXS constraints. The theoretical SAXS profile (green curve) is overlaid on experimental data (blue dots). The Guinier plot is given in the inset. Alternative possibilities leading to higher values are shown in supplemental Fig. S11. The zoom on the right highlights the proximity of residues 5 and 35 of PPR motifs 2 and 3 (analyzed by mutagenesis) with tRNA residues C56 and G18 at the corner of the tRNA (see open blue arrow). Abs., absorbance; a.u., arbitrary units.

Biophysical analysis of PRORP2-tRNA complex
length tRNA (11) as well as C56 and R57 in a minihelix corresponding to the tRNA acceptor domain (30).
3) Mutations also showed that the anticodon domain of tRNAs is completely dispensable for RNase P activity (11,30).
4) The PRORP2 catalytic pocket that includes Asp-421 and Asp-422 (and binds catalytic magnesium (12)) must be next to tRNA positions Ϫ1 and ϩ1. 5) PPR mutagenesis and AUC data determined here suggest that PPR motifs 2 and 3 are involved in tRNA binding accordingly at the opposite side of the acceptor arm, i.e. in the D and T loops.
6) The PPR code predicts that motifs PPR2 and 3 should bind a cytidine and a purine, respectively. Best candidates are positions G18, G19, C56, and R57.
Taking into account these spatial criteria, a series of models of the complex was generated from the crystal structure of PRORP2 and the model of L5T0 pre-tRNA Cys (prepared by homology modeling using Assemble (31) and based on Escherichia coli tRNA Cys(GCA) crystal structure, Protein Data Bank code 1U0B (32)). These models test different combinations of PPR-nucleotide interactions. Their theoretical scattering profiles were compared with experimental SAXS data to select the best model (i.e. with best goodness of fit or lowest value). Because PPR3 was found to be the most important for RNA interaction, models were first built with PPR3 interacting with either G18, G19, or R57. In the models involving G19 and R57, the orientation of the tRNA does not allow an additional interaction of PPR2 with a tRNA residue. In contrast, in the model involving PPR3 interaction with G18, PPR2 is close to C56. This model of the complex displayed in Fig. 4 is the only one satisfying all distance criteria: (i) motifs PPR2 and PPR3 binding two residues in the D/T loops of tRNA and (ii) nucleotides Ϫ1 and ϩ1 in the vicinity of metal binding aspartates. Most importantly, this model also gives the best fit with SAXS experimental data ( of 2.4). Other combinations involving a single PPR inter-action with base G19, C56, or G57 lead to a reorientation of the tRNA with respect to PRORP2 and higher values (ranging from 3.8 to 5.0), indicating that the shapes of these models are less representative of the objects present in solution (supplemental Fig. S11).
In our model, the backbone conformation of D/T loops was essentially kept as in the E. coli template tRNA, but the nucleobases C56 and G18 were flipped in syn conformation to point toward the protein. Indeed, in the unprocessed pre-tRNA transcript, these bases are certainly unpaired to keep D/T loops flexible and accessible to modification enzymes and thus to PPR modules as well. For instance, tertiary interactions that stabilize mature tRNAs (29), i.e. the C56 -G19 or the G18 -⌿55 interaction may hardly take place before PRORP binds pre-tRNA because RNase P cleavage is one of the first steps of pre-tRNA maturation after transcription (33). Among PRORP1 lysines shown in a recent mass spectrometry study to cross-link with the tRNA (28), the only two that are conserved in PRORP2 (Lys-42 and Lys-387, which are equivalent to Lys-109 and Lys-439 in PRORP1) are close to the tRNA backbone in our SAXS model of the complex. The comparison of the crystal structure and SAXS models of PRORP2 alone or in complex with pre-tRNA suggests that the enzyme core is quite flexible with a main hinge between the zinc-binding domain and the catalytic domain. In particular, structural differences between free and complexed forms of PRORP show that major conformational changes may take place upon binding of pre-tRNA. It appears that the plasticity of PRORP2 backbone allows opening of its ⌳-shape and a rotation of the catalytic domain to accommodate tRNA substrates (Fig. 5).

