Review Article Cellular and Biochemical Mechanisms of the...

Hindawi Publishing CorporationISRN BiochemistryVolume 2013 Article ID 728392 11 pageshttpdxdoiorg1011552013728392

Review ArticleCellular and Biochemical Mechanisms of the RetroviralRestriction Factor SAMHD1

Li Wu

Center for Retrovirus Research Department of Veterinary Biosciences and Department of Microbial Infection and ImmunityThe Ohio State University 1900 Coffey Road Columbus OH 43210 USA

Correspondence should be addressed to Li Wu wu840osuedu

Received 5 June 2013 Accepted 7 July 2013

Academic Editors I de la Serna and H Himeno

Copyright copy 2013 Li WuThis is an open access article distributed under theCreative CommonsAttribution License which permitsunrestricted use distribution and reproduction in any medium provided the original work is properly cited

Replication ofHIV-1 and other retroviruses is dependent on numerous host proteins in the cells Some of the host proteins howeverfunction as restriction factors to block retroviral infection of target cells The host protein SAMHD1 has been identified as thefirst mammalian deoxynucleoside triphosphate triphosphohydrolase (dNTPase) which blocks the infection of HIV-1 and otherretroviruses in non-cycling immune cells SAMHD1 protein is highly expressed in human myeloid-lineage cells and CD4+ T-lymphocytes but its retroviral restriction function is only observed in noncycling cells Recent studies have revealed biochemicalmechanisms of SAMHD1-mediated retroviral restriction In this review the latest progress on SAMHD1 research is summarized andthe mechanisms by which SAMHD1 mediates retroviral restriction are analyzed Although the physiological function of SAMHD1is largely unknown this review provides perspectives about the role of endogenous SAMHD1 protein inmaintaining normal cellularfunction such as nucleic acid metabolism and the proliferation of cells

1 Introduction

HIV-1 infection of human target cells is highly dependenton numerous host proteins to support viral replication [1 2]Genome-wide functional screens of HIV-1 cofactors haverevealed that over two hundreds of human proteins arerequired for efficient HIV-1 replication in the cells [3ndash6]However during the long history of host-retrovirus inter-actions primates including humans have evolved intrinsicimmunity to defend against viral infection through hostproteins called restriction factors which often can be coun-teracted by viral proteins via intracellular degradation [7ndash9]Studying those host restriction factors not only helps definethe unique function of certain viral proteins in the infectionand viral pathogenesis but also provides new insights intodeveloping more effective intervention strategies to blockretroviral infections

The major HIV-1 target cells supporting viral replicationare activated CD4+ T-lymphocytes [10] while other celltypes such as primary monocytes dendritic cells (DCs) andmacrophages also contribute to initial infection and viraltransmission [11 12] Nondividing CD4+ T-lymphocytes and

myeloid cells including monocytes DCs and macrophagesplay an important role in establishment of HIV-1 initialinfection and viral transmission [10 11] However thesecells are refractory to postentry HIV-1 infection due tomultifaceted cellular mechanisms [11 12] Resting CD4+ T-cells are essential for the establishment and maintenanceof HIV-1 latency as they can generate viral reservoirs inAIDS patients treated with antiretroviral drugs [13] As agroup of important antigen presenting cells to bridge theinnate and adaptive immune responses DCs can sense HIV-1infection and trigger innate immunity against HIV-1 [14] Itis conceivable that restriction to HIV-1 infection of myeloidcells and resting CD4+ T-lymphocytes is a means of innateor intrinsic immunity against retroviral infection in vivo [15]Recent studies have provided new insights into themolecularmechanisms underlying the viral defense in these importantimmune cells

Several previous studies have identified and character-ized three major retrovirus restriction factors includingAPOBEC3G [16] TRIM5120572 [17] and tetherin (as knownas BST2 CD317 or HM124) [18 19] Interestingly HIV-1has evolved effective ways to evade host restriction factors

2 ISRN Biochemistry

through its several accessory proteins such as Vif to counter-act APOBEC3G and Vpu to degrade tetherin [8 20] Recentstudies have identified a novel retroviral restriction factornamed SAMHD1 in non-cycling myeloid cells and restingCD4+ T-lymphocytes [21ndash25] Human SAMHD1 has beenshown to have a broad restriction of diverse retroviruses[26 27] In addition to HIV-1 SAMHD1 also blocks infectionof other retroviruses including HIV-2 feline immunodefi-ciency virus bovine immunodeficiency virus equine infec-tious anemia virus and murine leukemia viruses [26 27]However the mechanisms of SAMHD1-mediated retrovirusrestriction and its physiological functions remain to beelucidated Previously published reviews or commentarieshave summarized the role of SAMHD1 in restricting HIV-1 infection in nondividing human immune cells [15 28ndash34] In this review I would like to focus on the analysis ofthe cellular and molecular mechanisms by which SAMHD1mediates retroviral restriction I will also discuss potentialcellular function of SAMHD1 in nucleic acid metabolism andthe proliferation of cells

2 The Identification and Characterization ofSAMHD1 as an HIV-1 Restriction Factor

21 The Vpx Proteins from SIV and HIV-2 Enhance HIV-1Infection in Myeloid Cells Goujon et al showed thatpreincubation with virus-like particles derived from sootymangabey lineage SIV (SIVsm) significantly enhancestransduction efficiency of human monocyte-derived DCsusing HIV-1-derived lentiviral vectors [35] Similarly Vpxproteins from SIVsm and HIV-2 significantly promoteHIV-1 infection in human monocytes and derived DCs andmacrophages by facilitating the accumulation of full-lengthHIV-1 cDNA [36ndash40] It has also been known that Vpxis critical for HIV-2 infection in human macrophages byenhancing the efficiency of viral reverse transcription [41]Further studies indicated that Vpx from SIV or HIV-2 caninteract with the host protein DCAF1 in the CUL4ADDB1and E3 ubiquitin ligase complex [42 43] indicating that Vpxmay target a putative HIV-1 restriction factor for proteasomaldegradation in myeloid cells through the E3 ubiquitin ligasecomplex (for reviews please refer to [44 45]) These studieson Vpx and HIV-1 infection of myeloid cells have promptedthe identification of SAMHD1 as an HIV-1 restriction factorin human myeloid cells [21ndash23]

Although HIV-1 genome does not encode the Vpx pro-tein Vpx has been a useful tool in the design of lentiviralvectors to better transduce myeloid-lineage cell types that arerelatively resistant to lentiviral infection or transduction [46]Sunseri et al generated a modified HIV-1 to package SIVVpx which efficiently infects human primary macrophagesand DCs [47] Because Vpx is packaged into virions throughthe specific interaction with the p6 carboxy-terminal domainof Gag the authors introduced the Vpx packaging motif ofSIV p6 into an HIV-1 proviral DNA construct The chimericHIV-1 packaged Vpx in trans and showed significantly higherinfectivity in DCs andmacrophages compared with the wild-type virus Furthermore by introducing the Vpx coding

sequence into the proviral DNA to incorporate Vpx in cisthe modified HIV-1 can efficiently replicate in DCs andmacrophages [47] The Vpx-containing HIV-1 could be moreefficiently transmitted from DCs to cocultured CD4+ T cellssuggesting that Vpxmay facilitateDC-mediated transmissionof HIV-2 or SIV in vivo Given that the infection of DCswith theVpx-containingHIV-1 triggers activation ofDCs andcocultured CD4+ T-cells [47 48] these studies imply that thechimeric HIV-1 might be used to design DC-based vaccinesto induce an enhanced innate immune response

22 The Discovery of SAMHD1 as an HIV-1 Restriction FactorInteracting with Vpx The identification of SAMHD1 wasmainly dependent on the isolation and analysis of Vpx-interacting host proteins by mass spectrometry [21ndash23] Pre-vious studies have demonstrated that phorbol myristic acid-(PMA-) differentiated THP-1 cells can be rendered more per-missive to HIV-1 infection by transduction of SIVsmHIV-2 Vpx-containing virus-like particles derived from SIVmac[44] Based on these observations Laguette et al iden-tified SAMHD1 as a novel Vpx-interacting protein fromdifferentiated human monocytic THP-1 cells that ectopicallyexpress tagged Vpx [21] SAMHD1 is expressed in HIV-1nonpermissive cell types including THP-1 cells primarymonocytes monocyte-derived macrophages and DCs butnot in HIV-1 permissive CD4+ T-cell lines [21] Knockdownof endogenous SAMHD1 protein in non-permissive THP-1cells and primary DCs alleviates HIV-1 restriction By con-trast overexpression of SAMHD1 in monocytic U937 cellsand then differentiation with PMA inhibits HIV-1 infectionin the cells [21] Independently Hrecka and colleagues iden-tified SAMHD1 from the human embryonic kidney cell line(HEK 293T) expressing tagged Vpx in a proteomic screenusing multidimensional protein identification technology[22] The authors further demonstrated that the inhibition ofHIV-1 infection in monocyte-derived macrophages by Vpx-mediated proteasome degradation of SAMHD1 through thecellular E3 ubiquitin ligase complex [22] Together thesestudies confirmed that Vpx interacts with SAMHD1 andinduces proteasomal degradation of SAMHD1 in THP-1 cellsor macrophages which can be restored by treatment witha proteasome inhibitor [21 22] A recent analysis furtherdemonstrated that Vpx fromHIV-2 or SIV engages SAMHD1onto the E3 ubiquitin ligase complex CRL4DCAF1 to initiateproteasomal degradation of SAMHD1 [49] Moreover Wei etal identified a novel DCAF1-binding motif required for Vpx-mediated degradation of SAMHD1 protein [50] suggestingimportant interactions involved in the assembly of Vpx-hijacked DCAF1-DDB1-based E3 ubiquitin ligase complexThe molecular interactions between Vpx and SAMHD1 alsosuggest an evolutionary conflict between lentiviruses andhost restriction factors which lead to evolutionary studies ofprimate SAMHD1 and lentiviral Vpx proteins

