Endonuclease activity inhibition of the NS1 protein of parvovirus B19 as a novel target for antiviral drug development

Article abstract of DOI:10.1128/AAC.01879-18

Human parvovirus B19 (B19V), a member of the genus Erythroparvovirus of the family Parvoviridae, is a small nonenveloped virus that has a single-stranded DNA (ssDNA) genome of 5.6 kb with two inverted terminal repeats (ITRs). B19V infection often results in severe hematological disorders and fetal death in humans. B19V replication follows a model of rolling hairpin-dependent DNA replication, in which the large nonstructural protein NS1 introduces a site-specific single-strand nick in the viral DNA replication origins, which locate at the ITRs. NS1 executes endonuclease activity through the N-terminal origin-binding domain. Nicking of the viral replication origin is a pivotal step in rolling hairpin-dependent viral DNA replication. Here, we developed a fluorophore-based in vitro nicking assay of the replication origin using the origin-binding domain of NS1 and compared it with the radioactive in vitro nicking assay. We used both assays to screen a set of small-molecule compounds (n 96) that have potential antinuclease activity. We found that the fluorophore-based in vitro nicking assay demonstrates sensitivity and specificity values as high as those of the radioactive assay. Among the 96 compounds, we identified 8 which have an inhibition of 80% at 10 M in both the fluorophore-based and radioactive in vitro nicking assays. We further tested 3 compounds that have a flavonoid-like structure and an in vitro 50% inhibitory concentration that fell in the range of 1 to 3 M. Importantly, they also exhibited inhibition of B19V DNA replication in UT7/Epo-S1 cells and ex vivo-expanded human erythroid progenitor cells.

Full text of DOI:10.1128/AAC.01879-18

AAC Accepted Manuscript Posted Online 10 December 2018
Antimicrob. Agents Chemother. doi:10.1128/AAC.01879-18
Copyright © 2018 American Society for Microbiology. All Rights Reserved.
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Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target
for Antiviral Drug Development
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Peng Xu1, Safder S. Ganaie1, Xiaomei Wang1, Zekun Wang1, Steve Kleiboeker2, Nancy C.
Horton3, Richard F. Heier4, Marvin J. Meyers4,5, John E. Tavis6, and Jianming Qiu1
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1Department of Microbiology, Molecular Genetics and Immunology
University of Kansas Medical Center
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Kansas City, KS 66160
2ViraCor Eurofins Laboratories
Lee’s Summit, MO 64086
3Department of Molecular and Cellular Biology
University of Arizona
Tucson, AZ 85721
4Center for World Health and Medicine
5Department of Chemistry
6Department of Molecular Microbiology and Immunology
Saint Louis University
St. Louis, MO 63104
Running title: B19 NS1 endonuclease as a target for anti-B19 drugs
#Corresponding author:
Department of Microbiology,
Molecular Genetics and Immunology
University of Kansas Medical Center
Mail Stop 3029
3901 Rainbow Blvd.
Kansas City, KS 66160
Phone: (913) 588-4329
Fax: (913) 588-7295
E-mail: jqiu@kumc.edu
Version_11-15-18
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ABSTRACT
Human parvovirus B19 (B19V), a member of the genus Erythroparvovirus of the family
Parvoviridae, is a small non-enveloped virus that has a single-stranded DNA (ssDNA) genome
of 5.6 kilobases with two inverted terminal repeats (ITRs). B19V infection often results in severe
hematological disorders and fetal death in humans. B19V replication follows a model of rolling
hairpin-dependent DNA replication, in which the large non-structural protein NS1 introduces a
site-specific single strand nick in the viral DNA replication origins, which locate at the ITRs. NS1
executes endonuclease activity through the N-terminal origin binding domain. Nicking of the
viral replication origin is a pivotal step in rolling hairpin-dependent viral DNA replication. Here,
we developed a fluorophore-based in vitro nicking assay of the replication origin using the origin
binding domain of the NS1 and compared it with the radioactive in vitro nicking assay. We used
both assays to screen a set of small molecule compounds (96) that have potential anti-nuclease
activity. We found that the fluorophore-based in vitro nicking assay demonstrate sensitivity and
specificity values as high as the radioactive assay. Among the 96 compounds, we identified 8
which have an inhibition of >80% at 10 μM in both the fluorophore-based and radioactive in vitro
nicking assays. We further tested 3 compounds that have flavonoid-like structure for IC50 in vitro
that falls in the range of 1-3 μM. Importantly, they also exhibited inhibition of B19V DNA
replication in UT7/Epo-S1 cells and ex vivo expanded human erythroid progenitor cells.
KEY WORDS: Human parvovirus B19, in vitro nicking assay, anti-viral compounds
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INTRODUCTION
Human parvovirus B19 (B19V) was identified in 1975 when Yvonne Cossart screened
hepatitis B virus in a panel of human serum samples (1). B19V is a small, non-enveloped single-
stranded DNA (ssDNA) virus belonging to the genus Erythroparvovirus within the family
Parvoviridae (2). B19V exhibits a remarkable tropism for human erythroid progenitor cells
(EPCs) in the bone marrow and fetal liver (3-7). B19V most commonly causes Fifth disease or
slapped cheek syndrome in children (8,9); however, B19V infection can cause a series of
severe hematological disorders (10). B19V infection of the fetus can cause severe fetal anemia,
resulting in non-immune hydrops fetalis and fetal death (11-14). In certain circumstances, B19V
infection often results in bone marrow failure, most notably transient aplastic crisis in patients
with increased red blood cell turnover (e.g., sickle cell disease patients), and pure red-cell
aplasia in immunodeficient and immunocompromised patients (e.g., HIV/AIDS patients and
organ transplant recipients) (11,15). The clinical manifestations of B19V infection, as seen in
hydrops fetalis, transient aplastic crisis, and pure red-cell aplasia, are due to direct cytotoxicity
resulting from virus infection, which results in the death of EPCs in which B19V replicates (5,16-
22).
