REVIEW For reprint orders, please contact: reprints@futuremedicine.com Human parvovirus B19: a mechanistic overview of infection and DNA replication Yong Luo’ & Jianming Qiu®* ABSTRACT Human parvovirus 819 (819V) is a human pathogen that belongs to genus Erythroparvovirus of the Parvoviridae family, which is composed of a group of small DNA, viruses with a linear single-stranded DNA genome. B19V mainly infects human erythroid progenitor cells and causes mild to severe hematological disorders in patients. However, recent clinical studies indicate that B19V also infects nonerythroid lineage cells, such as myocardial endothelial cells, and may be associated with other disease outcomes. Several cell culture systems, including permissive and semipermissive erythroid lineage cells, nonpermissive human embryonic kidney 293 cells and recently reported myocardial endothelial cells, have been used to study the mechanisms underlying BI9V infection and BISV DNA replication. This review aims to summarize recent advances in BI9V studies with 2 focus on the mechanisms of BI9V tropism specific to different cell types and the cellular pathways involved in BI9V DNA replication including cellular signaling transduction and cell cycle arrest. Human parvovirus B19 (BI9V) was discovered in 1975 by Cossart and colleagues when screening for hepatitis B virus ina panel of human serum samples). The virus was described as 23 nm in diameter, a typical capsid size of a parvoviras. The virus came from the serum sample coded a: panel B number 19, and thereafter was named ‘Parvovirus B19’ Mose commonly, BIOV infection ‘causes erythema infectiosum or fifth disease (also named ‘slapped cheek syndrome’), which was firs identified by Anderson eral. in the early 1980s 2) In addition to infections in children, BIOV is also highly infectious and spreads easily among adules with a seropositive rate ranging from 60 to 90% 3). Ie eauses a spectrum of clinical complications in vulnerable populations, including arthropathy in healthy adults (particularly in middle-aged women), persistent anemia in immunosuppressed. patients, transient aplastic crisis in patients with increased erythropoiesis (ouch ar sickle-cell disease patients), and hydrops fetalis and congenital anemia in pregnant women ¢-«|. Noteworthy tion to these common manifestations, accumulating clinieal reports indicate that B19V infection is associated with cardiovascular diseases, for example, myocarditis, in both adults and children {7-12 The association of BISV infection to several liver diseases, for example, acure and chronic hepatitis, acute falminane liver failure and autoimmune hepatitis, has been also reported (3-16); however, only erychroid progenitor cells from fetal liver have been shown permissive to BISV replication (7. ‘And, therefore, che mechanism for BI9V infection ofliver cells will not be discussed in this review. BIOV is a member of the Erythvoparvovirus genus within the Parvovirinae subfamily of the Parvoviridae family (x). It asa linear ssDNA genome of 5596 nucleotides. Iris Hanked by two identical inverted terminal repeats (IRs) thae tom an imperfect palindrome at cach end (Figure 14) inaddi- KEYWoRDS + B19V « cell cycle « DDR + DNA damage response “+ DNA replication + Epo/EpoR signaling “human parvovirus 819 «hypoxia « tropism Future. VIROLOGY cornet cabo Hakan Gereert ences nvessyatinarhesolceve: olmepscnscroneen FULUTe Ss B Ee mags oven MedicineREVIEW Luo &aiu 156 Lar RaTR Oey gs ntiae Ber0 ee wt c (raya 5596 Banna} ¥ ee oy APRDR ART SARE (588) (208072210) Gass) @775) (484) onrt 30g veree 4960 onF2 8905174 onrs Sets" "TDs ala TAA ‘mRNA rts Protein (kDa) 1 2310 | NS1 (78) sat 7540 Ys 2611 1/NS1 (78) sof iat Rt sgiteg 2850 —Flua 806 1 75440875) Re ssitse 1107 1 7540875) ee R3 531586 2270 2840 66 a Ro soften sr? wee tt | 25KDATS) RO soikes 3a Sie Wer es)? 