23 Evolutionary Biology of SAMHD1 SIVsm SIVsm-derived SIV that infects rhesusmacaques (SIVmac) andHIV-2 that infects humans encode both Vpr and Vpx proteinsBy contrast SIVcpz that naturally infects chimpanzees and

ISRN Biochemistry 3

HIV-1 that infects humans only encode Vpr but not Vpx[44] The vpx gene has been suggested to be derived fromduplication of the primate lentivirus vpr gene while thesetwo related gene products may have different functions [44]Several studies have demonstrated the importance of Vpx inSIVpathogenesis sincemacaques infectedwithVpx-defectiveSIV had lower levels of viremia and viral replication aswell as slower AIDS progression compared to wild-type SIV-infected animals [51ndash53]The important role of Vpx in lentivi-ral infection in vivo suggests that Vpx-mediated SAMHD1degradationmay facilitate SIV replicationHowever it shouldbe pointed out thatVpxmay also have SAMHD1-independenteffects in the infected cells such as facilitating nuclear importof viral DNA [44] Thus the precise function of Vpx inlentiviral pathogenesis remains to be further investigated

Evolutionary analysis of human and primate SAMHD1sequences revealed strong positive selection of the SAMHD1genes [54ndash56] which is a common feature of all cur-rently identified retroviral restriction factors [20] Lim et alreported that the ability of primate lentiviruses to degradetheir host SAMHD1 proteins occurred prior to the birth ofthe lentiviral protein Vpx [55] The authors observed thatnot only multiple Vpx but also some SIV Vpr proteins areable to degrade SAMHD1 which likely led to strong positiveselection of SAMHD1 in the primate subfamily Cercopitheci-nae [55] Thus the authors suggested that the function ofVpr-mediated degradation of SAMHD1 occurred before thebirth of the separated vpx gene in SIV thereby initiatingan evolutionary arms race between primate lentiviruses andSAMHD1

Similarly Laguette and colleagues performed evolution-ary and functional analyses of the interactions betweenmultiple primate SAMHD1 and lentiviral Vpx proteins andfound that SAMHD1 restriction of HIV-1 is evolutionarilymaintained among different primates while antagonismof SAMHD1 by Vpx appears to be species-specific [54]The authors also suggested that strong positive selectionof SAMHD1 during primate evolution occurred in theCatarrhini ancestral branch before the separation betweenhominoids andOldWorldmonkeys Furthermore the identi-fication of SAMHD1 residues under positive selection guidedmapping the shortVpx-interaction domain to theC-terminusof SAMHD1 [49 54] (Figure 1)

Using ancestral host state reconstruction and temporaldynamic analyses Zhang and colleagues suggested thatcoevolution of primate SAMHD1 and lentivirus Vpx leadsto the loss of the vpx gene in HIV-1 ancestor [56] Theiranalyses indicated that the most recent common ancestor ofSIV and HIV-1 was a SIV that had a vpx gene however thevpx gene of SIVcpz was lost approximately 3643 to 2969years ago during the SIVcpz infection of chimpanzees [56]HIV-1 therefore could not inherit the lost vpx gene from itsancestor SIV infecting chimpanzees The lack of Vpx in HIV-1 likely results in restricted infection in myeloid cells that areimportant for antiviral immunity which may contribute tothe AIDS pandemic due to immune evasion of HIV-1 [56]Taken together these evolutionary and functional analysesof SAMHD1 and Vpx proteins not only confirmed the armsrace between host restriction factors and lentiviruses but also

provided a pathogenic explanation why HIV-1 genome doesnot contain a vpx gene It is conceivable that losing the vpxgene in HIV-1 ancestor might contribute to its cross-speciestransmission into humans and avoiding protective immuneresponses to viral infection

24 Structure and Enzymatic Activities of Human SAMHD1Protein TheHD domains in proteins have putative nucleoti-dase and phosphodiesterase activities [57] and the highlyconserved histidine (H) and aspartic acid (D) residues in theHD domain of SAMHD1 are critical for its dNTPase activity[58] Laguette et al first showed that overexpression of theHDAA (aa 206-207) mutant SAMHD1 in U937 cells is notable to restrict HIV-1 suggesting that the phosphodiesteraseactivity of the HD domain is important for the restrictionfunction of SAMHD1 Further analysis revealed that the HDdomain of SAMHD1 is responsible for its dGTP-stimulateddNTPase activity [58 59] (Figure 1)

The nuclear localization signal (NLS) of human SAMHD1protein has been identified to residues 11KRPR14 in theN-terminus of the protein [60 61] Mutagenesis of theseresidues of SAMHD1 changed its nuclear distribution tothe cytoplasm SAMHD1 mutants localized to the cyto-plasm can potently restrict HIV-1 and SIV similar to thewild type protein but the nuclear Vpx proteins cannotdegrade SAMHD1 mutants that localize to the cytoplasm[60 61] These studies suggest that cytoplasmic localizationof SAMHD1 does not affect its dNTPase activity and anti-HIV function in nondividing cells Of note using confocalmicroscopy and subcellular fractionation techniques Baldaufet al demonstrated that endogenous SAMHD1 protein canlocalize in both the nucleus and cytoplasm in primarymacrophages and CD4+ T-lymphocytes [24] However thephysiological function of nuclear and cytoplasmic SAMHD1remains unclear and it is unclear whether retroviral infectionmay alter the intracellular localization of SAMHD1 in pri-mary monocytes DCs and macrophages

25 SMAHD1 Gene Expression and Regulation SAMHD1mRNA is highly expressed in primary B-lymphocytes CD4+T-lymphocytes and CD14+ monocytes isolated from healthyblood donors Moreover 2- to 6-fold variations of the levelsof SAMHD1 mRNA were observed among different blooddonors [62] SAMHD protein expression has been reportedin many types of primary immune cells from healthy donorsincluding B-cells CD4+ and CD8+ T-cells CD14+ mono-cytes monocyte-derived macrophages or DCs [21ndash25 63ndash66]However the physiological function of SAMHD1 in theseimmune cell types remains unclearWelbourn and colleaguesidentified naturally occurring splice variants of SAMHD1 inseveral cell lines which lack exons 8-9 and 14 respectively[67] These splice variants localize primarily to the nucleusand are sensitive to Vpx-dependent degradation Howevercompared to full-length SAMHD1 these splice variants lackmetabolic stability and catalytic activity to block HIV-1infection [67] Furthermore the expression and potential roleof the splice variants of SAMHD1 in HIV-1 primary targetcells remain to be established

4 ISRN Biochemistry

1 45 110 115 562 595 626

PV

Enzymatic sites

Vpxinteracting

T592

(167H 206HD207 311D)

SAM HD domain

NLS(11KRPR14)

Figure 1 Schematic illustration of SAMHD1 protein and its functional domains Numbers indicate amino acid positions of human SAMHD1protein The SAM domain HD domain and the C-terminal variable region of SAMHD1 are indicated Other critical residues and motifsinclude nuclear localization signal (NLS) four critical residues in SAMHD1 NLS (11KRPR14) four residues in the enzymatic HD domain(H167 H206 D207 and D311) and phosphorylation of threonine at residue 529 (T592 indicated with the letter P)

A recent study by de Silva and colleagues reported thatpromoter methylation regulates SAMHD1 gene expressionin human CD4+ T cells [65] The SAMHD1 promoter ismethylated in CD4+ Jurkat and Sup-T1 T-cell lines but notprimary CD4+ T lymphocytes which express high levels ofSAMHD1 These data indicate a direct correlation betweenthe methylation of the SAMHD1 promoter and transcrip-tional repression [65] Further SAMHD1 protein expressioncan be induced in CD4+ T cell lines by blocking the activity ofDNA methyltransferase and histone deacetylase suggestingthat promoter methylation and histone deacetylation areepigenetic mechanisms by which regulate SAMHD1 geneexpression [65] Epigenetic mechanisms are critical for can-cer initiation and progression through modulation of geneexpression and transcriptional gene repression via epigeneticmodifications have been reported in many cancers [68]Therefore it is conceivable that downregulation of SAMHD1to reduce its dNTPase activity can contribute to cancer devel-opment because cancer cells can maintain a high dNTP poolto support their rapid DNA replication and cell proliferation