B19V has a linear ssDNA genome of about 5.6 kilobases (kb), which has identical
terminal repeats (ITRs) of 383 nucleotides at both ends. The double-stranded replicative form
(RF) DNA of the B19V genome contains a P6 promoter at the left hand (10). The left side of the
RF genome encodes a large non-structural protein (NS1) and a small non-structural protein 7.5-
kDa protein, whereas the right side of the genome encodes two capsid proteins (VP1 and VP2),
along with a small nonstructural protein 11-kDa using a different open reading frame (23). B19V
NS1, 671 amino acids (aa) long, has a molecular weight of approximate 78 kDa (Fig. 1A)
(24,25). NS1 predominantly localizes in the nucleus of infected cells as it contains nuclear
localization signals at aa residues 177-180 (KKPR) and 316-321 (KKCGKK), respectively
(25,26). The N-terminus (aa 1-176) of the NS1 contains a DNA replication origin-binding domain
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(OBD) that also exhibits endonuclease activity (27,28); the central region contains ATPase and
nucleoside triphosphate binding motifs (20); and the C-terminus contains transactivation
domains (20,29). NS1 is essential for replication of viral DNA through its endonuclease and
helicase activities (30). NS1 also binds the P6 promoter of the viral RF genome to regulate viral
gene expression (31). In addition, NS1 has been reported to transactivate several other host
genes (29,32,33).
B19V is an autonomous parvovirus, replicating itself in the host cells without a helper
virus (10), as are the majority of the members in the Parvoviridae family except for adeno-
associated viruses (AAVs) (34). In contrast, AAVs, whose genome also contains their unique
ITRs of 144 nucleotides, requires coinfection of a helper virus, such as adenovirus, herpesvirus,
or human bocavirus, for replication (35,36). B19V replication arrests cell cycle at late S phase,
hijacks cellular DNA replication factors present in the S phase, and replicates its genome
following a rolling hairpin model of DNA replication (37-39). In principle, the B19V ssDNA
genome uses 3′-end hairpin as a self-primer (3’OH) to extend viral ssDNA into the dsDNA
genome by cellular replication proteins (40), a step called first-strand DNA synthesis (39). The
extended 3′ end is presumably ligated to the 5′ end of the genome to form a partial circular DNA
genome. NS1, which has site-specific endonuclease activity and DNA helicase activity, nicks
ssDNA at the terminal resolution site (trs) between the 5’-end hairpins and the newly
synthesized viral DNA to form a novel 3’ primer that initiates hairpin transfer, followed by the
strand displacement (39). The elongated viral genomes (both RF and double RF intermediates)
are also resolved by NS1 nicking to release ssDNA that is finally packaged into the capsid.
The minimal replication origin (Ori) of B19V DNA has been identified to be 67-nt, which
includes a STAT5-binding element (STAT5BE), a trs or nicking site, two copies of NS1-binding
element (NS1BE), and the putative cellular factor-binding site (CFBE) (Fig. 2A) (25,27,41,42).
Specific cleavage of the ssDNA Ori has been demonstrated in an in vitro nicking assay using
purified B19V NS1 N-terminus (aa 1-176) and a 5’ end [32P] labelled oligo that contain the trs
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(28). In this study, we further demonstrated the specificity of the radioactive nicking assay, and
developed a non-radioactive nicking assay using an oligo of 20 nucleotides labelled with 6-
carboxyfluorescein (FAM) and the quencher Iowa Black® FQ (IBFQ) at the 5’ and 3’ ends,
respectively. The fluorophore-based nicking assay recapitulates the same sensitivity and
specificity as found in the radioactive assay. We further validated the assay using a small library
of 96 compounds, where 8 compounds inhibited NS1 nicking activity by over 80%, and out of
which 3 compounds also inhibited viral DNA replication in B19V RF DNA-transfected UT7/Epo-
S1 cells and B19V-infected CD36+ EPCs.
RESULTS
Purified NS1N, but not the endonuclease motif mutant, cleaves B19V Ori ssDNA, and
does not cleave the mutant minimal replication origin (Ori).
B19V NS1 has the endonuclease activity at the N terminus aa 1-176 (NS1N, Fig. 1A).
NS1N has been proved to bind the Ori and executes its cleavage in vitro (27,28). We first tested
the specific Ori cleavage of the NS1N. To this end, we expressed and purified both the wild-type
NS1N and NS1NmEndo that has alanine substitutions in the endonuclease motif (residues 140-
143) (20) (Fig. 1A). Purified NS1N and NS1NmEndo, which contain a 6 x histidine tag at the C-
terminus, show a purity of >90% (Fig. 1B&C). We used a short version of the B19V ssDNA form
Ori, which is 30-nt, as a template for cleavage, because the NS1-binding elements (NS1BEs)
are not required for nicking of the ssDNA Ori in the condition of the in vitro nicking assay that
contains high concentrations of NS1N (28). Ori30 and Ori30mut (Fig. 2A) were 5’ end labeled
with 32P and then used at a final concentration of 2 nM in the nicking buffer (see MATERIALS
AND METHODS) with 2 μM of the purified NS1N. After incubation of 16-18 h at 37°C, the
reactions were analyzed in a denaturing urea polyacrylamide gel. We found that NS1N cleaved
Ori30 at the trs and produced a cleaved DNA band at 19-nt (Fig. 2B, lane 4), while NS1N did
not cleave the trs mutant Ori30mut (Fig. 2B, lane 5). Moreover, we asked whether the NS1N nick
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is endonuclease motif-specific. We used the purified NS1NmEndo in the nicking assay. The result
showed that while the wild-type NS1N was capable of cleaving the Ori-30 at trs and released a
band of 19-nt, the NS1NmEndo did not (Fig. 2C, lanes 3&4). We next determined the minimal
concentration of NS1N required for nicking of the Ori. We found there was a dose-dependent
increase in the level of the nicked band (19-nt) (Fig. 2D). At 2 μM of the NS1N, greater than 70%
of the probe was cleaved. We chose to use NS1N at 2 μM in subsequent experiments.