Discussion
Since the first descriptions of protein-only RNase P (3, 4), crystal structures and biophysical studies of Arabidopsis and human PRORP have revealed the functional organization

Biophysical analysis of PRORP2-tRNA complex
and structural diversity of these enzymes (for a review, see Schelcher et al. (20)). Initial investigations of PRORP mode of action showed which tRNA residues are in contact with PRORP (11), but the way tRNA recognition is achieved by PRORP was unknown, and no data were available on the structure of the PRORP-tRNA complex and on the dynamics of the enzyme.
Mutagenesis of PPR motifs and the model derived from SAXS data acquired on the PRORP2-tRNA complex suggest that only the PPR2 and -3 motifs of PRORP are involved in tRNA recognition. Besides PRORP, all characterized PPR proteins bind single-stranded RNA (16), and the current trend is that tandem arrays of PPR motifs specifically bind unpaired RNA bases in a sequence-specific manner (24). Because PRORP binds any tRNA of canonical structure, it was proposed that PRORP diverged from the general mode of action of PPR proteins and developed a specific RNA recognition mode based on structural features rather than on linear sequences (2,30). Our results suggest that this is not completely the case at least for motifs PPR2 and -3 that appear to follow the canonical base recognition by PPR motifs. Indeed, PPR2 could recognize C56, and PPR3 could recognize a purine at the corner of the tRNA. This purine could be either G18 or G57, although our SAXS model indicates that G18 is closer to motif PPR3 and is thus more likely recognized by this motif when PRORP interacts with a full tRNA. However, in the context of a minihelix mimicking the acceptor arm with a less constrained single-stranded T loop region, as used by Brillante et al. (30), PPR3 might also be able to bind G57, whereas PPR2 interacts with C56.
In contrast, motifs PPR4 and -5 do not seem to participate in tRNA binding, and their function, besides maintaining the PPR architecture, remains elusive. They might be involved in the recognition of yet unidentified non-tRNA substrates of PRORP2. Alternatively, the role of these motifs might be to position PPR2/3 at the right distance from the active site of the NYN domain. The overall PRORP fold would then act as a ruler that selects the cleavage site with respect to the tRNA corner held by PPR2/3. However, although positions 5 and 35 of PPR motifs are recognized as the most important for specific RNA binding, the functional relevance of other positions such as position 2, which was also proposed to be involved in RNA interaction, for PRORP/tRNA recognition remains unexplored.
In addition to the PRORP RNA recognition process, we determined and compared the crystal and different solution models of PRORP2 to capture various conformations of PRORP and look at the conformational landscape of PRORP proteins. To explore possible domain reorientations, we performed a NMA, which is known to provide a good description of low-frequency collective motions in proteins (34). The comparison of NMA perturbed models with our SAXS data in solution suggests that both PRORP1 and PRORP2 can adopt a variety of conformations because of the presence of a hinge between the catalytic NYN domain and the central zinc-binding domain. Related movements correspond to the opening of the ⌳-shape of PRORP and the rotation of its catalytic domain around the central hinge (supplemental Fig. S3). Models were further refined against SAXS data with the genetic algorithm DADIMODO and led to an excellent fit for PRORP2xs, again showing multiple compatible conformations with a reorientation of the catalytic domain (supplemental Fig. S5). The presence of long and floppy extensions in the full-length enzyme led to slightly higher goodness of fit (), but the trend remained the same. The superposition of the PRORP2 crystal structure and solution models alone or in complex suggests the existence of a continuum of conformations that allow the accommodation of RNA substrates and possibly with a longer helical domain than a classical tRNA acceptor arm (Fig. 5). This adaptability is exemplified by the capacity of PRORP to cleave substrates such as tRNA His , which has an 8-base pair acceptor stem and can be processed by PRORP at positions Ϫ1 and ϩ1 (35) or by the cleavage of tRNA-like substrates as observed for mitochondrial nad6 and orf291 mRNAs (7,36). Overall, the intrinsic plasticity of PRORP2, which likely applies to all members of the PRORP family, may be crucial to bind RNA substrates and to release matured RNA products.
Such type of conformational adaptation has been observed in other monomeric tRNA-binding enzymes. In the case of the human mitochondrial phenylalaninyl-tRNA synthetase, the crystal structure of the isolated enzyme revealed a very compact conformation. SAXS data on the complex with tRNA indicated a large rigid-body motion of the anticodon-binding domain upon tRNA binding (37). Other examples are the bacterial elongation factor Tu and the conformational transition following GTP hydrolysis that leads to ribosome translocation and elongation factor Tu release (38); the bacterial TruB, which catalyzes pseudouridine formation at U55 in tRNA and undergoes a large rigid body displacement of its C-terminal region upon tRNA binding (39); and the archaeal O-phosphoseryl-tRNA kinase, the enzyme that phosphorylates Ser-tRNA Sec to produce O-phosphoseryl-tRNA Sec , which displays a Ͼ60-Å movement of its C-terminal domain to bind the variable region of tRNA Sec (40). Large conformational movements thus seem to be a general feature to modulate the recognition, binding, or release of tRNAs.