3 The Role of SAMHD1 inthe Innate Immunity

The immunological function of SAMHD1 remains to bedefined SAMHD1 mutations are involved in Aicardi-Goutieres syndrome (AGS) a genetic encephalopathy mim-icking congenital viral infection [69] SAMHD1 was firstcloned from human DCs as an interferon (IFN)-120574-induciblegene [70] and has been proposed to act as a negative regulatorof the IFN response [69] Yan et al reported that the cellularexonuclease TREX1 binds and degrades excess cytosolicHIV-1 DNA that could activate type I IFN expression and triggerinnate immune responses against viral infection [71] ThusHIV-1 may evade innate immunity in the infected individ-uals through TREX1-mediated viral DNA degradation It ispossible that a similar role of SAMHD1 may play in HIV-1immune evasion Similar to SAMHD1 TREX1 mutations inhumans are associated with autoimmune and inflammatorydiseases such as AGS [71] It is currently unknown whetherAGS patients are more susceptible to HIV-1 or other viralinfections Two studies demonstrated that peripheral bloodmononuclear cells and CD4+ T-lymphocytes from AGS

patients are more susceptible to HIV-1 infection in vitrocompared to healthy donorsrsquo cells [23 24] Developing aSAMHD1 knockout mouse model will likely provide a usefultool to study the role of SAMHD1 in the innate immunityand to examinewhether SAMHD1 can blockmouse retroviralinfection or endogenous retrotransposons in vivo

4 Potential Role of SAMHD1 inBlocking HIV-1 Infection In Vivo

Genetic analysis of the SAMHD1 gene polymorphismshas been applied to determine whether the expression ofSAMHD1 mRNA is affected by single nucleotide polymor-phisms (SNPs) in SAMHD1 and whether the SNPs are associ-ated with HIV-1 infection status [62] Using a tagging SNPapproach Coon et al determined the association betweeneight tagging SNPs in the SAMHD1 gene and the levelsof SAMHD1 mRNA expression in 70 healthy white donorsand identified one SNP that is significantly associated withSAMHD1 mRNA expression However analysis of the avail-able dataset of the genome-wide association study including857 HIV-1 controllers and 2088 HIV-1 progressors from theEuropean and African-American cohorts indicate that thereis no significant association between SNPs in the SAMHD1gene and HIV-1 infection status [62] It will be interestingand informative to analyze whether the polymorphism ofSAMHD1 is associated with HIV-2 infection or diseaseprogression in humans

In order to investigate the role of Vpx-mediated SAMHD1antagonism in the virological and clinical outcome of HIV-2 infection Yu et al analyzed the SAMHD1 degradationactivity of vpx alleles derived from seven viremic and fourlong-term aviremic HIV-2-infected individuals [72] Theauthors found that the efficiency of Vpx-mediated SAMHD1antagonism is not associated with the potency of viral controlin HIV-2-infected individuals [72] The functions of Vpx-mediated SAMHD1 degradation and enhancement of HIV-1 infection of myeloid cells are observed in most HIV-2-infected individuals including all seven patients who devel-oped AIDS [72] Although a small numbers of samples fromHIV-2 infected individuals were analyzed this study suggeststhat the activation of antiviral innate immune responses afterVpx-dependent infection ofmyeloid cells cannot fully explain

ISRN Biochemistry 5

less pathogenic effects in HIV-2-infected individuals whocontrol viral replication and become long-term survivors

SAMHD1-mediated HIV-1 restriction in resting CD4+ T-lymphocytes might be important for AIDS immunopatho-genesis Doitsh et al reported that abortive HIV-1 reversetranscription in resting tonsil CD4+ T-lymphocytes causesproapoptotic cell death and proinflammatory response [73]Of note these detrimental immune responses after HIV-1infection are triggered by accumulation of incomplete viralreverse transcripts which likely contribute to the depletion ofCD4+ T-cells inHIV-1 infected individuals [73] It is currentlyunknown whether SAMHD1-mediated restriction of HIV-1in resting CD4+ T-lymphocytes may trigger innate immunerecognition of HIV-1 cDNA and thereby result in cell deathand T-cell depletion [15]

HIV-1 cell-to-cell transmission is an efficient and rapidway to disseminate viral infection betweenDCandCD4+cells[10ndash12] or between CD4+ T-cells [74ndash76] Puigdomenechet al recently reported that SAMHD1 restricts HIV-1 cell-to-cell transmission and limits immune detection in monocyte-derived DCs [77]This study was focused onHIV-1 transmis-sion from infected CD4+ T-cells to SAMHD1-expressing orknocking-down DCs [77] It is conceivable that SAMHD1-restriced HIV-1 infection in DCs would limit viral spread-ing from DCs to CD4+ T-cells although the experimentalevidence is lacking More recently Pauls et al reported thatupregulation of SAMHD1 in macrophages accounts for atleast in part restriction of HIV-1 replication in macrophagesby IL-12 and IL-18 [78] This study suggests that cytokine-induced upregulation of SAMHD1 in macrophages or DCscan further protect these important antigen-presenting cellsfrom direct HIV-1 infection in vivo Additional ex vivo viro-logical and immunological studies using myeloid cells andCD4+ T-cells fromHIV-1 infected individuals are required tobetter understand the role of SAMHD1 in viral infection

5 The Mechanisms by Which SAMHD1 BlocksRetroviral Infection in Cells

51 Limiting Retroviral Reverse Transcription by DecreasingIntracellular dNTP Pool Previous studies using differentretrovirus as a model system have demonstrated that intra-cellular nucleotide levels can regulate retroviral infection effi-ciencies in cells (reviewed in [79]) It has been known for overtwo decades that low levels of deoxynucleotides in peripheralblood lymphocytes could be ameans to inhibit HIV-1 replica-tion [80] Efficient HIV-1 infection of macrophages dependson efficient cellular dNTP utilization by reverse transcriptase[81] Likely due to low levels of the dNTP pool in resting cellsHIV-1 entry into quiescent primary lymphocytes can initiatereverse transcription of viral cDNA but cannot complete thefull-length HIV-1 cDNA synthesis [82]

Using purified recombinant SAMHD1 expressed in Ecoli Goldstone et al and Powell et al independently reportedthat SAMHD1 is a dGTP-stimulated dNTPase which con-verts deoxynucleoside triphosphates (dNTPs) into to the con-stituent deoxynucleotides and inorganic triphosphate in vitro[58 59] Subsequent work by Lahouassa et al indicated that

SAMHD1 restricts HIV-1 replication in human macrophagesby maintaining low levels of the intracellular dNTP pool[83] SAMHD1-mediated HIV-1 restriction in resting CD4+T-cells is also dependent on low dNTP levels in the cells [24]Furthermore St Gelais and colleagues demonstrated thatSAMHD1 restricts HIV-1 infection inmonocyte-derivedDCsby dNTP depletion since Vpx-mediated SAMHD1 degrada-tion significantly increased intracellular dNTP concentra-tions and enhanced single-cycle and spreading HIV-1 infec-tion in DCs [66] These studies indicate a strong correlationbetween low levels of intracellular dNTPs and restriction ofHIV-1 infection however addition of deoxynucleotides intoresting CD4+ T-cells or macrophages to increase the intracel-lular dNTP pool cannot fully restore HIV-1 or SIV infections[24 64] suggesting that decreasing the intracellular dNTPpool by SAMHD1may not be the sole mechanism underlyingretroviral restriction in nondividing cells (Figure 2)

Gramberg and colleagues recently reported that proto-type foamy virus (PFV) and humanT cell leukemia virus typeI (HTLV-1) are not restricted by human SAMHD1 [27] Giventhat the reverse transcription of PFV mainly occurs beforeviral entry which may not be affected by the dNTP level inthe target cell The complex human retrovirus HTLV-1 mayhave evolved a mechanism to counteract SAMHD1-mediatedrestriction which may be due to a Vpx function-like viralprotein encoded by HTLV-1 genome Another possibility isthat the reverse transcriptase of HTLV-1 may support theviral replication cycle in nondividing cells with low dNTPconcentrations Indeed Jones et al demonstrated that cell-free HTLV-1 can infect human blood myeloid DCs [84] Itwould be interesting to compare HTLV-1 and HTLV-2 toexamine whether HTLV-2 is sensitive to SAMHD1-mediatedrestriction function

52 Phosphorylation of SAMHD1 Negatively Regulates ItsHIV-1 Restriction Function Two recent studies reported thatphosphorylation of SAMHD1 impairs its HIV-1 restrictionfunction [85 86] White et al showed that SAMHD1-mediated HIV-1 restriction function is regulated by thephosphorylation of threonine at residue 529 (T529) howevermutagenesis studies indicated that T529 phosphorylation ofSAMHD1 does not affect its dNPTase activity [85] MoreoverSAMHD1 contains a target motif for cyclin-dependent kinase1 (CDK1) and CDK1 activity is responsible for SAMHD1phosphorylation [85] In agreement with these findings Cri-bier et al recently reported that phosphorylation of SAMHD1by Cyclin A2CDK1 regulates its HIV-1 restriction activity[86] SAMHD1 phosphorylation regulates restriction but notthe cellular levels of dNTPs [85] Thus SAMHD1-mediatedHIV-1 restrictionmay be independent of its dNTPase activitysuggesting additional cellular co-factors may contribute toSAMHD1-mediated HIV-1 restriction function (Figure 3)The role of Cyclin A2CDK1 in regulating SAMHD1 anti-HIV-1 function remains to be investigated given that phos-phorylation of T529 in human SAMHD1 does not affect itsdNPTase activity [85] One possibility is that phosphorylationof SAMHD1 may alter its structure or conformation andthereby regulate its HIV-1 restriction function