Taken together, these results confirmed NS1N specifically and effectively cleaves the
Ori (at 2 nM) at trs at a concentration as low as 2 μM, and that the endonuclease motif is
important for the nicking activity.
Small molecule compounds inhibit cleavage of the ssDNA Ori by NS1N.
To identify small molecule compounds that can inhibit the NS1N-mediated Ori cleavage,
we used 96 compounds (Table S1 in Supplemental Materials), which we have used
previously to screen inhibitors for RNase H nuclease activity of the human hepatitis B (HBV)
polymerase (43-47), to test them for inhibitory effects on the NS1N-mediated Ori cleavage using
32P-labeled Ori30 template. We first tested them at 100 μM, and found that 25 compounds
inhibited the nicking of NS1N by greater than 80% as shown in Fig. 3A. Next, we tested the
positive 25 compounds at 10 μM. Eight compounds (#7, #9, #12; #135, #151, #153, #201, and
#328) appeared to nearly abolish cleavage of the Ori by NS1N, a >80% inhibition compared
with the DMSO control (Fig. 3B).
Fluorophore FAM-based nicking assay reproduces the sensitivity and specificity of the
radioactive nicking assay.
We next developed a method that could screen a large number of compounds easily.
We synthetized one fluorescent Ori oligo: FAMOri20Q, a 20-nt ssDNA oligo, in which the 5’ end is
labeled with FAM and the 3’ end is labeled with a quencher IBFQ (Fig. 4A). The quencher dye
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quenches the fluorescence emitted by the fluorophore via Förster resonance energy transfer
(FRET) when the two dyes are in close proximity, and the intact FAMOri20Q did not show
significant fluorescence. When NS1N was added, cleavage occurred at the trs, which resulted in
an increase in fluorescence to a level of 72% of the fluorescence from the same oligomer but
without a quencher present (FAMOri20), and the fluorescence intensity difference with or without
NS1N reached 18.38-fold (Fig. 4B). We further tested the optimal concentration of the oligo. We
found that at 25 to 200 nM of FAMOri20Q, the assay displayed >20-fold difference in the
fluorescence intensity with and without NS1N (Fig. 4C). Therefore, we chose FAMOri20Q at 200
nM in subsequent experiments based on the high fold change (20-fold) and the maximum
fluorescence intensity (reading). We noticed that 2 μM of NS1N was enough to saturate the
nicking of the Ori at concentrations ranging from 25 to 800 nM as increased fluorescence
intensities were detected (Fig. 4C).
We next used the FAMOri20Q probe to repeat screening the 71 compounds (at 10 μM)
which did not show >80% inhibition at 100 μM by the radioactive assay (Fig. 3A). We set up a
cutoff value of 80% inhibition as in the radioactive assay, compared with the DMSO control. We
found none of the 71 compounds showed an inhibition of >80% at 10 μM (Fig. 5A). We next
carefully tested the 25 compounds, which showed an inhibition of >80% at 100 μM in the
radioactive assay (Fig. 3B), at 10 μM with FAM-based nicking assay using FAMOri20Q probe. We
found that 8 compounds (#7, 9, 12, 135, 151, 153, 201, and 328) were positive (>80% inhibition)
in the inhibition of NS1N nicking activity (Fig. 5B). The results confirmed that the fluorescent
assay detected all the 8 positive compounds from the radioactive assay.
We established the correlation coefficient value (r) between the two assays, which is
0.91 (Fig. 5B), indicating the two methods match well. We also generated the scatter plot and
trendline of the two assays (Fig. 5C). The coefficient R squared value (R2) is 0.83, representing
smaller differences between the two assays.
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Cheminformatics analysis showed that compounds #7, #9, #135, #201, and #328 have
flavonoid structures, while compounds #12, #151, and #153 possess naphthyridinone structures
(Fig. 6). We chose flavonoids compounds #7, #135, and #201 for further in-cell study below.
Flavonoid compounds inhibit B19V DNA replication in UT7/Epo-S1 cells.
We first determined the IC50 (the half inhibition of in vitro nicking) of the three flavonoids
compounds #7, #135, and #201, which are 3.1±0.8 μM, 2.9±0.8 μM, and 1.1±0.4 μM,
respectively (Fig. 7). We next asked whether they inhibited B19V DNA replication in B19V-
permissive cells. For a direct result of inhibition of viral DNA replication, we tested them in
UT7/Epo-S1 electroporated with a RF DNA of the B19V genome, the B19V infectious M20 DNA
(48). B19V infects UT7/Epo-S1 cells poorly (41,49) and electroporation using the nucleofector
(Lonza) directly delivers the viral double-stranded RF DNA into the nucleus, where the RF DNA
is nicked by NS1 to initiate viral DNA replication (39). After electroporation of the cells with
linearized M20 DNA, a compound was added at various concentrations. At two days post-
electroporation under hypoxic conditions, cells were collected for flow cytometry using an anti-
B19V capsid antibody to select capsid-expressing cells.
The half maximal effective concentration (EC50) of B19V replication inhibition was
calculated compared with the vehicle control (DMSO). Compounds #7, #135, and #201 have
EC50 values of 44.2±18.6 μM, 61.1±0.3 μM, and 55.1±7.9 μM, respectively (Fig. 8A). The CC50
(drug concentration that affects viability of 50% of cells in culture) values of compounds #7,
#135, and #201 as determined in UT7/Epo-S1 cells are 194.0±22.3 μM, 227.0±21.0 μM, and
180.9±20.5 μM, respectively (Fig. 8B). Therefore, the selective index (SI=CC50/EC50) for
compounds #7, #135, and #201 is 4.4, 3.7, and 3.3, respectively.