PRORP2 and tRNA purification
Expression of the full-length enzyme in the wild-type and catalytically impaired (PRORP2mut) versions as well as of PPR mutants and their purification by immobilized metal affinity chromatography and SEC were performed as described by Pinker et al. (21) A more compact and catalytically inactive form (PRORP2xs) was designed, and the region of PRORP2 cDNA coding for residues 25-516 was introduced in a pTYB1 plasmid (New England Biolabs) to produce the protein Cterminally fused to self-cleavable intein and chitin-binding domain. In both PRORP2mut and PRORP2xs, two essential aspartate residues (421 and 422) were mutated to alanines to inhibit the nucleolytic activity and facilitate the formation of stable complexes, and PPR motifs were mutated to abolish RNA interaction. Mutations were introduced with the QuikChange Site-Directed Mutagenesis kit (Stratagene) using PCR primers designed with the manufacturer's qcprimerdesign online tool.
The expression of PRORP2xs was performed in E. coli BL21 (DE3) strain at 17°C in 2 liters of LB medium containing ampi-Biophysical analysis of PRORP2-tRNA complex cillin and induced with isopropyl 1-thio-␤-D-galactopyranoside. The cells were harvested by centrifugation, resuspended in a buffer containing protease inhibitors (Sigma, catalogue number S8830; one tablet per 100 ml), 50 mM HEPES-Na, pH 7.5, 250 mM NaCl, 15% (w/v) glycerol, and 1 mM TCEP and were disrupted by ultrasonication. Cell debris was removed by centrifugation, and the supernatant was applied onto a 50-ml chitin affinity column (New England Biolabs) equilibrated with the same buffer including 500 mM NaCl. To trigger intein cleavage and PRORP2xs release, 50 mM DTT was added to the buffer, and the protein was incubated on the column for 16 h at 4°C. After elution, PRORP2xs was further purified by SEC on a Superdex 200 10/300 GL column (GE Healthcare) and stored in 50 mM HEPES-Na, pH 7.5, 250 mM, NaCl, 15% (w/v) glycerol, and 1 mM TCEP. To reduce the NaCl concentration prior to complex formation, this storage buffer was exchanged by ultrafiltration or dialysis with a buffer containing 150 mM NaCl, 30 mM HEPES-Na, pH 7.5, 5% (w/v) glycerol, and 1 mM TCEP (buffer P). Sample quality was systematically assessed by SDS-PAGE and DLS as described previously (11,21). Arabidopsis mt-tRNA Cys(GCA) L5T0 and L51T30 pre-tRNA cDNAs (with leader sequences of 5 or 51 nucleotides as well as trailer sequences of 0 or 30 nucleotides) were amplified from Arabidopsis seedling total cDNA, cloned in pUC19, and transcribed in vitro by T7 RNA polymerase as described by Gobert et al. (11).

Determination of PRORP2 crystal structure
Crystallization and crystallographic analysis were performed as described by Pinker et al. (21). In brief, PRORP2 in buffer P was crystallized in 2-l batch drops at 4°C by mixing (1:1 ratio) with a crystallant solution containing 200 mM sodium malonate, pH 6, and 20% (w/v) PEG 3350. Diffraction data were collected at 100 K with a wavelength of 1 Å using a PILATUS 2M detector on the X06DA beamline at the Swiss Light Source (SLS Synchrotron, Paul Scherrer Institute, Villigen, Switzerland). Search models for molecular replacement were derived from the structure of organellar PRORP1 (Protein Data Bank code 4G23) using MODELLER (41). A molecular replacement search was performed with Phaser and the PHENIX package (42,43). Models of single PRORP2 monomer were split in three domains (PPR, ZBD, and NYN), and one copy of each was placed in the P1 unit cell. As the self-correlation function indicated the presence of 2-fold non-crystallographic symmetry, the second molecule was localized using a second copy of the catalytic domain. Two complete copies of PRORP2 were then refined at 3.05-Å resolution using a maximum likelihood target as implemented in PHENIX (42). Non-crystallographic symmetry constraints between the two monomers were applied during refinement as well as a translation-libration-screw-rotation (TLS) model for atomic displacement parameters. Manual inspection and rebuilding were performed with Coot (44). The final structure (Protein Data Bank code 5FT9) describes two monomers of PRORP2 including residues 28 -515. N-terminal (1-27) and C-terminal (516 -536) tails and two loops (312-321 and 499 -514) are not visible in the electron density map. Data collection and refinement statistics are given in supplemental Table S1.

Isothermal titration calorimetry
ITC was performed on a MicroCal ITC instrument (Malvern Instruments, Malvern, UK) at 20°C with 187 M PRORP2 in buffer P with 5 mM MgCl 2 . 19 samples of 3 l of protein solution were injected with a 120-s interval in the cell containing 280 l of 22 M L5T0 tRNA Cys precursor dialyzed in the same buffer. Data analysis was performed with MicroCal Origin7 software.