6 ISRN Biochemistry

Myeloid cell orresting CD4+T-cell

Binds and degradesHIV-1 RNA

HIV-1 RNA

HIV-1 cDNA

HIV-1

CCR5CXCR4

CD4

Nucleus

dNTPs

dN + PPP

SAMHD1

RT

Figure 2 Proposed mechanisms of SAMHD1-mediated retroviralrestriction The diagram shows the proposed mechanisms by whichSAMHD1 restricts HIV-1 infection in nondividing cells such asmyeloid cells and resting CD4+ T-cells SAMHD1 possesses nucleaseand dNTPase activities which are dependent on its HD domainbut the SAM domain is required for maximal activity and nucleicacid binding SAMHD1 functions as a dNTPase to decrease intra-cellular dNTP pool and thereby limits HIV-1 DNA synthesis duringreverse transcription Furthermore SAMHD1may directly bind anddegrade retroviral RNA or viral DNA products generated in theinfected cell

HIV-1 infection

PMAP

Activation(proliferation)

Cycling cells

T592

CDK1

CycA2dNTPuarr dNTPdarr

Noncycling cells

SAMHD1SAMHD1

Figure 3 Phosphorylation of SAMHD1 negatively regulates itsHIV-1 restriction function SAMHD1 only exhibits antiretroviralactivity when expressed in non-cycling cells because SAMHD1 isunphosphorylated in non-cycling cells Phosphorylated threonineat residue 529 (T529) of human SAMHD1 is repressive for HIV-1restriction in cycling cells CDK1 cyclin-dependent kinase 1 CycA2Cyclin A2 The letter P in the diagram indicates phosphorylation ofT592 of human SAMHD1 PMA phorbol 12-myristate 13-acetate

53 Structure of SAMHD1 Can Affect Its HIV-1 Restric-tion Function The crystal structure of the catalytic core ofSAMHD1 reveals that the N-terminus truncated protein (aa110-626) is dimeric and indicates a structural basis for dGTPstimulation of catalytic activity hydrolyzing dNTPs [58] Thestructure of full-length SAMHD1 has not solved perhapsdue to insolubility of the full-length protein In contrastto the dimeric model suggested from the structural work

[58] functional analyses of catalytic and anti-HIV activi-ties of SAMHD1 have demonstrated that tetramerization ofSAMHD1 is critical for its dNTPase activity and restrictionof HIV-1 infection in nondividing cells [87] By contrastSAMHD1 mutagenesis studies by White et al suggestedthat oligomerization of SAMHD1 is not correlated with itsHIV-1 restriction activity [26 85] It is possible that distinctassays used in these studies may yield different results whichremains to be confirmed

A recent study by Delucia et al revealed that Vpx bindingto SAMHD1 inhibits the catalytic activity of SAMHD1 beforeleading to SAMHD1 recruitment to the E3 ubiquitin ligasecomplex for proteasome-dependent degradation [88] Thisstudy indicated that Vpx proteins interface with the C-terminus of primate SAMHD1 proteins with high affinityand this interaction inhibits SAMHD1 catalytic activityand induces disassembly of a dGTP-dependent oligomer[88] Further studies of the structure and functionalityof SAMHD1-Vpx complex will provide important insightinto mechanisms underlying SAMHD1-mediated retroviralrestriction

54 Nuclease Activity of the Human SAMHD1May Contributeto HIV Restriction In addition to the dNTPase activityof SAMHD1 recent studies suggest that SAMHD1 canbind to and degrade HIV-1 mRNA which can be a novelmechanism blocking retroviral infection [89ndash91] Goncalveset al identified SAMHD1 as a nucleic-acid-binding proteindisplaying a preference for RNA over DNA [89] SAMHD1is mislocalized due to AGS-associated mutations Tungleret al demonstrated that single-stranded RNA and DNA canpromote the formation of the SAMHD1 complex [90] Theyprovided first direct evidence that SAMHD1 associates withendogenous nucleic acids in situ Using fluorescence cross-correlation spectroscopy the authors demonstrated thatSAMHD1 specifically interacts with single-stranded RNAand DNA They also found that nucleic-acid-binding andformation of SAMHD1 complexes appear to be mutuallydependent The interaction with nucleic acids and complexformation requires the HD domain and the C-terminalregion of SAMHD1 but not the SAM domain Further-more mutations associated with AGS exhibit both impairednucleic acid-binding and complex formation implicating thatinteraction with nucleic acids is an important function ofSAMHD1 [90] These results highlight an important role ofSAMHD1 in nucleic acid metabolism which can associatewith cell proliferation and cell cycle regulation

Notably Beloglazova et al have reported exonucleaseactivity of the human SAMHD1 protein [91] They foundthat purified full-length human SAMHD1 protein possessesmetal-dependent 31015840-51015840exonuclease activity digesting single-stranded DNA and RNA substrates in vitro In double-stranded DNARNA substrates SAMHD1 preferentiallycleaves 31015840-overhangs and RNA in blunt-ended DNARNAduplexes Full-length SAMHD1 also exhibits strong bindingto DNA and RNA substrates with complex secondary struc-tures such as HIV-1 gag and tat RNA or cDNA synthesized invitro The authors concluded that the nuclease and dNTPase

ISRN Biochemistry 7

activities of SAMHD1 are dependent on itsHDdomain whilethe SAMdomain is required formaximal exonuclease activityand nucleic acid binding [91]

Together these biochemical analyses of the interactionsbetween nucleic acids and SAMHD1 and its exonucleaseactivity suggest that SAMHD1 may directly bind to anddegrade retroviral genomic RNA or transcribed viral mRNAas well as viral cDNA products generated from reversetranscription in the infected cells However this hypothesisremains to be tested in HIV-1 infected cells Further delin-eation of the mechanisms underlying SAMHD1-mediatedHIV-1 RNA and DNA binding and degradation is importantfor fully understanding the HIV-1 restriction in non-cyclingcells

6 SAMHD1-Independent HIV-1 Restriction inMyeloid Cells and Resting CD4+ T-Cells

It is possible that additional cellular restriction factorsexpressed in myeloid cells can also contribute to blockingHIV-1 in these cells Pertel et al demonstrated that Vpxrescues HIV-1 transduction of DCs from the antiviral statecaused by type 1 IFN independent of SAMHD1 [92] Inagreement with this finding a recent study by Dragin et alshowed that IFNblock toHIV-1 transduction inmacrophagesdespite SAMHD1 degradation and high dNTP levels [93]More recent studies also showed evidence for IFN alpha-induced but SAMHD1-independent cellular inhibitors ofearly HIV-1 infection in several cell lines [94] Given thata diverse range of gene products are effectors of the type IIFN antiviral response [95] it is plausible that many otherHIV restriction factors can account for the type I IFN-induced antiviral state in DCs Thielen et al suggest thatdeaminase activity of APOBEC3A isoforms in monocytesandmacrophagesmay play an important role in restriction ofHIV-1 infection in these cells [96] Furthermore APOBEC3Ahas been suggested to be a specific inhibitor of the earlyphases of HIV-1 infection in myeloid cells likely functioningtogether with SAMHD1 to block HIV-1 replication withdifferent mechanisms [97]

HIV-1 restriction in myeloid cells mainly occurs at thereverse transcription step due to SAMHD1 however HIV-1 gene transcription in myeloid cells can be further blockedDong et al have demonstrated blocking ofHIV-1 gene expres-sion at the transcriptional level due to lack of host factorsthat are required for viral gene transcription (such as cyclinT1) in primary undifferentiated monocytes or monocyte-derived DCs [98 99] These studies suggest that multifacetedmechanisms may contribute to HIV-1 restriction in humanmyeloid cells

Additional host factor may contribute to HIV-1 restric-tion in resting CD4+ T-cells For example the host proteinMurr1 involved in copper regulation can inhibit HIV-1 repli-cation in unstimulated CD4+ T cells in part through its abilityto inhibit basal and cytokine-stimulated nuclear factor-kappaB activity [100] Goujon et al also showed that HIV-1infection can be blocked by type I IFN-induced proteinsin primary macrophages and CD4+ T-cells [101] Indeed

a diverse range of host proteins have been identified to act aseffectors of the type I IFN antiviral response [95] Moreovera recent study revealed a novel anti-HIV mechanism withinthe innate immune response in which Schlafen 11 (SLFN11)selectively inhibits viral protein synthesis in HIV-infectedcells by means of codon-bias discrimination [102] SLFN11inhibits the late stages of HIV-1 production by preventingthe expression of viral proteins Schlafen genes are a subsetof IFN-stimulated early response genes (ISGs) that are alsoinduced directly by pathogens via the IFN regulatory factor 3pathway [103] It remains to be established whether SLFN11-mediated HIV-1 restriction plays a role in myeloid cells andresting CD4+ T-cells and whether HIV-1 has evolved a viralantagonist to counteract SLFN11

7 Conclusion Remarks and Future Directions

The discovery of SAMHD1 as a host restriction factorblocking HIV-1 and other retroviruses has revealed novelmolecular and cellular mechanisms to study intrinsic immu-nity against retroviruses and possibly other viral pathogensNewfindings aboutmechanisms of SAMHD1-mediatedHIV-1 restriction provide a platform towards better understandingthe important role of SAMHD1 and its interactions withthe viral antagonist (such as Vpx from SIVsmHIV-2 andVpr from some SIV lineages) in lentiviral pathogenesisFurther studies of SAMHD1 function and its mechanisms ofblocking retroviral pathogens will benefit the design of moreeffective vaccines and antiretroviral interventions againstHIV-1 infection