We further analyzed viral DNA replication in UT7/Epo-S1 cells using Southern blotting.
The results showed that at the concentrations close to their EC50, compounds #7, #135, and
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#201 inhibited at least half of the level of the RF DNA (Fig. 9). These results suggested that the
inhibition of B19V replication is likely due to the inhibition of viral DNA replication.
Flavonoid compounds inhibit B19V infection in CD36+ EPCs.
To mimic the B19V infection in human bone marrow, we used ex vivo expanded primary
CD36+ EPCs cultured under hypoxic conditions which were differentiated from CD34+
hematopoietic stem cells isolated from human bone marrow. We infected the CD36+ EPCs with
B19V plasma, and at the same time, the cells were treated with various concentrations of the
compounds. At 48 h post-infection, the cells were collected for determining B19V infection by
flow cytometry using an anti-capsid antibody. We found that for compounds #7, #135, and #201,
the EC50 is 37.6±3.6, 53.9±7.1, and 33.5 ±1.4 μM, respectively (Fig. 10A); the CC50 is 55.9±2.1,
89.8±7.8, and 60.0±2.4 μM, respectively (Fig. 10B). Therefore, the SI for compounds #7, #135,
and #201 is 1.5, 1.7, and 1.8, respectively. For compound #7 at 40 μM, there were >95% living
cells, whereas B19V infection was inhibited at an efficiency of >60%. For compound #135, at 50
μM, there were >95% living cells, whereas B19V infection was inhibited at an efficiency of 50%.
For compound #201, at 40 μM, there were >80% living cells, whereas B19V infection was
inhibited at an efficiency of 70% (Fig. 10). We next analyzed viral DNA replication in drug-
treated infected cells at 48 h post-infection using Southern blotting. It was obvious that
compounds #7, #135, and #201 inhibited viral DNA replication by over 10-fold at a concentration
of 40, 50, and 50 μM, respectively (Fig. 11).
Furthermore, using quantitative PCR (qPCR), we were able to determine the inhibition of
viral DNA replication by the compounds at early time (18 h) post-infection, compared to that at
middle (30 h) and late times (48 h) during infection, respectively. At 18 h, 30 h and 48 h post-
infection, cells were collected for extraction of total DNA which was subjected to qPCR for
copies of viral genome relative to mitochondrial (mt) DNA, which was used to calculate EC50.
Compound #7 has EC50 values of 6.43±0.12 μM, 20.84±0.88 μM and 20.5±3.17 μM at 18 h, 30
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h and 48 h post-infection, respectively (Fig. 12A); compound #135 has EC50 values of
20.47±1.11 μM, 31.28±1.95 μM and 38.35±4.68 μM at 18 h, 30 h and 48 h post-infection,
respectively (Fig. 12B); and compound #201 has EC50 values of 20.47±1.11 μM, 16.15±0.54 μM
and 24.51±0.1μM at 18 h, 30 h and 48 h post-infection, respectively (Fig. 12C). These results
suggest all the three compounds showed a similar level of inhibition on the B19V DNA
replication during early infection as effective as during middle and late time points of infection.
Of note, compound #7 has a relatively effective EC50 of 6.4 μM at 18 h post-infection. Therefore,
the selective indexes for compounds #7, #135, and #201 as determined by quantification of viral
DNA at 48 h post-infection are 2.7, 2.4, and 2.5, respectively.
Collectively, these results demonstrated that compounds #7, #135, and #201 inhibited
B19V infection and DNA replication in CD36+ EPCs at a concentration that does not significantly
kill cells.
DISCUSSION
In B19V infection-caused transient aplastic crisis, pure red-cell aplasia, chronic anemia
and hydrops fetalis, the clinical manifestations are due to the direct cytotoxicity of the virus
infection (11,15,21), a direct outcome of the cell cycle arrest and cell death of human erythroid
progenitor cells (EPCs) that host B19V replication (5,16,18,19). To date, a vaccine against
B19V infection and specific treatments for B19V infection-caused transient aplastic crisis,
chronic anemia and pure red-cell aplasia, hydrops fetalis and congenital anemia in infants are
not available. Although intravenous immunoglobulin (IVIG) can be administrated to patients with
B19V-associated chronic anemia and pure red-cell aplasia (50-52), the symptoms often recur
when IVIG treatment is interrupted (53-56). The repeated applications of IVIG and the
maintenance therapy that are required to fully eliminate B19V-associated pure red-cell aplasia
are cost prohibitive (51,57-63). As all these complications of hematological disorders caused by
B19V infection are the direct outcome of B19V infection of EPCs, diminishing B19V replication
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in EPCs is expected to be an effective approach for treating B19V infection-induced
hematological disorders. Cidofovir, an acyclic nucleoside phosphonate broadly against dsDNA
viruses, has shown some promises in antiviral effect on B19V infection (64,65). Small molecule
compounds that can specifically inhibit B19V replication of ex vivo expanded CD36+ EPCs will
be ideal candidates to test as drugs for the treatment of hematological disorders caused by
B19V infection.
In the current study, we identified eight compounds that exhibit inhibition of NS1 nicking
of the B19V Ori from screening of 96 small molecule compounds, which have been used to
screen inhibitors for RNase H nuclease activity of the HBV polymerase (43-47), using an in vitro
nicking assay. We chose three compounds #7, #135, and #201, that share a flavonoid similar
chemo structure for detailed study. All three flavonoid compounds inhibited B19V DNA
replication in UT7/Epo-S1 cells and exhibited a selective index of 3-4. However, the compounds
showed higher cytotoxicity in primary CD36+ EPCs with a therapeutic index of >1.5, although
they inhibited B19V infection at concentrations without showing significant cytotoxicity. These
data indicate that the 8 screening hits identified here are candidates for future medicinal
chemistry campaigns to improve efficacy and reduce cytotoxicity.