3034 | VP (84) RS 5siSé6 a Sia3 RO gg fSSOS —$ ee UNE HORLTS) soe Zag 2028 Biss | vas) 7 2173.1 vp2 (58) RT siee 220363 3228 Biss we ae | 28 ta 5) FS sites 20807563 wiersies “ V teouthiy> 514 1 114081 RO ssite6 220568 Tareas FDA) Figure 1. Structure of the human parvovirus B19 genome and the human parvovirus B19 genetic ‘map. (A) Schematic diagram ofthe minus strand of the BI9V ssDNA genome. Identical ITRs are present at each end of the genome, and these are depicted with unpaited or mismatched bases in the palindromes represented by ‘bulges’ or bubbles; respectively (B) Schematic diagrams of the duplex RF of the BI9V genome (819V J35 strain; GenBank accession number: AY386330), which has the capability to express viral genes, replicate and produce progeny virions. The left and right-hand TTRs (L-ITR and RIT), P6 promoter, RNA initiation site, splice donors (D1 and D2}, splice acceptors (41-1, 1.2, A2-1 and A22) and proximal and distal polyadenylation sites (pA)pV/2 and (pA)d) are indicated, along with the nine major mRNAs (R1-8) and three minor mRNAs (R13). The numbers of ‘nucleotides (nt) are indicated in each case. The proteins encoded by each mRNA are shown on the right, while the question mark (} denotes that the protein translaced from the mRNA is curcently ‘unknown. The size ofthe ITRs and the NSI- and VP1-encoding regions diagrammed are not to scale. 319V: Human parvovirus 819; ITR Inverted terminal repeat; NSI: Nonstructural proteins; AF: Replicative form; VPI: Capsid proteins. tavescerce soup MB ature Virol. 2015)ivy), a Feature also shared by adeno-associ ated virus 2 (AAV2) and human parvovirus 4 (PARVA) fx, By conteas, all other members of the Parvovirinae subfamil terminal repeats (23). Under the P6 promocee located at map anit 6, che zeplicative form (RF) ‘of the BI9V genome cranscribes nine major vital mRNAs (R1-9) that encode capsid proteins (VPI and VP2) and nonstructural proteins (NS1, 11 and 75 kDa) Figure 1B) 24-26). ABIOV DNA infectious clone (B19V RF DNA M20; Figure 18) has been constructed (27), based on which, mutagenesis studies bave confirmed INS1 isthe only protein essential for BI9V DNA replication; wheteas VPL/2 and 11-Da proteins are required for progeny virus production (2 BI9V isan autonomous parvovirus, represent ing a msjorty of the Parvoviridae family mem: bers chat can replicate by themselvesin host cells, in contrast to AAV that requires coinfection of helper virus, such as adenoviews, for replication 29). Generally, DNA replication of autonomous parvoviruses happens in the S phase of the host cell cycle .0-2) and follows a ‘rolling hairpin’ ‘model of DNA replication (435), Because an in vitro reconstitution BIOV DNA replication system as not been established, current knovl- ‘edge on BIOV DNA replication is largely derived from AAV, whose genome bas two ITRs, and the auconomoue parvovirus minute virus of mice. A hairpin-primed ssDNA replication model of BISV is proposed here (Figure 2). In principle, the BISV ssDNA genome uses 3-end hairpin asa selfiprimer (3/OH) co extend viral ssDNA the deDNA genome by cellular replication, proteins, a sep in so-called frsestrand DNA syn- thesis Figure 28). The extended 3 ends presum- ably ligated to the 5 end of the genome by an ‘unknown cellular ligase ro form a parcial circular DNA structure (Figure 2¢) (36-3). The dsDNA form of the virus transcriber viral mRNAs for the expression of viral proteins. The largest non= structural protein (NSI), a maltiple-functional protein with site-specific endonuclease activity and DNA helicase activity, nicks the junction at the so-called erminal resolution site (sr) berween the Send hairpin (Figure 20) and the newly synthesized viral DNA to form a novel 3 primer that inciates the strand displacement and rolling hairpin-dependent DNA replication (Figure 2-H). The elongated vieal genomes (both replicative and double-replicative intermediates) are resolved by NSI nicking and release DNA thac is finally packaged into the capsid. have asymmecie Human parvovirus 819: a mechanistic overview REVIEW Understanding the mechanism underlying BIOV DNA replication, a ertical step to devel ‘oping antivirus strategies has been impeded by the difficulty of efficiently propagating BISV in an sn vitro cell euleute system, Several break throughs have been made recently to improve and expand BIOV infection in in vitro euleates of cells, including permissive, semipermissive and nonpermissive cells, which have greatly advanced. the understanding of B19V infection and DNA replication, This article will summarize these studies and discuss the cellular requirements for BI9V tropism and BI9V DNA replication in these different cell systems 819V tropism for erythroid lineage cells BIOV infection and viral DNA replication ate restricted by the natrow tropists of the virus. In astute, BIIV mainly infects human erythroid progenitor celle (KEFC®) liver (40-4), although restricted infection of her tissues has been frequently reported. BIOV DNA replication was Bist observed in suspen sion culkates of bman erythroid bone matrow from patients with hemolytic anemia), and