Manel and colleagues have suggested that HIV-1 restric-tion in DCs allows HIV-1 to avoid the antiviral immuneresponses derived from DCs which are critical antigen pre-senting cells bridging the innate and adaptive immunity [48]SAMHD1-mediated HIV-1 restriction in myeloid-lineagecells protects these cells from efficient HIV-1 infection whichlikely prevents an innate immune response triggered by HIV-1 infection [29 48] By contrast SIVsm and HIV-2 encodeVpx to overcome SAMHD1-mediated restrictionwhich likelyinduces protective innate immunity to confine viral infectionin natural hosts Thus the interactions between SAMHD1and Vpx may contribute to different consequences of HIV-1 and HIV-2 infection in humans Future in vivo studiesusing animalmodels includingmacaqueSIV and humanizedmouseHIV-1 would be able to test this hypothesis

How does an HIV-1 target cell sense viral infection andtrigger the innate immune response is not fully understoodand remains an important topic in the field [14 29] Studyingretroviral restriction factors can help understand the mech-anisms of anti-HIV innate immune responses and immuneevasion The retroviral restriction factor TRIM5 has beenreported as an innate immune sensor for the retrovirus capsidlattice [104] Galao et al and Tokarev et al have reported thatthe IFN-induced viral restriction factor tetherin stimulatesnuclear factor-kappa B signaling and induces innate pro-inflammatory responses in cells [105 106] Recent studiesby Wu and colleagues identified that cyclic GMP-AMP(guanosine monophosphate-adenosine monophosphate or

8 ISRN Biochemistry

cGAMP) is a metazoan second messenger in innate immunesignaling by cytosolic DNA [107] Furthermore it hasbeen demonstrated that cellular cyclic GMP-AMP synthase(cGAS) is a cytosolic DNA sensor that activates the type IIFN pathway [108 109] It would be interesting to investigatewhether cGAS can sense cytosolic HIV-1 DNA through thesecondmessenger cGAMP in the infected cells and the poten-tial role of SAMHD1 in this process In summary a betterunderstanding of the mechanisms of host restriction factorsand their additional physiological functions can facilitate thedevelopment of a more effective intervention against HIV-1or other viral infections

Abbreviations

SAMHD1 Sterile alpha motif domain- and HDdomain-containing protein 1

HIV-1 Human immunodeficiency virus type 1HIV-2 Human immunodeficiency virus type 2SIV Simian immunodeficiency virusDDB1 Damage-specific DNA binding protein 1CUL4A Cullin-4ADCAF1 DDB1- and CUL4A-associated factor-1DCs Dendritic cellsAPOBEC3G Apolipoprotein B mRNA-editing

enzyme-catalytic polypeptide-like 3GTRIM5120572 Tripartite motif-containing protein 5120572

Conflict of Interests

Theauthor reports no conflict of interests associated with thispaper

Acknowledgments

The research in the authorrsquos laboratory was supported byGrants AI098524 and AI102822 from the National Institutesof Health and by the Center for Retrovirus Research and thePublic Health Preparedness for Infectious Diseases Programof The Ohio State University The author apologizes to allcolleagues whose important publications could not be citedas a result of space limitations

References

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[2] A Engelman and P Cherepanov ldquoThe structural biology ofHIV-1 mechanistic and therapeutic insightsrdquo Nature ReviewsMicrobiology vol 10 no 4 pp 279ndash290 2012

[3] A L Brass D M Dykxhoorn Y Benita et al ldquoIdentification ofhost proteins required for HIV infection through a functionalgenomic screenrdquo Science vol 319 no 5865 pp 921ndash926 2008

[4] R Konig Y Zhou D Elleder et al ldquoGlobal analysis of host-pathogen interactions that regulate early-stage HIV-1 replica-tionrdquo Cell vol 135 no 1 pp 49ndash60 2008

[5] H Zhou M Xu Q Huang et al ldquoGenome-scale RNAi screenfor host factors required for HIV replicationrdquo Cell Host andMicrobe vol 4 no 5 pp 495ndash504 2008

[6] F D Bushman N Malani J Fernandes et al ldquoHost cell factorsin HIV replication meta-analysis of genome-wide studiesrdquoPLoS Pathogens vol 5 no 5 Article ID e1000437 2009

[7] P D Bieniasz ldquoIntrinsic immunity a front-line defense againstviral attackrdquo Nature Immunology vol 5 no 11 pp 1109ndash11152004

[8] D Wolf and S P Goff ldquoHost restriction factors blockingretroviral replicationrdquo Annual Review of Genetics vol 42 pp143ndash163 2008

[9] R S Harris and M T Liddament ldquoRetroviral restriction byAPOBEC proteinsrdquo Nature Reviews Immunology vol 4 no 11pp 868ndash877 2004

[10] LWu ldquoBiology ofHIVmucosal transmissionrdquoCurrentOpinionin HIV and AIDS vol 3 no 5 pp 534ndash540 2008

[11] C M Coleman and L Wu ldquoHIV interactions with monocytesand dendritic cells viral latency and reservoirsrdquo Retrovirologyvol 6 article 51 2009

[12] L Wu and V N KewalRamani ldquoDendritic-cell interactionswith HIV infection and viral disseminationrdquo Nature ReviewsImmunology vol 6 no 11 pp 859ndash868 2006

[13] E Eisele and R F Siliciano ldquoRedefining the viral reservoirs thatprevent HIV-1 eradicationrdquo Immunity vol 37 no 3 pp 377ndash388 2012

[14] J Luban ldquoInnate immune sensing of HIV-1 by dendritic cellsrdquoCell Host Microbe vol 12 no 4 pp 408ndash418 2012

[15] L Wu ldquoSAMHD1 a new contributor to HIV-1 restriction inresting CD4+ T-cellsrdquoRetrovirology vol 9 no 1 article 88 2012

[16] A M Sheehy N C Gaddis J D Choi and M H MalimldquoIsolation of a human gene that inhibits HIV-1 infection and issuppressed by the viral Vif proteinrdquo Nature vol 418 no 6898pp 646ndash650 2002

[17] M Stremlau C M Owens M J Perron M Kiessling PAutissier and J Sodroski ldquoThe cytoplasmic body componentTRIM5120572 restricts HIV-1 infection in old world monkeysrdquoNature vol 427 no 6977 pp 848ndash853 2004

[18] S J D Neil T Zang and P D Bieniasz ldquoTetherin inhibitsretrovirus release and is antagonized byHIV-1VpurdquoNature vol451 no 7177 pp 425ndash430 2008

[19] N van Damme D Goff C Katsura et al ldquoThe interferon-induced protein BST-2 restricts HIV-1 release and is downreg-ulated from the cell surface by the viral Vpu proteinrdquo Cell Hostand Microbe vol 3 no 4 pp 245ndash252 2008

[20] R S Harris J F Hultquist and D T Evans ldquoThe restrictionfactors of human immunodeficiency virusrdquo The Journal ofBiological Chemistry vol 287 no 49 pp 40875ndash40883 2012

[21] N Laguette B Sobhian N Casartelli et al ldquoSAMHD1 is thedendritic- and myeloid-cell-specific HIV-1 restriction factorcounteracted by Vpxrdquo Nature vol 474 no 7353 pp 654ndash6572011

[22] K Hrecka C Hao M Gierszewska et al ldquoVpx relievesinhibition of HIV-1 infection of macrophages mediated by theSAMHD1 proteinrdquoNature vol 474 no 7353 pp 658ndash661 2011

[23] A Berger A F R Sommer J Zwarg et al ldquoSAMHD1-deficientCD14+ cells from individuals with Aicardi-Goutieres syndromeare highly susceptible to HIV-1 infectionrdquo PLoS Pathogens vol7 no 12 Article ID e1002425 2011

[24] H M Baldauf X Pan E Erikson et al ldquoSAMHD1 restrictsHIV-1 infection in resting CD4+ T cellsrdquo Nature Medicine vol18 no 11 pp 1682ndash1689 2012

[25] B Descours A Cribier C Chable-Bessia et al ldquoSAMHD1restricts HIV-1 reverse transcription in quiescent CD4+ T-cellsrdquoRetrovirology vol 9 no 1 article 87 2012

ISRN Biochemistry 9

[26] T E White A Brandariz-Nunez J C Valle-Casuso et alldquoContribution of SAMandHDdomains to retroviral restrictionmediated by human SAMHD1rdquo Virology vol 436 no 1 pp 81ndash90 2012

[27] T Gramberg T Kahle N Bloch et al ldquoRestriction of diverseretroviruses by SAMHD1rdquoRetrovirology vol 10 article 26 2013

[28] C St Gelais and L Wu ldquoSAMHD1 a new insight into HIV-1restriction inmyeloid cellsrdquoRetrovirology vol 8 article 55 2011

[29] N Manel and D R Littman ldquoHiding in plain sight how HIVevades innate immune responsesrdquo Cell vol 147 no 2 pp 271ndash274 2011

[30] V Planelles ldquoRestricted access to myeloid cells explainedrdquoViruses vol 3 no 9 pp 1624ndash1633 2011

[31] YH Zheng K T Jeang andK Tokunaga ldquoHost restriction fac-tors in retroviral infection promises in virus-host interactionrdquoRetrovirology vol 9 article 112 2012