Microscale thermophoresis
MST was performed with a Monolith NT 115 instrument (NanoTemper Technologies GmbH, Munich, Germany). 10-l aliquots of a serial dilution of PRORP2 (188 M to 6 nM) in buffer P with 5 mM MgCl 2 and 0.3 mg/ml BSA were added to 10 l of a 100 nM solution of 5Ј-Cy5-labeled L5T0 tRNA Cys (IBA GmBH, Göttingen, Germany). The light-emitting diode laser power was set to 20%, the IR laser power was set to 40%, and the variation of fluorescence related to molecule and complex mobility was monitored at 20°C according to the manufacturer's instructions. Data analysis was performed with Monolith MO.Affinity analysis software.

Analytical ultracentrifugation
Sedimentation velocity experiments were performed in a Beckman Coulter ProteomeLab XL-I instrument at 20°C and 50,000 rpm with absorbance detection. 400 l of different ratios of PRORP2 and L5T0 tRNA Cys were prepared in buffer P with 5 mM MgCl 2 . tRNA concentration was kept constant at 0.7 M, and protein was added to final concentrations varying from 0.25 to 10 M. Samples of 0.7 M tRNA alone and 10 M PRORP2 alone were also measured. The fitting of data was performed using SEDFIT software (www.analyticalultracentrifugation.com/) 6 (55) and a continuous sedimentation coefficient distribution model. The distributions obtained for each sample were integrated to determine the weight average sedimentation coefficient effective particle theory swfast as a function of protein concentrations and to generate swfast isotherms (isotherms of the fast boundary component of rapidly interacting systems based on the Gilbert-Jenkins theory (45). The isotherms were loaded into SEDPHAT for fitting with the heteroassociation model A ϩ B 7 AB to obtain an estimate of the K D . In the analysis, s A and s B were fixed at the experimentally determined values for PRORP2 and tRNA, respectively, whereas K AB and s AB were subject to optimization through nonlinear regression. The error surface projection analysis was exploited to determine the error intervals of the best fit K D values at a 95% confidence level. Buffer density, buffer viscosity, and protein partial specific volumes were calculated using SEDNTERP software. The software GUSSI was used to plot and integrate the sedimentation coefficient distributions and to generate the isotherms (46).

RNase P activity assays
RNase P cleavage assays were performed with three replicates using 0.5 M transcript and 0.15 M PRORP2 protein in buffer containing 30 mM Tris-HCl, pH 8, 40 mM NaCl, 4.5 mM MgCl 2 , 20 g⅐ml Ϫ1 BSA, and 100 nM DTT for 15 min at 25°C as described previously (4). RNA fragments were separated by denaturing PAGE, visualized by ethidium bromide staining, and quantified as described (4).

Small-angle X-ray scattering analysis
SAXS experiments were performed on the SWING beamline at Synchrotron SOLEIL (Saint Aubin, France) as described previously (11). In brief, samples were loaded onto a size exclusion column (Bio SEC-3 with 150-Å pore size, Agilent Technologies), and SAXS measurements were performed throughout elution. A mixture of 30 l of 320 M catalytically inactive PRORP2xs and 25 l of 185 M L5T0 pre-tRNA Cys in buffer P with 5 mM MgCl 2 was injected in the column equilibrated with buffer P and 5 mM MgCl 2 supplemented with 1.5 M PRORP2xs to prevent dissociation of partners during elution. PRORP2 and PRORP2xs were also characterized alone in buffer P. Data processing, interpretation, and R g evaluation over elution profiles were performed using Foxtrot (47), and data analysis (determination of R g and d max ) was performed with the ATSAS package (48). Based on NMA with ElNemo (49), crystal structures of PRORP1 and -2 were perturbed to select models that better represented the SAXS profile of PRORP2 because of a goodness of fit calculated with CRYSOL (50,51). Complete models of PRORP2 and PRORP2xs (including their respective N-and C-terminal extensions not described in the crystal structure) were generated under SAXS constraints using DADIMODO (52), a genetic algorithm-based refinement program. Models of PRORP2-tRNA complex were built with SASREF (53) under SAXS constraints using this PRORP2xs solution structure with a flexible hinge between the zinc-binding and catalytic domains and taking into account distance restraints between above mentioned residues in PPR2/3 modules, the catalytic domain, and nucleotides in the tRNA. SASREF models were manually inspected to remove steric clashes and adjusted in PyMOL using the SAXS plug-in SASpy (54).
Author contributions-P. G. and Cl. S. designed and coordinated the experiments. F. P., Ce. S., P. F.-M., A. G., C. B., A. T., P. R., P. G., and Cl. S. performed the experiments and analyzed the results. P. G. and Cl. S. wrote the manuscript.