replication eflcieney was greatly enhanced dr ing infection of isolated hematopoictic progeni- tor cll fom normal human bone marrow Identification of EPCs as BIOV cargo celle wa fist observed by Mortimer eta) and Young and colleagues (0). Farther studies showed chat BIOV infection cases reduction of i sero-cule tured colony-forming wni erythroid and burst forming unit erythroid cll, but only in the fist few days in cukure (4, indicating that efficient BISV DNA replication equires cern diferen- tiation stages of the hEPCs. In particular, BI9V only infects KEPCs with surface marker CD36* Aiffeeosiaed from CD34" human hematopoietic stem calls (FISCs), but not the HSCs and myeloid lineage celle 6-47 The extablishment of ex vivo expanded CD36" hEPCs from HSCs by Wong tal bas greatly advanced studies of BIDV infee- sion CD36" EPCs are highly permissive 0 IV infection with productive BISV DNA repli- cation occurring on afew interval days during the petiod of pose-differeniatin from CD34" HSCs t0 CD36" HEPCs 5), Therefore, productive BISV infection onl occurs fora shor time during hEPC ditferenviation, lee not clear whether the diferen- ‘ation satus of CD36" MEPCs aces BIDV enry us aficking and what are the cellular fae- tors uctuatng during the dfferesiation affect productive BISV infection sm bone marrow and Brvescene sow vwwnv faturemedicine com 187REVIEW Luo &aiu 158 Lire 5 RAR @ eo to tee ets RF DNA anit(7s7) | a eT (eek ca RN SS : | ® Strand-displacement Figure 2. Relling-hairpin model of human parvovirus B19 DNA replication, (A &B) The BI9V ssONA {genome as shown is fist converted into RF ONA thats primed by the 3/0H of the LTR, a process that may not require viral proteins s-(C) The 3 end of the newly synthesized complementary strands likely ligated to the R-ITR, resuking inthe formation of cAF ONA as the major conversion product cRF DNA, tavescerce soup MBHuman parvovirus 819: a mechanistic overview REVIEW Figure 2. Rolling-hairpin model of human parvovirus 819 DNA replication (cont). (D) Further replication of RF DNA requires NSI to specifically bind the Ori and nicks the top strand atthe ts (E) This event creates a new 3‘0H to lead DNA synthesis following melting ofthe hairpinned ITR, Which subsequently repairs the ITR and results in an open-ended duplex replication intermediate. (6) The repaired TRis then denatured, which ikely requires the helicase activity of the NS, and reannealed, in a process termed reinitiation, to form a double-hairpinned intermediate, which creates a new 3 primer (30H) (H) to initlate around of strand displacement synthesis. 819V: Human parvovirus B18; cRF: Closed replicative form; ITR: Inverted terminal repeat; LTR: Left hand inverted terminal repeat; NSI: Nonstructural proteins; RF: Replicate form; R-ITR: Right-hand inverted terminal repeat A few megakatyoeyte-erythroid lineage call, lines were documented to support BIV infee tion, including erythroid leukemic cell lines (KUSI2Ep6 and JK-11) 8-50), and human megakaryocytic leukemia cell lines (MB-02, UT7IEpo and UT7IEpo-S1) [31-53]. A com: parison study evaluating their differences in petmissiveness to BIV infection showed that the UT7IEpo-S1 cel tive cell line to BIOV infection, based on detec tion ofthe viral NSI protein and inereased viral DNA production js). Although BISV infection in UT7/Epo-SI cells is less efficient than that in EPCs 5, i ie particularly useful for the transfection of the BIV infectious clone and subsequent mutagenesis studies, since trans- fection is extremely difficult in EPCs due to the nature of thie type of primary cele. ‘At least two major factors have been iden- tified ¢o account for the tropism of BIOV for REPCs and these megakaryocyte lineage leukemic cell lines. First all these types of cells express globoside (erythrocyte P anti- gen), which is the primary receptor for BISV 5s). BIOV capsid directly interacts with the glo- boside on erythroid cells, and pre-incubation antigloboride antibody or purified glaboside prevents BISV infection of human bone marrow cells) Individuals whose erythrocytes do not have globoside are naturally resistant co BISV infection (se). Globoside is also present in red blood cells and some nonerythroid cells, such as fetal myocytes, placenta, megakaryocytes and some endothelial cells 7-0), which might explain the diverse clinical manifestations in different tissues, Besides che primary receptor globoside, integrin 451 and Ku80 were pro- posed to be coreceptors for BIOV (6.4; however, cell surface expression of Ku80 was shown to be very low in ex vivo-expanded CD36" hEPCs (£5%) and other B19V-permissive cells (5.4 Therefore, the erythroid tropism of BISV can- not be simply explained by the presence of the 1e was the most sensi scceptor/coreceptors, The second explanation is ‘hatall these megakaryocyce—crythroid lineage cells requite erythropoietin (Epo) for growth, and Epo receptor (EpoR) signaling is equired for B19V DNA replication In EPCs, Epo binds to EpoR and activates the Janus kinase 2 (Jak2) by autophosphorylation (6s). Activated Jak? furcher phosphorylates EpoR and initiates a kinase eascade with three major pathways, including the signal transducer and activator of ‘ranscription 5A (STATSA), mitogen-activated protein kinase (ERK/MAPK) kinase (MEK) and phosphatidylinositol-3 kinase (P13K). BI9V infection of EPCs is Epo concentra dependent ss). Unlike globoside, EpoR is not required for BI9V entry because Epo exposure rus entry sill enables BIOV DNA rep- lication. In fact, the EpoR signaling of Jak2 phosphorylation and the MEKJERK activa- tion play a key role in facilitating BI9V DNA replication in hEPCS (6 In addition to these findings, a recent study showed that the VPI unique region (VPlu) of the BI9V capsid prosein VPI is essential for BISV binding and internalization during BIOV infection of in UT7/Epo-S1 cells 6 Purified-recombinane VPIa ean also be int alized in UT7/Epo-S1 cells. The N 29 amino acids of the VPlu have been shown to be essential for binding and internaliza- tion of the VPlu, which is independene of the PLA2 activity chat was thought to be ert cal co BISV infectivity (6. Interestingly, the \VPtu-ineeracting cellular partner was uniquely expressed on UT7/Epo-S1 and KUSI2Ep6 cells, bur not on nonerythroid lineage cells such as HeLa, HEK 293 and HepG2 cell 6 indicating the unique role of VPtu in facli- tating BI9V binding and inter erythroid lineage cells. Although the specific \VPtu-interatcing molecule (receptor) has not been idenvified, this study presents a novel parvovirus internalization mechanism. erminal lization in Brescene sow wen faturemedicine com 159REVIEW Luo &aiu In conclusion, in in visro culcures, BI9V is mainly permissive co BEPCs and a few megakaryocyce—erythroid lineage leukemic cell lines, The tropism of BI9V for erythroid lineage cellsis largely due tothe BISV receptor globoside and Epo/EpoR signaling, as well at VPI that facilitates BIOV internalization B19V infection of myocardial endothe cells & monocytic cells, A few cher nonerythroid lineage cells have been seported to support BISV infection (67-4, BISV infection has been suggested co be associated with, myocarditis and acute and chronic inflamma. ory cardiomyopathies, since BI9V DNA was frequenely detected in patients who have these symptoms 676-71, BIOV was alzo shown tobe che ‘most Irequent pathogen detected in patients with. normal coronary anatomy that clinically mim- ies acute myocardial infarction (6). In addition, in vivo seadies have demonsteated that BIOV can, also productively infect endod 2) and placental tissues, cells could be a natural target for BI9V infection. A recent study by Kietzell etal. identified a new route for the BIOV infection of myocardial endothelial cells (6. There are no major dif. races in surface expression of BISV recep ‘or/coreceptors among UT7/Epo-SI and primary «endothelial cells, which are isolated from the pul- ‘monary arteria (human pulmonary artery celle [HPAEC), the umbilical vein (human umbilical vein endothelial cells (HUVEC]) and che aorea (suman aortic endochelial cell [HAoEC]). BIV binds primary endoclial celle ata similar level at that of UT7/Epo-SI cells. However, BIOV inter- nalization is deficient in these endothelial cells, deficiency which is significantly enhanced by pre- incubation of che virwe with anti-BI9V antibodies. Mechanistcally, the BI9V-antibody complex ‘might enter the cells through endacytasis medi ated by the direct interaction of antibody-bound complement factor Clg with its receptor CD93 fon the cell surface. Kietell er als study explains shar che BI9V genome and virus were decected in ‘myocardial samples from patients with cardiac diseases (7), and provides an explanation for che frequent prevalence of BIOV in endothelial cells from a variety of tissues, which may be related to che spread of BISV to other cell cypes i However, the scudy does not provide evidence of BI9V DNA replication in endothelial ell ‘An early study