[32] T Schaller C Goujon and M H Malim ldquoAIDSHIV HIVinterplay with SAMHD1rdquo Science vol 335 no 6074 pp 1313ndash1314 2012

[33] N Laguette and M Benkirane ldquoHow SAMHD1changes ourview of viral restrictionrdquo Trends in Immunology vol 33 no 1pp 26ndash33 2012

[34] D Ayinde N Casartelli and O Schwartz ldquoRestricting HIV theSAMHD1 way through nucleotide starvationrdquo Nature ReviewsMicrobiology vol 10 no 10 pp 675ndash680 2012

[35] C Goujon L Jarrosson-Wuilleme J Bernaud D Rigal J-Darlix and A Cimarelli ldquoWith a little help from a friendincreasing HIV transduction of monocyte-derived dendriticcells with virion-like particles of SIVMACrdquo Gene Therapy vol13 no 12 pp 991ndash994 2006

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[37] C Goujon V Arfi T Pertel et al ldquoCharacterization ofsimian immunodeficiency virus SIVSMhuman immunodefi-ciency virus type 2 Vpx function in human myeloid cellsrdquoJournal of Virology vol 82 no 24 pp 12335ndash12345 2008

[38] S Schule B Kloke J K Kaiser et al ldquoRestriction of HIV-1replication in monocytes is abolished by Vpx of SIVsmmPBjrdquoPLoS ONE vol 4 no 9 Article ID e7098 2009

[39] R Kaushik X Zhu R Stranska Y Wu and M StevensonldquoA cellular restriction dictates the permissivity of nondividingmonocytesmacrophages to lentivirus and gammaretrovirusinfectionrdquo Cell Host and Microbe vol 6 no 1 pp 68ndash80 2009

[40] N Sharova Y Wu X Zhu et al ldquoPrimate lentiviral Vpxcommandeers DDB1 to counteract a macrophage restrictionrdquoPLoS Pathogens vol 4 no 5 Article ID e1000057 2008

[41] M Fujita M Otsuka M Miyoshi B Khamsri M Nomaguchiand A Adachi ldquoVpx is critical for reverse transcriptionof the human immunodeficiency virus type 2 genome inmacrophagesrdquo Journal of Virology vol 82 no 15 pp 7752ndash77562008

[42] S Srivastava S K Swanson N Manel L Florens M P Wash-burn and J Skowronski ldquoLentiviral Vpx accessory factor targetsVprBPDCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligaseto enable macrophage infectionrdquo PLoS Pathogens vol 4 no 5Article ID e1000059 2008

[43] A Bergamaschi D Ayinde A David et al ldquoThe humanimmunodeficiency virus type 2Vpx protein usurps the CUL4A-DDB1DCAF1 ubiquitin ligase to overcome a postentry block in

macrophage infectionrdquo Journal of Virology vol 83 no 10 pp4854ndash4860 2009

[44] D Ayinde C Maudet C Transy and F Margottin-GoguetldquoLimelight on two HIVSIV accessory proteins in macrophageinfection is Vpx overshadowing Vprrdquo Retrovirology vol 7article 35 2010

[45] M H Malim and M Emerman ldquoHIV-1 accessory proteinsmdashensuring viral survival in a hostile environmentrdquo Cell Host andMicrobe vol 3 no 6 pp 388ndash398 2008

[46] G Berger S Durand C Goujon et al ldquoA simple versatileand efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1-derived lentiviral vectorsrdquoNature Protocols vol 6 no 6 pp 806ndash816 2011

[47] N Sunseri M OrsquoBrien N Bhardwaj and N R LandauldquoHuman immunodeficiency virus type 1 modified to pack-age Simian immunodeficiency virus Vpx efficiently infectsmacrophages and dendritic cellsrdquo Journal of virology vol 85 no13 pp 6263ndash6274 2011

[48] N Manel B Hogstad Y Wang D E Levy D Unutmaz and DR Littman ldquoA cryptic sensor forHIV-1 activates antiviral innateimmunity in dendritic cellsrdquo Nature vol 467 no 7312 pp 214ndash217 2010

[49] J Ahn C Hao J Yan et al ldquoHIVSimian ImmunodeficiencyVirus (SIV) accessory virulence factor Vpx loads the hostcell restriction factor SAMHD1 onto the E3 ubiquitin ligasecomplex CRL4DCAF1rdquoThe Journal of Biological Chemistry vol287 no 15 pp 12550ndash12558 2012

[50] W Wei H Guo X Han et al ldquoA novel DCAF1-binding motifrequired for Vpx-mediated degradation of nuclear SAMHD1and Vpr-induced G2 arrestrdquo Cellular Microbiology vol 14 no11 pp 1745ndash1756 2012

[51] J S Gibbs A A Lackner SM Lang et al ldquoProgression toAIDSin the absence of a gene for vpr or vpxrdquo Journal of Virology vol69 no 4 pp 2378ndash2383 1995

[52] V M Hirsch M E Sharkey C R Brown et al ldquoVpx is requiredfor dissemination and pathogenesis of SIV(SM)PBj evidence ofmacrophage-dependent viral amplificationrdquo Nature Medicinevol 4 no 12 pp 1401ndash1408 1998

[53] M Belshan J T Kimata C Brown et al ldquoVpx is critical forSIVmne infection of pigtail macaquesrdquo Retrovirology vol 9article 32 2012

[54] N Laguette N Rahm B Sobhian et al ldquoEvolutionary andfunctional analyses of the interaction between the myeloidrestriction factor SAMHD1 and the lentiviral Vpx proteinrdquo CellHost and Microbe vol 11 no 2 pp 205ndash217 2012

[55] E S Lim O I Fregoso C O McCoy F A Matsen H SMalik and M Emerman ldquoThe ability of primate lentiviruses todegrade themonocyte restriction factor SAMHD1 preceded thebirth of the viral accessory protein Vpxrdquo Cell Host and Microbevol 11 no 2 pp 194ndash204 2012

[56] C Zhang S de Silva J Wang and L Wu ldquoCo-evolution ofprimate SAMHD1 and Lentivirus Vpx leads to the loss of thevpx gene in HIV-1 ancestorrdquo PLoS ONE vol 7 no 5 Article IDe37477 2012

[57] M D Zimmerman M Proudfoot A Yakunin and W MinorldquoStructural insight into the mechanism of substrate specificityand catalytic activity of an HD-domain phosphohydrolase the51015840-deoxyribonucleotidase YfbR fromEscherichia colirdquo Journal ofMolecular Biology vol 378 no 1 pp 215ndash226 2008

[58] D C Goldstone V Ennis-Adeniran J J Hedden et al ldquoHIV-1restriction factor SAMHD1 is a deoxynucleoside triphosphate

10 ISRN Biochemistry

triphosphohydrolaserdquo Nature vol 480 no 7377 pp 379ndash3822011

[59] R D Powell P J Holland T Hollis and F W PerrinoldquoAicardi-Goutieres syndrome gene and HIV-1 restriction factorSAMHD1 is a dGTP-regulated deoxynucleotide triphosphohy-drolaserdquoThe Journal of Biological Chemistry vol 286 no 51 pp43596ndash43600 2011

[60] A Brandariz-Nunez J C Valle-Casuso T E White et al ldquoRoleof SAMHD1 nuclear localization in restriction of HIV-1 andSIVmacrdquo Retrovirology vol 9 article 49 2012

[61] H Hofmann E C Logue N Bloch et al ldquoThe Vpx lentiviralaccessory protein targets SAMHD1 for degradation in thenucleusrdquo Journal of Virology vol 86 no 23 pp 12552ndash125602012

[62] S Coon DWang and LWu ldquoPolymorphisms of the SAMHD1gene are not associated with the infection and natural controlof HIV type 1 in Europeans and African-Americansrdquo AIDSResearch and Human Retroviruses vol 28 no 12 pp 1565ndash15732012

[63] B Kim L A Nguyen W Daddacha and J A HollenbaughldquoTight interplay among SAMHD1 protein level cellular dNTPlevels andHIV-1 proviral DNA synthesis kinetics in human pri-marymonocyte-derivedmacrophagesrdquoTheJournal of BiologicalChemistry vol 287 no 26 pp 21570ndash21574 2012

[64] P Reichard ldquoInteractions between deoxyribonucleotide andDNA synthesisrdquo Annual Review of Biochemistry vol 57 pp349ndash374 1988

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[66] C St Gelais S de Silva S M Amie et al ldquoSAMHD1 restrictsHIV-1 infection in dendritic cells (DCs) by dNTP depletion butits expression inDCs and primary CD4+ T-lymphocytes cannotbe upregulated by interferonsrdquo Retrovirology vol 9 no 1 article105 2012

[67] S Welbourn E Miyagi T E White F Diaz-Griffero andK Strebel ldquoIdentification and characterization of naturallyoccurring splice variants of SAMHD1rdquo Retrovirology vol 9 no1 article 86 2012

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[69] G I Rice J Bond A Asipu et al ldquoMutations involved inAicardi-Goutieres syndrome implicate SAMHD1 as regulator ofthe innate immune responserdquoNature Genetics vol 41 no 7 pp829ndash832 2009

[70] N Li W Zhang and X Cao ldquoIdentification of human homo-logue of mouse IFN-120574 induced protein from human dendriticcellsrdquo Immunology Letters vol 74 no 3 pp 221ndash224 2000