Flavonoids are a class of ubiquitous secondary plant metabolites. They have been
reported to have functions in a wide range of biological activity, including anti-microbe,
antioxidant, anti-cancer, anti-inflammatory and eukaryotic enzyme inhibition properties (66,67).
As antivirals, flavonoids been shown to execute antiviral activities against many human viruses,
including DNA viruses, RNA viruses, and retroviruses, observed in cell culture and animals (68).
Mechanistically, flavonoids function in various pathways of virus life cycle, including virus entry
(69), RNA transcription of viral genome (70,71), and viral RNA translation (72,73). Flavonoids
can inhibit viral protease activity (74,75), viral helicase activity (76,77), and viral (HIV) reverse
transcriptase (78,79). They also have been shown to suppress cellular pathway that are
essential to virus replication (80). Our studies expand the range of antiviral mechanisms
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possessed by the flavonoids to include endonuclease activity of viral proteins (68). Flavonoids
inhibited B19V infection, assessed for viral capsid expression, and B19V DNA replication in
UT7/Epo-S1 cells nucleofected with M20 B19V infectious DNA (Figs. 8&9), as well as for B19V
DNA replication during early infection of CD36+ EPCs (Fig. 12), suggesting that the inhibitions
likely take place in steps of viral DNA replication. As the nicking of the Ori by NS1 is an
essential step for B19V DNA replication (81), we believe that flavonoids specifically target the
nicking step. Another important class of small molecules that inhibits NS1N nicking is
napthyridinone. Compounds #12 and #153 have been shown to inhibit the RNase H activity of
the HBV polymerase (45,82). Naphthyridinones also have been shown to inhibit nuclease
activity of the HIV reverse transcriptase (83). As a next step, we will test compounds #12, #151,
and #153 for their activity against B19V infection in cells.
Identification of multiple hits in two chemotypes provides clues regarding the structure-
activity relationships of these compounds. All of the flavonoid and napthyridinone hits share a
hydrophilic two-ring heteroatom aromatic structure with a single appendage containing one or
more benzyl moieties extending from similar spots on the bi-ring structure. Converting the
benzylic extensions seen in the naphthyridinones #151 and #153 to a hydroxyl in #152 and
#148 (Fig. 6) ablated activity against NS1N activity (Figs. 3&5). Similarly, ablating the hydroxyl
on the nitrogen heteroatom in #12 to create #150 (Fig. 6) also ablated activity, possibly by
removing the potential for chelation of the Mg++ ions in the NS1N active site that is known to be
important as the naphthyridinones bind in this way against the HIV RNase H (84). These
similarities will be valuable during future medicinal chemistry optimization of both the flavonoid
and napthyridinone scaffolds.
An important contribution of this study is the establishment of a fluorescent in vitro
nicking assay. We have identified the 67-nt Ori that supports B19V DNA replication in B19V-
permisive UT7/Epo-S1 cells (41) and characterized the NS1BEs of the Ori in vitro (27). Based
on this information, an in vitro nicking assay using a 32P-labelled DNA probe, which spans the
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trs in Ori, and purified B19V NS1N, was established (28). This radioactive nicking assay is not
appropriate for high throughput screening of small molecule compounds that inhibit the B19V
NS1N-driven nicking of the Ori. The NS1N cleaves FAMOri20Q probe efficiently and releases a
significant fluorescent signal. Compared with the non-NS1N control, NS1N increases the
fluorescence intensity by >18-fold, reaching ~72% of the signal from a non-quenched probe
FAMOri20. Notably, the FAM-based nicking assay possesses the same sensitivity and specificity
in detecting inhibition of NS1N cleavage of the Ori as the radioactive nicking assay. The two
assays exhibit a good correlation and minimal difference in detection of the NS1N-effected
nicking of the B19V Ori. We expect this FAM-based fluorescent nicking assay could be utilized
in high-throughput screening of large libraries of small molecule compounds for anti-B19V drug
candidates.
MATERIALS AND METHODS
Compounds.
Compounds used in this study were as follows: #1-5, #7, #9-12, #19, #20, #23, #34 #49-
52, #54, #60, #62, #64, #65, #67-69, #121, #125-130, #132, #134-139, #148-158, #197, #198,
#200-209, #214, #215, #217, #218, #304, #307, #321-329, #338-346, #348-350, #352-357.
Their structures are shown in Table S1. They were acquired commercially or synthesized
(described in the Supplemental Materials) (85-87). Synthesized compounds were purified ≥95%
purity as analyzed by HPLC. All compounds were dissolved in DMSO (Sigma, St. Louis, MO)
and stored at -80°C.
Ethics statement.
Primary human CD34+ hematopoietic stem cells (HSCs) were isolated from bone
marrow of a healthy human donor. We purchased them from AllCells LLC. (www.allcells.com,
Alameda, CA), and obtained B19V-containng plasma samples from ViraCor Eurofins
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Laboratories (Lee’s Summit, MO). Both the cell and virus samples were deidentified, and
therefore institutional review board (IRB) reviews were waived.
Cell line and primary cells.
Primary human CD36+ erythroid progenitor cells (CD36+ EPCs) (49,88): We ex vivo
expanded CD34+ hematopoietic stem cells under normoxic conditions (21% O2; 5% CO2) at
37°C until Day 4 (the day of procurement was designated Day 0). On Day 4, the expanded cells
were frozen in liquid nitrogen as “Day 4 cells”. For each experiment, Day 4 cells were thawed
and cultured for 2 to 3 days in Wong expansion medium under normoxic conditions. On Day 6
or 7, the cells were cultured under hypoxic conditions (1% O2 and 5% CO2) for 2 days; these
cells expressed CD36+ (a marker for erythroid cells), and so we named them CD36+ erythroid
progenitor cells (CD36+ EPCs).
UT7/Epo-S1 cells: UT7/Epo-S1 cells, a human megakaryoblastoid cell line obtained
from Dr. Kevin Brown with permission from Dr. Kazuo Sugamura (89), were cultured under
normoxic conditions in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and erythropoietin (2 U/ml; Amgen, Thousand Oaks, CA).