bas shown an antibody-depen entenhancement of BI9V infection in monocytic cell line U937 cells), BIV DNA was devected, in infected U937 cells but wich abortive BIOV DNA replication, By addition of anti-B19V IgG, BI9V DNA replication was significancly increased, However, the antibody-enhance ment pathway appeats to be differene beween, endothelial and monocytic cells, as monocytic cells ate speeulaced to be Fe seceptor-mediated. enhancement of BIOV internalization [ In conclusion, collective evidence indicates chat BIOV enters myocardial endothelial cells, butlacks sufficient support to replicat There is a long road ahead to prove BIOV is a causative agent of myocardial diseases, Ie is tractive to speculate that the endocytosed BI9V genome could be sensed by cytosol nor nuclest innate immunity DNA sensors, which induce proinflammatory cytokine secretion [74], and. subsequently inflammatory cardiomyopathies. ‘Additionally, che fact that BIDV infects both the monocytic cell line and myocardial endothelial cells through an antibody-enhancement path- way suggests thatthe antibody-mediated BIOV entry might be a common mechanism for BIOV infection of nonerythroid lineage cell. in them, Identification of the B19V minimum DNA replication origin “Abortion of BIDV infection was thoughé previ- coudly to be due toa block in full-lengeh transcrip- sion maturation, as wel as the in the conversion. of vital seDNA to double-stranded replicative Intermediates (75.77). With the available infectious DNA of BIOV [2 recent studies have suggested, that the abortive BI9V infection in nonpermissive celle is largely due to the inefficient replication. or nonreplication of BI9V DNA in these cells. With the help of adenovirus, BI9V DNA repli- cates in nonpermissive human embryonic kid- ney 293 cells (293 cells) (1573). 293 cells either infected with adenovirus or sransfected with the Helper plasmid, which contains the adenovirus genes E2A, Eorf6 and VA RNA, suppore BISV. DNA replication (33. In line with this, adenovi- rut infection also enhances BI9V DNA replica ion in UT7/Epo-SI cells. One explanation for jis phenomenon is chat adenovirus ELA protein srancactivates the BIOV promoter in nonpermis- sive cells 91. Also, both BIOV and AAV have symmetric ITRs ar each end of che viral genome; thus, helped by adenovirus, B19V DNA replica tion in 293 cells may share the same mechanism. as that in adenovirus-helped AAV DNA repli- cation. A detailed examination of the funecion 180 tavescerce soup MBof the adenovirus gene products in BIOV DNA replication confirmed that the EAor6 protein and VARNA functioned similarly co help BI9V DNA, replication as they do during adeno-associated vitus 2 replication, while E2A had no stimula toty effect on BIIV DNA replication or gene ‘expression 8}. More specifically, the E4oxf6 pro: tein serves as a scaffold co form a cullin 5-based E3 ubiquitin ligase complex chat targets cellar proteins, such ae p53 and Mee, for degradation, while che VA RNA binds and inactivates pro: tein kinase PKR, a (ds) RNA-dependent protein kinase (75). Notably, adenovirus gene products are ‘not requited for the DNA replication of human Docavirus and the canine virus analogue, min: ute virus of canines (MVC) (aos). Both human bocaviruses and MVC have asymmettc terminal Inairpin structures, suggesting the differences of ‘terminal hairpin structures may account for the different replication mechanisms of parvovirus. By using the 293 cell transfection system, a BIOV DNA replication origin has been identi fied [is). The minimum origin of B19V DNA replication is only 67 nucleotides (nucleotide 5214 to 5280 of GenBank: AY386330), which covers a NSI ers and four repeated NSI binding elements (NSBEs) (Figure 3). BIO NSI spe- cifically binds to the NSBEI-NSBE2 region in an in vitro binding assay, while NSBE3 and NSBE4 may provide binding sites for potential cellular factors (¢2|, which should assemble a nucleoprotein complex involving cellular fac- tors to separate the dsDNA strand and enable NSI to nick the top strand atthe trs (Figure 20) Surprisingly. the mutant BI9V infectious DNA with deletion of either the lefi-hand oF the righthand ITR sill replicates in 293 with adenovirus infection or pHelper transfec- tion. In addition, transfection of a BI9V DNA fragment containing the NSI expression cas. sette and only the Ori replicates in 293 cells in the presence of the three adenovirus gene products, as well as in UT7/Epo-SI cells (5. Based on these result, a