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[73] G Doitsh M Cavrois K G Lassen et al ldquoAbortive HIVinfection mediates CD4 T cell depletion and inflammation inhuman lymphoid tissuerdquo Cell vol 143 no 5 pp 789ndash801 2010

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[75] P Chen W Hubner M A Spinelli and B K Chen ldquoPredomi-nant mode of human immunodeficiency virus transfer betweenT cells is mediated by sustained Env-dependent neutralization-resistant virological synapsesrdquo Journal of Virology vol 81 no22 pp 12582ndash12595 2007

[76] WMothes N M Sherer J Jin and P Zhong ldquoVirus cell-to-celltransmissionrdquo Journal of Virology vol 84 no 17 pp 8360ndash83682010

[77] I Puigdomenech N Casartelli F Porrot and O SchwartzldquoSAMHD1 restricts HIV-1 cell-to-cell transmission and limitsimmune detection inmonocyte-derived dendritic cellsrdquo Journalof Virology vol 87 no 5 pp 2846ndash2856 2013

[78] E Pauls E Jimenez A Ruiz et al ldquoRestriction of HIV-1replication in primary macrophages by IL-12 and IL-18 throughthe upregulation of SAMHD1rdquo Journal of Immunology vol 190no 9 pp 4736ndash4741 2013

[79] S M Amie E Noble and B Kim ldquoIntracellular nucleotidelevels and the control of retroviral infectionsrdquoVirology vol 436no 2 pp 247ndash254 2013

[80] W Y Gao A Cara R C Gallo and F Lori ldquoLow levels ofdeoxynucleotides in peripheral blood lymphocytes a strategyto inhibit human immunodeficiency virus type 1 replicationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 90 no 19 pp 8925ndash8928 1993

[81] T L Diamond M Roshal V K Jamburuthugoda et alldquoMacrophage tropism of HIV-1 depends on efficient cellulardNTP utilization by reverse transcriptaserdquo The Journal ofBiological Chemistry vol 279 no 49 pp 51545ndash51553 2004

[82] J A Zack S J Arrigo S RWeitsmanA S GoAHaislip and IS Y Chen ldquoHIV-1 entry into quiescent primary lymphocytesmolecular analysis reveals a labile latent viral structurerdquo Cellvol 61 no 2 pp 213ndash222 1990

[83] H Lahouassa W Daddacha H Hofmann et al ldquoSAMHD1restricts the replication of human immunodeficiency virustype 1 by depleting the intracellular pool of deoxynucleosidetriphosphatesrdquo Nature Immunology vol 13 no 3 pp 223ndash2282012

[84] K S Jones C Petrow-Sadowski Y K Huang D C Bertoletteand F W Ruscetti ldquoCell-free HTLV-1 infects dendritic cellsleading to transmission and transformation of CD4+ T cellsrdquoNature Medicine vol 14 no 4 pp 429ndash436 2008

[85] T E White A Brandariz-Nunez J C Valle-Casuso et alldquoThe retroviral restriction ability of SAMHD1 but not itsdeoxynucleotide triphosphohydrolase activity is regulated byphosphorylationrdquo Cell Host and Microbe vol 13 no 4 pp 441ndash451 2013

[86] A Cribier B Descours A L Valadao N Laguette and MBenkirane ldquoPhosphorylation of SAMHD1 by cyclin A2CDK1regulates its restriction activity toward HIV-1rdquo Cell Reports vol3 no 4 pp 1036ndash1043 2013

[87] J Yan S KaurMDelucia et al ldquoTetramerization of SAMHD1 isrequired for biological activity and inhibition of HIV infectionrdquoThe Journal of Biological Chemistry vol 288 no 15 pp 10406ndash10417 2013

[88] M Delucia J Mehrens Y Wu and J Ahn ldquoHIV-2 and SIVmacaccessory virulence factor Vpx down-regulates SAMHD1 catal-ysis prior to proteasome-dependent degradationrdquo The Journalof Biological Chemistry 2013

ISRN Biochemistry 11

[89] A Goncalves E Karayel G I Rice et al ldquoSAMHD1 is anucleic-acid binding protein that is mislocalized due to aicardi-goutieres syndrome-associated mutationsrdquo Human Mutationvol 33 no 7 pp 1116ndash1122 2012

[90] V Tungler W Staroske B Kind et al ldquoSingle-stranded nucleicacids promote SAMHD1 complex formationrdquo Journal of Molec-ular Medicine vol 91 no 6 pp 759ndash770 2013

[91] N Beloglazova R Flick A Tchigvintsev et al ldquoNuclease activ-ity of the human SAMHD1 protein implicated in the Aicardi-Goutieres syndrome and HIV-1 restrictionrdquo The Journal ofBiological Chemistry vol 288 no 12 pp 8101ndash8110 2013

[92] T Pertel C Reinhard and J Luban ldquoVpx rescues HIV-1 trans-duction of dendritic cells from the antiviral state established bytype 1 interferonrdquo Retrovirology vol 8 article 49 2011

[93] L Dragin L A Nguyen H Lahouassa et al ldquoInterferonblock to HIV-1 transduction in macrophages despite SAMHD1degradation and high deoxynucleoside triphosphates supplyrdquoRetrovirology vol 10 article 30 2013

[94] C Goujon T Schaller R P Galao et al ldquoEvidence for IFN120572-induced SAMHD1-independent inhibitors of earlyHIV-1 infec-tionrdquo Retrovirology vol 10 article 23 2013

[95] J W Schoggins S J Wilson M Panis et al ldquoA diverse rangeof gene products are effectors of the type i interferon antiviralresponserdquo Nature vol 472 no 7344 pp 481ndash485 2011

[96] B K Thielen J P McNevin M J McElrath B V S HuntK C Klein and J R Lingappa ldquoInnate immune signalinginduces high levels of TC-specific deaminase activity in primarymonocyte-derived cells through expression of APOBEC3Aisoformsrdquo The Journal of Biological Chemistry vol 285 no 36pp 27753ndash27766 2010

[97] G Berger S Durand G Fargier et al ldquoApobec3a is a specificinhibitor of the early phases of hiv-1 infection in myeloid cellsrdquoPLoS Pathogens vol 7 no 9 Article ID e1002221 2011

[98] C Dong A M Janas J Wang W J Olson and L WuldquoCharacterization of human immunodeficiency virus type 1replication in immature and mature dendritic cells revealsdissociable cis- and trans-infectionrdquo Journal of Virology vol 81no 20 pp 11352ndash11362 2007

[99] C Dong C Kwas and L Wu ldquoTranscriptional restrictionof human immunodeficiency virus type 1 gene expression inundifferentiated primary monocytesrdquo Journal of Virology vol83 no 8 pp 3518ndash3527 2009

[100] L Ganesh E Burstein AGuha-Niyogi et al ldquoThe gene productMurr1 restrictsHIV-1 replication in restingCD4+ lymphocytesrdquoNature vol 426 no 6968 pp 853ndash857 2003

[101] C Goujon and M H Malim ldquoCharacterization of the 120572interferon-induced postentry block to HIV-1 infection in pri-mary human macrophages and T cellsrdquo Journal of Virology vol84 no 18 pp 9254ndash9266 2010

[102] M Li E Kao X Gao et al ldquoCodon-usage-based inhibition ofHIV protein synthesis by human schlafen 11rdquo Nature vol 491no 7422 pp 125ndash128 2012

[103] W Sohn D Kim K Lee et al ldquoNovel transcriptional regulationof the schlafen-2 gene in macrophages in response to TLR-triggered stimulationrdquo Molecular Immunology vol 44 no 13pp 3273ndash3282 2007

[104] T Pertel S Hausmann D Morger et al ldquoTRIM5 is an innateimmune sensor for the retrovirus capsid latticerdquo Nature vol472 no 7343 pp 361ndash365 2011

[105] R P Galao A Le Tortorec S Pickering T Kueck and S J NeilldquoInnate sensing of HIV-1 assembly by Tetherin induces NF120581B-dependent proinflammatory responsesrdquo Cell Host and Microbevol 12 no 5 pp 633ndash644 2012

[106] A Tokarev M Suarez W Kwan K Fitzpatrick R Singh and JGuatelli ldquoStimulation of NF-120581B activity by the HIV restrictionfactor BST2rdquo Journal of Virology vol 87 no 4 pp 2046ndash20572013

[107] J Wu L Sun X Chen et al ldquoCyclic GMP-AMP is an endoge-nous secondmessenger in innate immune signaling by cytosolicDNArdquo Science vol 339 no 6121 pp 826ndash830 2013

[108] L Sun J Wu F Du X Chen and Z J Chen ldquoCyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the typeI interferon pathwayrdquo Science vol 339 no 6121 pp 786ndash7912013

[109] P Gao M Ascano Y Wu et al ldquoCyclic [G(21015840 51015840)pA(31015840 51015840)p]is the metazoan second messenger produced by DNA-activatedcyclic GMP-AMP synthaserdquo Cell vol 153 no 5 pp 1094ndash11072013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology

2 ISRN Biochemistry

through its several accessory proteins such as Vif to counter-act APOBEC3G and Vpu to degrade tetherin [8 20] Recentstudies have identified a novel retroviral restriction factornamed SAMHD1 in non-cycling myeloid cells and restingCD4+ T-lymphocytes [21ndash25] Human SAMHD1 has beenshown to have a broad restriction of diverse retroviruses[26 27] In addition to HIV-1 SAMHD1 also blocks infectionof other retroviruses including HIV-2 feline immunodefi-ciency virus bovine immunodeficiency virus equine infec-tious anemia virus and murine leukemia viruses [26 27]However the mechanisms of SAMHD1-mediated retrovirusrestriction and its physiological functions remain to beelucidated Previously published reviews or commentarieshave summarized the role of SAMHD1 in restricting HIV-1 infection in nondividing human immune cells [15 28ndash34] In this review I would like to focus on the analysis ofthe cellular and molecular mechanisms by which SAMHD1mediates retroviral restriction I will also discuss potentialcellular function of SAMHD1 in nucleic acid metabolism andthe proliferation of cells

2 The Identification and Characterization ofSAMHD1 as an HIV-1 Restriction Factor

21 The Vpx Proteins from SIV and HIV-2 Enhance HIV-1Infection in Myeloid Cells Goujon et al showed thatpreincubation with virus-like particles derived from sootymangabey lineage SIV (SIVsm) significantly enhancestransduction efficiency of human monocyte-derived DCsusing HIV-1-derived lentiviral vectors [35] Similarly Vpxproteins from SIVsm and HIV-2 significantly promoteHIV-1 infection in human monocytes and derived DCs andmacrophages by facilitating the accumulation of full-lengthHIV-1 cDNA [36ndash40] It has also been known that Vpxis critical for HIV-2 infection in human macrophages byenhancing the efficiency of viral reverse transcription [41]Further studies indicated that Vpx from SIV or HIV-2 caninteract with the host protein DCAF1 in the CUL4ADDB1and E3 ubiquitin ligase complex [42 43] indicating that Vpxmay target a putative HIV-1 restriction factor for proteasomaldegradation in myeloid cells through the E3 ubiquitin ligasecomplex (for reviews please refer to [44 45]) These studieson Vpx and HIV-1 infection of myeloid cells have promptedthe identification of SAMHD1 as an HIV-1 restriction factorin human myeloid cells [21ndash23]

Although HIV-1 genome does not encode the Vpx pro-tein Vpx has been a useful tool in the design of lentiviralvectors to better transduce myeloid-lineage cell types that arerelatively resistant to lentiviral infection or transduction [46]Sunseri et al generated a modified HIV-1 to package SIVVpx which efficiently infects human primary macrophagesand DCs [47] Because Vpx is packaged into virions throughthe specific interaction with the p6 carboxy-terminal domainof Gag the authors introduced the Vpx packaging motif ofSIV p6 into an HIV-1 proviral DNA construct The chimericHIV-1 packaged Vpx in trans and showed significantly higherinfectivity in DCs andmacrophages compared with the wild-type virus Furthermore by introducing the Vpx coding

sequence into the proviral DNA to incorporate Vpx in cisthe modified HIV-1 can efficiently replicate in DCs andmacrophages [47] The Vpx-containing HIV-1 could be moreefficiently transmitted from DCs to cocultured CD4+ T cellssuggesting that Vpxmay facilitateDC-mediated transmissionof HIV-2 or SIV in vivo Given that the infection of DCswith theVpx-containingHIV-1 triggers activation ofDCs andcocultured CD4+ T-cells [47 48] these studies imply that thechimeric HIV-1 might be used to design DC-based vaccinesto induce an enhanced innate immune response

22 The Discovery of SAMHD1 as an HIV-1 Restriction FactorInteracting with Vpx The identification of SAMHD1 wasmainly dependent on the isolation and analysis of Vpx-interacting host proteins by mass spectrometry [21ndash23] Pre-vious studies have demonstrated that phorbol myristic acid-(PMA-) differentiated THP-1 cells can be rendered more per-missive to HIV-1 infection by transduction of SIVsmHIV-2 Vpx-containing virus-like particles derived from SIVmac[44] Based on these observations Laguette et al iden-tified SAMHD1 as a novel Vpx-interacting protein fromdifferentiated human monocytic THP-1 cells that ectopicallyexpress tagged Vpx [21] SAMHD1 is expressed in HIV-1nonpermissive cell types including THP-1 cells primarymonocytes monocyte-derived macrophages and DCs butnot in HIV-1 permissive CD4+ T-cell lines [21] Knockdownof endogenous SAMHD1 protein in non-permissive THP-1cells and primary DCs alleviates HIV-1 restriction By con-trast overexpression of SAMHD1 in monocytic U937 cellsand then differentiation with PMA inhibits HIV-1 infectionin the cells [21] Independently Hrecka and colleagues iden-tified SAMHD1 from the human embryonic kidney cell line(HEK 293T) expressing tagged Vpx in a proteomic screenusing multidimensional protein identification technology[22] The authors further demonstrated that the inhibition ofHIV-1 infection in monocyte-derived macrophages by Vpx-mediated proteasome degradation of SAMHD1 through thecellular E3 ubiquitin ligase complex [22] Together thesestudies confirmed that Vpx interacts with SAMHD1 andinduces proteasomal degradation of SAMHD1 in THP-1 cellsor macrophages which can be restored by treatment witha proteasome inhibitor [21 22] A recent analysis furtherdemonstrated that Vpx fromHIV-2 or SIV engages SAMHD1onto the E3 ubiquitin ligase complex CRL4DCAF1 to initiateproteasomal degradation of SAMHD1 [49] Moreover Wei etal identified a novel DCAF1-binding motif required for Vpx-mediated degradation of SAMHD1 protein [50] suggestingimportant interactions involved in the assembly of Vpx-hijacked DCAF1-DDB1-based E3 ubiquitin ligase complexThe molecular interactions between Vpx and SAMHD1 alsosuggest an evolutionary conflict between lentiviruses andhost restriction factors which lead to evolutionary studies ofprimate SAMHD1 and lentiviral Vpx proteins

23 Evolutionary Biology of SAMHD1 SIVsm SIVsm-derived SIV that infects rhesusmacaques (SIVmac) andHIV-2 that infects humans encode both Vpr and Vpx proteinsBy contrast SIVcpz that naturally infects chimpanzees and

ISRN Biochemistry 3









4 ISRN Biochemistry

1 45 110 115 562 595 626

PV

Enzymatic sites

Vpxinteracting

T592

(167H 206HD207 311D)

SAM HD domain

NLS(11KRPR14)









ISRN Biochemistry 5










6 ISRN Biochemistry



HIV-1 RNA

HIV-1 cDNA

HIV-1

CCR5CXCR4

CD4

Nucleus

dNTPs

dN + PPP

SAMHD1

RT


HIV-1 infection

PMAP


Cycling cells

T592

CDK1


Noncycling cells

SAMHD1SAMHD1







ISRN Biochemistry 7












8 ISRN Biochemistry


Abbreviations






Acknowledgments


References


























ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biochemistry 3









4 ISRN Biochemistry

1 45 110 115 562 595 626

PV

Enzymatic sites

Vpxinteracting

T592

(167H 206HD207 311D)

SAM HD domain

NLS(11KRPR14)









ISRN Biochemistry 5










6 ISRN Biochemistry



HIV-1 RNA

HIV-1 cDNA

HIV-1

CCR5CXCR4

CD4

Nucleus

dNTPs

dN + PPP

SAMHD1

RT


HIV-1 infection

PMAP


Cycling cells

T592

CDK1


Noncycling cells

SAMHD1SAMHD1







ISRN Biochemistry 7












8 ISRN Biochemistry


Abbreviations






Acknowledgments


References


























ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 ISRN Biochemistry

1 45 110 115 562 595 626

PV

Enzymatic sites

Vpxinteracting

T592

(167H 206HD207 311D)

SAM HD domain

NLS(11KRPR14)









ISRN Biochemistry 5










6 ISRN Biochemistry



HIV-1 RNA

HIV-1 cDNA

HIV-1

CCR5CXCR4

CD4

Nucleus

dNTPs

dN + PPP

SAMHD1

RT


HIV-1 infection

PMAP


Cycling cells

T592

CDK1


Noncycling cells

SAMHD1SAMHD1







ISRN Biochemistry 7












8 ISRN Biochemistry


Abbreviations






Acknowledgments


References


























ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biochemistry 5










6 ISRN Biochemistry



HIV-1 RNA

HIV-1 cDNA

HIV-1

CCR5CXCR4

CD4

Nucleus

dNTPs

dN + PPP

SAMHD1

RT


HIV-1 infection

PMAP


Cycling cells

T592

CDK1


Noncycling cells

SAMHD1SAMHD1







ISRN Biochemistry 7












8 ISRN Biochemistry


Abbreviations






Acknowledgments


References


























ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 ISRN Biochemistry



HIV-1 RNA

HIV-1 cDNA

HIV-1

CCR5CXCR4

CD4

Nucleus

dNTPs

dN + PPP

SAMHD1

RT


HIV-1 infection

PMAP


Cycling cells

T592

CDK1


Noncycling cells

SAMHD1SAMHD1







ISRN Biochemistry 7












8 ISRN Biochemistry


Abbreviations






Acknowledgments


References


























ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biochemistry 7












8 ISRN Biochemistry


Abbreviations






Acknowledgments


References


























ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 ISRN Biochemistry


Abbreviations






Acknowledgments


References


























ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biochemistry 9
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Cellular and Biochemical Mechanisms of the...

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