Virus and infection.
Plasma sample #404 that contains B19V at 1 × 1012 viral genomic copies per ml (vgc/ml)
as quantified by qPCR was provided by ViraCor Eurofins Laboratories (Lee’s Summit, MO).
CD36+ EPCs were infected with B19V at a multiplicity of infection (MOI) of 1,000 vgc per cell.
After 2 days under hypoxia incubation, the infected cells were collected for flow cytometry and
extraction of low-molecular weight (Hirt) DNA (41). In addition, at 18 h, 30 h, and 48 h post-
infection, total DNA was extracted from cells for qPCR.
Electroporation.
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Two million of UT7/Epo-S1 cells were electroporated with 3 μg of Sal I-linearized B19V
infectious clone pM20 (48) in Solution V using Amaxa Nucleofector (Lonza, Basel, Switzerland),
as described previously (29). After electroporation, the cells were cultured under hypoxia (1%
O2 and 5% CO2) for two days.
Southern blot analysis.
Hirt DNA was extracted from either B19V-infected CD36+ EPCs or B19V duplex genome
(M20)-transfected UT7/Epo-S1 cells and was analyzed by Southern blotting as described
previously (41). M20 excised from Sal I-digested pM20 was used as a probe. The blots were
reprobed for mitochondrial (mt) DNA using a specific probe (90)
Protein expression and purification.
NS1 N terminal aa 1-176 (NS1N) and NS1N endonuclease motif mutant (NS1NmEndo
)
coding sequences were ligated into vector pET-30a (GE Health Life Science) through Nde I and
Xho I sites. Both plasmids were transformed into BL21(DE3)pLyss competent cells. Protein
expression was induced by addition of IPTG in the bacterial culture at 1 mM. Protein was
purified with a Ni-NTA affinity agarose (QIAGEN) as described previously (42).
In vitro DNA nicking assays.
Radioactive nicking assay: Oligonucleotide (oligo) was 5’ end-labeled with and [γ-
32P]ATP (PerkinElmer, Inc.) using T4 polynucleotide kinase (NEB). The labelled oligo was
purified using MicroSpin G-50 columns (GE Healthcare). 32P-labeled oligo and NS1N at final
concentrations of 2 nM and 2 μM, respectively, were added in the nicking buffer (50 mM
HEPES-NaOH, pH 7.0, 150 mM NaCl, and 10 mM CoCl2) in a total volume of 20 μL. The
reaction was incubated at 37°C for 16-18 h, and was quenched by the addition of 20 μL of
quenching solution (80% formamide, 50 mM EDTA, 1 mg/mL xylene cyanol FF dye, and 1
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mg/mL bromophenol blue dye), before electrophoresis on denaturing polyacrylamide [20%
acrylamide:bis (acrylamide) (19:1 ratio), 4 M urea, 89 mM Tris base, 89 mM boric acid, and 2
mM EDTA] gels. The gels were autoradiographed without drying against a phosphor screen at
4°C for 12−18 h. Images were obtained by scanning the phosphor screen on a GE Typhoon
FLA 9000 scanner (GE Healthcare) and processed with ImageQuant TL (GE Healthcare)
software for quantification.
Fluorophore-based nicking assay: Fluorescein (FAM) labeled oligos were synthesized
with 6-carboxyfluorescein (6-FAM) at the 5’ end and Iowa Black® FQ quencher at the 3’ end at
Integrated DNA Technologies, Inc. (Coralville, Iowa). The labeled oligo was diluted to 100 μM as
a stock solution. The in vitro nicking reaction was set up at 60 μL, which was composed of
labelled oligo at 200 nM and purified NS1N at 2 μM in the nicking buffer. After incubation for 16-
18 h, the samples were transferred to a black 96-well plate (Corning, cat. #3991). The
fluorescence intensity of each sample was detected with excitation at 492 nm and emission at
518 nm on a microplate reader (Synergy H1 BioTek).
IC50, EC50, and CC50 determinations.
Half maximal inhibitory concentration (IC50): In the fluorophore-based nicking assay,
various concentrations of each inhibitor were used in the in vitro nicking reaction with Ori20
oligo. Fluorescence intensity of reactions with FAMOri20Q but without NS1N were set up as
background. Fluorescence readings of the reactions with each concentration of the compound
were compared with the DMSO (1%) control. The IC50 of each compound was calculated using
GraphPad Prism.
Half maximal effective concentration (EC50) based on viral capsid expression: This
is the concentration at which half of the virus replication is inhibited in cells. For UT7/Epo-S1
cells, the cells were cultured under hypoxic conditions for 2 days, and then the cells were
transfected with B19V duplex genome M20. Transfected cells were diluted with medium to 0.4
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million/ml and transferred 1 ml to 12-well plates, followed by addition of a compound at various
concentrations. After 2 days under hypoxic conditions, the cells were collected for flow
cytometry to detect the capsid-expressing cells. For CD36+ EPCs, compounds were added to
B19V-infected CD36+ EPCs at various concentrations. After 2 days, the infected cells were
collected for flow cytometry to detect the capsid positive cells. The final EC50 was calculated
using GraphPad Prism. DMSO (0.1%) was used as a vehicle control.
EC50 determined based on levels of viral DNA: Day 6 CD36+ EPCs were cultured
under hypoxic condition for 2 days, then the cells were infected with B19V for 1 h and washed
three times with the culture medium. The infected cells were diluted with the medium to 0.4
million/ml, of which cells of 0.1 ml were transferred to wells of a 96-well plate, followed by
addition of compounds at various concentrations. At 18 h, 30 h and 48 h post-infection,
infected cells were collected for extraction of total DNA using the DNeasy Blood&Tissue Kit
(Qiagen) following manufacturer’s instructions. A multiplex qPCR assay using a set of probe
and primers targeting the B19V VP1 unique region-coding sequence and a set of probe and
primers targeting cellular mitochondrial DNA was used to quantify the viral DNA and cellular
DNA, respectively. The final EC50 was calculated using GraphPad Prism with the ratio of B19V
DNA/mt DNA related to the DMSO (0.1%) control group.