haispin-independent replication model has been proposed for BIOV DNA replication (ss. Ie was expected that NSt has the abiliey to reverse the direction of the replication (Figure 2F & 6), regardless of the repairing/annealing of the IR scrucrure asi, for second-strand replacement synthesis (Figure 3H). However, the in-depth mechanism is not understood yet. Despite of these interesting observations in BI9V-permissive UT7/Epo-SI cells and Human parvovirus 819: a mechanistic overview REVIEW B19V nonpermissive 293 cells, ic is not cleat whether the hairpin-independent replication is employed during BI9V infection of hEPCs Alto, litle is known about its selevance to nat ‘ural infection, The faec that BI9V DNA repli cates in nonpermissive 293 cells with the helper function of adenovirus provides a possibility that BIDV coinfection with other viruses in nonerythtoid lineage cells, for example, myo cardial endothelial cells, facilitates a high level of BI9V DNA replication, which may resale in some disease outcomes and awaits more in vivo Hypoxic conditions promote productive BI9V infection of erythroid lineage cells Alchough the ¢* vivo-expanded EPCs enable BISV DNA replication a¢ a high efficiency, there is still a buge discrepancy in the produc tion of progeny virions from infected EPCs and during viremia of BI9V-infected patients (10 genomic copies per milliliter of plasma), indicating th fied « human bone marrow in patients in vitro, One of these factors could be the oxygen level in human bone marrow, which ie much lower than, that in in vitro cell calsure conditions (8, In fact, a significant enhancement of BISV DNA replication az well as progeny virus production har been observed during BIOV infection of hEPCs when they were culeared under 1% O, (hypoxia) [444-45 Interestingly, hypoxia actually reduces the differentiation potential and the proliferation rate of the EPCs (54). The productive B19V infection under hypoxia is facilitaced neither through an increase in virus entry or ineracellular traf. ficking nor shrough the network regulated by hypoxia-inducible factor, a common sign- sling sensor for hypoxia-induced signaling transduction (54). Strikingly, hypoxia regu- lares Epo/EpoR receptor signaling, which is essential for BI9V DNA replication 1s), and. thereby, enhances BI9V DNA replication. Two pathways mediated by the Epo/EpoR/TAK2 pathways, including upregulation of STATS signaling and downregulation of MEK/ERK signaling, boost BI9V DNA replication in both EPCs and UT7/Epo-S1 cells 54 ‘A promising application of this finding is to study BI9V DNA replication by cransfect- ing the BI9V infectious clone in UT7/Epo-St cells preculcured under hypoxia. The inoculated « other factors remain unidenti recapture the in vive BIOV infection of Brvescene sow vwwnvfaturemedicine com 161REVIEW Luo &aiu Bre RATR Bie on (er) " "S NSBEL NSBEZ noses usaee MME eee ETS soe Fp fae 7 ‘bubbles bulge 5410 Flo bulge bubbles bulge 5410 Figure 3. Structure of the human parvovirus 819 right-han human parvovirus 819 minimal ONA replication o inverted terminal repeat and the jin (Or). The 819V right-hand ITR (R-ITR) at 355 nis depicted in both the lip’ and the ‘lop’ orientation, andthe trs and NSBEs thought to comprise the NSI binding site are indicated, The ITRis a neatly perfect palindromic structure; the exceptions are the three unpaired bases at two sites shown as bulges) and three mismatched bases at three sites (¢hown as ‘bubbles’. The nucleotide sequences of the minimal 819V DNA replication origin (Ori) are indicated. The ITR sequence refers tothe B19V J35 strain (GenBank accession number: AY386330), 319V: Human parvovirus 819; TR: Inverted terminal repeat; NSBE:NSI-binding element; ts: Terminal resolution site infectious clone replicated efficiently and pro- duced progeny viions that were highly infectious in EPCs (34), which holds promise co perform mutagenesis study of BIOV DNA replication, DNA damage response-facilitated BI9V. DNA replication A large number of DNA viruses have been shown, to induce a DNA damage responce (DDR), and some of chem, including parvovirue ss), require the DDR for efficient viral DNA replication ras). DDR is triggered by damaged DNA struc- sues, such as ssIDNA breaks, dsDNA breaks and stalled replication forks. The signaling transduc- sion is conducted by ase of host defense machin- cry that is composed of a number of signaling sensors, transducers and effectors. Three major DDR sensors have been