Drug concentration that affects viability of 50% of cells in culture (CC50): UT7/Epo-
S1 cells or Day 6 CD36+ EPCs were cultured under hypoxic condition for 2 days, and then the
cells were diluted with medium to 0.4 million/ml and transferred 0.1 ml to 96-well plates.
Compounds were added with different concentrations. After 2 days, the cell viability was
determined using CytoTox-Glo™ Cytotoxicity Assay kit (Promega) followed manufacturer’s
instructions. We used GraphPad Prism to calculate the CC50.
Flow cytometry.
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To determine B19V capsid-expressing cells, infected or transfected cells were collected,
fixed and permeabilized as described previously (29). A mouse anti-B19V capsid monoclonal
antibody (clone 521-5D; MilliporeSigma, cat no.: MAB8292) was used to detect capsid-
expressing cells on a 3-laser flow cytometer (LSR II; BD Biosciences, San Jose, CA). All flow
data were analyzed using FACS DIVA software (BD Biosciences).
Quantification of viral and mt DNA using multiplex qPCR.
The primers and FAM-labelled probe for quantification of B19V DNA are VP1u forward
primer (5’-CCT GGG CAA GTT AGC GTA C-3’; nt 2975-2993), VP1u reverse primer (5’-ATG
AAT CCT TGC AGC ACT GTC A-3’; nt 3082-3061), and the VP1u probe (5’-/6-FAM/CCG GTA
CTA/ZEN/ACT ATG TTG GGC CTG GCA A/3IABkFQ/-3’; nt 3000-3027). The primers and JOE-
labelled probe for quantification of mt DNA are the mt forward primer (5’-TCA AAC TCA AAC
TAC GCC CTG-3’; nt 3673-3693), the mt reverse primer (5’-GTT GTG ATA AGG GTG GAG
AGG-3’; nt 3809-3789), and the mt probe (5’/6-JOEN/TGC GAG CAG/ZEN/TAG CCC AAA CAA
TCT/3IABkFQ/-3’; nt 3704-3727). Multiple PCR was performed as described previously (41,49)
on the 7500 Real-Time Fast PCR System (Applied Biosystems). Nucleotide sequences of B19V
and mitochondrial DNA refer to GenBank accessions AY386330.1 and GU170821.1,
respectively.
Statistical analyses.
GraphPad Prism8 software was used to calculate IC50, EC50 and CC50 using inhibitor vs.
response-variable slope (four parameters).
For the correlation (r value) between data of two methods (the two assays) was
analyzed by Pearson Correlation using SPSS software (IBM Corp, Armonk, NY). When
comparing two methods through SPSS analysis, the coefficient of correlation was p < 0.01,
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which was considered to be significant. The two methods are considered to be highly correlated
as long as r ı 0.8. Scatter plot, trendline and R2 were calculated at the same time.
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ACKNOWLEDGMENTS
We would like to thank members in the Qiu and Tavis labs for technical support and
valuable discussions. We acknowledge the Flow Cytometry Core Laboratory, The University of
Kansas Medical Center, for help in flow cytometry analysis. We also would like to thank Sarah
McNitt and Max Bourdillon for the synthesis of compounds. This study was supported by PHS
grant R01 AI070723 (to JQ), R01 AI104494 (to JET), and R21 AI124672 (to JET&MJM) from
the National Institute of Allergy and Infectious Diseases. The funders had no role in study
design, data collection and interpretation, or the decision to submit the work for publication.
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FIGURE LEGENDS
Fig. 1. Functional domains of B19V NS1 and purification of NS1N and NS1NmEndo proteins.
(A) A schematic diagram of the B19V NS1 protein. The B19V NS1 is depicted with
the Ori-binding (OBD/Endonuclease), Helicase, and Transactivation domains. NS1N has the
NS1 aa 1-176, and NS1NmEndo has alanine substitutions in the endonuclease motif (aa 140-143)
(20). The Walker boxes, NTP-binding sites, and the zinc finger motifs are indicated. The C-
terminal region (shown in yellow) contains a transactivation domain 2 (TAD2) (523SSFFNLITP531
(20). (B&C) Purification of NS1N and NS1NmEndo proteins. One liter of IPTG-induced bacteria
was collected, sonicated, and lysed. The cleared lysate was mixed with ~1 ml NTA beads
(Qiagen) and loaded onto a column. The beads were then washed with Wash buffer, followed
)
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by elution buffer. Each fraction was collected at ~1ml, and 20 μl was loaded for SDS-15%-
PAGE. The gels were stained with Coomassie blue. Dialyzed fraction F2 or F3 was used in the
in vitro nicking assay.
Fig. 2. Purified B19V NS1N specifically cleaves the B19V ssDNA Ori at the terminal
resolution site.
(A) The sequences of Ori30, Ori30mut and a size marker of 19-nt (M19). The core
sequence of the Ori is shown with the STAT5BE, terminal resolution site (trs), and NS1-binding
elements (NS1BEs). The Ori30 probe and the Ori30mut probe that has three mutations (in blue)
at the trs site are shown. The nicked DNA fragments from the Ori30 are shown under the
arrowhead. (B-C) Radioactive in vitro nicking assay. Reactions of in vitro nicking were
analyzed on denaturing polyacrylamide gel, together with labeled probes alone and a labeled
oligo of 19-nt as a size marker of the cleaved band. (B) NS1N cleaved Ori30 but not Ori30mut
.