identified, inchuding ataxia telangiectasia-murated kinase (ATM), ATM- and Rad3-related kinase (ATR) and DNA-dependent prosein kinase catalytic subu- nit (DNA-PKcs). Each of these DDR sensors recognizes specitc cypes of damaged (cellular) DNA seruerures and transduces a kinase cascade 0 numerous downstream mediacors/effectors, which result in cither cell cycle arrest for DNA. repairing ot apoptosie 9 BI9V infection of hEPCs activates the DDR signaling for the facilication of viral DNA genome replication (|. The BIOV genome hae ‘wo identical ITRs separated by a large ssDNA sap (Figure 2A), which isa perfect trigger to acti- vate the ATR signaling (:). In addition, BI9V infection activates ATM and DNA-PKes, por sibly due co the intermediate replicative viral genome (Figure 2G8H) during BOV replication he crost-interaction between DDR signaling. Notably, BI9V hijacks ATR and DNA-PKcs, but not ATM, for viral DNA replication \9o.. The DDR activation is associated wih viral DNA. replication status but not individual BIOV struc ral or nonstructural proteins (1, suggesting sat the DDR involving proteins direcely interact with the replicating BI9V genome, Irisnotclearhow BIOV replication is facilitated by che DDR signaling. Possibly, the ATR signal- ing plays a role in the firststrand DNA synthesis, which could be a DNA repairassociated DNA replication, and the DNA-PKes activation recruits DNA ligase IV to ligate che RF DNA (igure 20) 182 tavescerce soup MBLate S-phase-dependent 819V DNA replication Cell-eyele attest is required for a number of DNA viruses to modulate host micraenviton: ment in such a way that favors vital DNA rep: lication. Eatly scadies have shown that BISV infections of both EPCs and UT7/Epo-S1 cells induced G2/M artes, status of 4N DNA con: tent (5392-94), However, & mote careful exami nation of BI9V-infeeted cells wsing 5-bromo: 2-deoxyutidine (BrdU) incorporation combined with 46°-diamidino-2-phenylindole bydrochlo: ride (DAPI) staining demonstrated that BIOV. infection actually induces a cell-cycle status with 4N DNA content at well as with BrdU- incorporation, suggesting a late § phase arrest 20). Expression of NSI alone induces a teue G2/M arrest, a status with 4N DNA content and without BrdU incorporation (so). The NS1 induced G2/M arrest har been reported to be ‘caused by the deregulation of E2F family tan: sctiption (9), and ie not caused by BI9V infee tion induced DDR (9). BISV infection induced late S-phase arrest suggests tha replication ofthe BI9V DNA genome, az with other autonomous parvoviruses whore DNA replication depends fon host cells arrested at § phase (1 0}, requizes cellular replication factors expressed in $ phase Similarly, during early infection of parvovirus MVC, MVC DNA replication induces a DDR, which in curn arrests the cells at $ phase [9536 ‘The S phase arrest furcher facilitates MVC DNA replication. We speculate chat BIOV DNA rep- lication-induced DDR causes cells arrested at S phase, while expression of che NSI solely arrests cells ata statue with 4N DNA content, and that the compromising of these two arrests confers the infected cells a late § pha. Specific cellular replication factors, such ae the DNA polymerase, have not been identified for 9V DNA replication, largely because of the ‘essential role of there factors for proliferation and survival, and che lack of an in vitro DNA replica tion system of BIDY. Nevertheles, several § phase factors, inchuding DNA polymerase delta, prolif: crating cell nuclear antigen (PCNA), replication factor complex 1 (RFCI), chromosome maintenance complex 2 (MCM2) are found to appearin the BI9V DNA replication center {0}. Knockdown of MCM2 and MCMS to a level tha does not affece cell proliferation blocks BI9V DNA replication, confirming that the S-phase cellular DNA replication factors ace recruited by an unknown mechanism for B19V lin A and mini- Human parvovirus 819: a mechanistic overview REVIEW DNA plication, Ie would be importane co know whether these factors are recruited theough a DDR-dependent pathway 97 Conclusion B19Vis the only member of the Erythroparvovirus genus of the Parvoviridae family, which infects humans, Although initially BI9V was identi fied to infect erythroid lineage celle in bone ‘marrow ot fecal liver and co cause several mild to severe human hemalogical disorders, accu: smulating clinical reports indicate that BIOV also infects nonerythtoid lineage cells and say be associated with other diseases, such as myocarditis. ‘The selective BIOV infection of erythroid lin cage cells is due co the primaty receptor globo. side and Epo/EpoR signaling. BIOV alo infects ‘myocardial endothelia cells and a monocytic cell. line through an antibody-mediated enhance ment pathway. In the presence of adenovirus gene products, BIOV replicates in nonpermi sive 293 cells and produces infectious progeny “Mechanistic study of BI9V DNA replication in erythroid lineage cells is greatly facilitated by the establishment of ex vive-expanded CD36" EPCs and hypoxic conditions for B19V infec- tion, BIOV replicate in late § phase of infected cells by recruiting both cellular DNA replica sion/tepairing factors (possibly through DDR signaling) to facilitate fit strand synthesis. Thereafter, the deDNA genome becomes con petent for gene expression, NSI binding and nicking at the replication origin, which initi- ates strand displacement of viral DNA syn thesis, likely, chrough a haiepin-independene mechanism. Meanwhile, EpoR signaling, which is further enhanced under hypoxia is crucial for BI9V DNA replication and progeny virion production Future perspective Studies of BI9V infection and DNA replication hhave greatly increased our underscanding of the BIOV life eycle, which will shed lig tifying anti-BLOV strategies and eventually a therapeutic approach ro BLOV infection-catsed severe hematological disorders. Alehough an # vitvo cell euleure system. of BIOV has heen improved recently, the sys tem still does nor recapitulace B19V infec tion in vive, which produces progeny virions at a high yield. A number of questions about on iden- Brvescene sow vwwnv faturemedicine com 163REVIEW Luo &aiu the detailed mechanisms involved in B19V infection await exploration, For example, it is necessary to understand how EpoR signaling (STATS and MEK) affects BI9V replication, what minimal cellular replication factors ate involved in BISV DNA synthesis and whether these replication factors ate recruited theough, a DNA tepait- and S phase-dependent path: way. BIOV infection arrests erythroid lineage cells at late $ phase, Whether this represents a compromised condition forced by the NST protein and DDR signaling requires Further study. Also, the «ransmission for transmission ‘of BIOV from che respiratory tract co human bone mattow, along with its infection of che myocardial system, liver system and possi- bly even more unidentified organs, warrants furcher investigation Financial & competing interests disclosure The seudy was supported by NUIT ROL AIO70723. The authors have me other relevaneafiatens ov financial Anvlvemens with any enganizason or emisy witha finan tal ner in on fnanca confi with the subject matter er materiale dcused in the manuscript apart frm thie iced. [No writing aittance was wrlized in the production of hit menssript EXECUTIVE SUMMARY B19V infection of erythroid lineage cells ‘+ The primary receptor globoside and likely the coreceptor integrin a5 have been indicated for the Human parvovirus £19 (B19V) tropism for erythroid lineage cells: ‘* _ Erythropoietin/erythropoietin receptor signaling is essential for B19V tropism and productive DNA replication. * Hypoxia boosts productive B19V infection through enhancement of erythropoietin receptor signaling, ‘+ B19V infection hijacks DNA damage response signaling to facilitate virus DNA replication. ‘+ B19V infection induces late S phase, which favors virus DNA replication, B19V infection of nonpermissive cells '* Inthe presence of adenovirus gene products, B19V DNA replicates in 293 cells. '* AG7-nucleotide region of the 819V minimum DNA replication origin has been identified and a hairpin-independent ‘model of 819V DNA replication has been proposed, ‘© B19Vinfects myocardial cells and monocytic cel ine U937 through an antibody-mediated entry pathway. References 6 Heegeard ED, Brown KE, Human infection and sete myocarditis late Pape of special note have been highlighted as: parvowicus BIB, Cli. Micebial. Reo 15(3), Med. $910),79 (2010) + afinterer * of considerable ivees, 485-505 (2002) 12 PapedogiannakisN, Tlfeasam T Facer 1 Cowart YE, Field AM, Cant B, Widdows D, 7 Molina KM Garcia X, Denilé SW ea B, NotbeekO, Broliden K. Active, flminas, Parvovrurlike parties in human sera, Parvovsus B19 myocarditis causes Iechal myocarditis anoited with patvovieus Lanes (7898), 2-73 (1975) 2 Anderton Mj, Jones SE, Fisher Hoch SP era Human parvovirus, he cae of erythems Infecosu (fh disease)? 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