32P-labeled Ori30 or Ori30mut was incubated with NS1N in the nicking buffer. (C) NS1N, but not
NS1NmEndo, cleaved Ori30. 32P-labeled Ori30 was incubated with NS1N or NS1NmEndo in the
nicking buffer. (D) Determination of a minimal concentration of NS1N used in the nicking
reactions. 2 nM 32P-labeled Ori30 oligo was incubated with NS1N at a final concentration of 0.1,
0.5, 2 and 4 μM, respectively, in the nicking buffer.
Fig. 3. Compound screening for inhibition of cleavage of the 32P-labeled B19V Ori30 oligo.
A total of 96 small molecule compounds at 100 μM (A) or the chosen 25 compounds at
10 μM (B) were incubated with 32P-labeled Ori30 and NS1N in the nicking buffer. The reactions
were analyzed on denaturing polyacrylamide gel. Relative nicking efficiencies are shown. The
ratio of the signals at 19-nt vs. 19-nt plus 30-nt in the DMSO vehicle control group is set up 1. (A)
Compounds inhibited all nicking activity are shown in blank as no obvious nicked bands at 19-nt
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were detected. (B) Autoradiography gel images for the 25 compounds assessed at 10 μM are
shown.
Fig. 4. Establishment of a 6-carboxyfluorescein (FAM)-based in vitro nicking assay.
(A) A diagram of the FAM-labeled oligos. Sequences of the Ori20 is shown with FAM
and Iowa Black® FQ quencher (Q) at 5’ and 3’ ends, respectively. After incubation with NS1N,
Ori20 is cleaved into two shorter oligos, and then FAM-linked short oligo of 9-nt is released for
fluorescence detection. (B) FAMOri20Q-based nicking assay. 200 nM FAMOri20Q were incubated
with 2 μM NS1N protein in the nicking buffer. The fluorescence intensity of each sample was
detected on a microplate reader. FAMOri20Q without NS1N, and FAMOri20 without a quencher
were used as controls. (C) Optimization of the probe concentration. Various concentrations
of the FAMOri20Q probe were used in the nicking assay. Fluorescence intensity was determined
with or without NS1N as indicated. The fold changes in the presence of NS1N compared with no
NS1N are shown.
Fig. 5. Screening of compounds for inhibition of cleavage of the B19V Ori by NS1N using
FAM-labeled B19V Ori20.
(A) FAM-based in vitro nicking assay. Each of the 71 compounds were incubated with
NS1N at 10 μM in the nicking buffer at 37°C for 16-18 h. The fluorescence intensity of each
sample was detected on a microplate reader. Ori20 without NS1N was set up as background,
and Ori20 with NS1N and DMSO was set up as positive control. We set up an inhibition of 80%
of the relative fluorescence intensity as a cutoff value for the inhibition of NS1N nicking. (B)
Comparison of the 25 chosen compounds between the radioactive and fluorescent
nicking assays. At the final concentration of 10 μM, 8 compounds showed inhibition of >80% in
the NS1N nicking of the 32P-labeled Ori30 (in red), and 8 compounds showed positive in the
NS1N nicking of the FAM-labeled Ori20 (in green). The correlation coefficient (r) between the
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radioactive and fluorescent nicking assays was calculated using SPSS software. (C) Scatter
plot and trendline of the radioactive nicking assay with the fluorescent nicking assay.
Scatter plot, trendline and R2 of the two assays were calculated using SPSS software.
Fig. 6. Chemo structures of the eight screened compounds.
(A) Flavonoids. (B) Naphthyridinones.
Fig. 7. IC50 determination of compounds #7, #135 and #201.
In the FAM-based nicking assay, various concentrations of the compound were added to
the nicking assay with FAMOri20Q and NS1N protein. The fluorescence intensity of each reaction
was detected. FAMOri20Q without NS1N was set up as background. Each concentration was
compared with DMSO. The IC50 of each inhibitor was calculated using GraphPad Prism.
Fig. 8. EC50 and CC50 determination of compounds #7, #135 and #201.
(A) EC50 determination. UT7/Epo-S1 cells were transfected with B19V duplex genome
M20. Each inhibitor was added with different concentrations as labeled. After 2 days, the cells
were collected for flow cytometry to detect the capsid-expressing cells. DMSO was used as a
vehicle control. The final EC50 was calculated with GraphPad Prism. (B) CC50 determination.
Compounds, at various concentration as shown, were added to UT7/Epo-S1 cells in 96-well
plates. After 2 days, the percentage of viable cells were determined using CytoTox-Glo™
Cytotoxicity Assay kit (Promega), and the CC50 was calculated using GraphPad Prism.
Fig. 9. Compounds #7, #135 and #201 inhibit B19V DNA replication in UT7/Epo-S1 cells.
UT7/Epo-S1 cells were electroporated with B19V duplex genome M20, followed by
addition of the compound #7 (A), compound #135 (B), or compound #201 (C) at various
concentrations as shown. At two days post-transfection, Hirt DNA samples prepared from
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treated cells were digested with Dpn I and subjected to Southern blotting with the M20 probe.
DMSO was used a vehicle control. UT7/Epo-S1 cells without transfection were set up as Mock.
pM20 digested with Sal I was loaded as size marker. Mitochondrial (mt) DNA was detected as
loading control using a specific mt DNA probe (91). *mRF DNA, monomer replicative form DNA,
and **ssDNA, single-strand DNA.
Fig. 10. EC50 and CC50 determination of compound #7, compound #135 and compound
#201.
(A) EC50 determination. CD36+ EPCs were infected with B19V. Each compound was
added with different concentrations as shown. At 2 days post-infection, the cells were collected
for flow cytometry to detect the capsid-expressing cells. DMSO was used as a vehicle control.
The final EC50 was calculated with GraphPad Prism. (B) CC50 determination. EPCs in 96-well
plates were treated with each compound at various concentrations as shown. At 2 days post-
infection, percentages of viable cells were determined, and the CC50 was calculated using
GraphPad Prism.
Fig. 11. Compounds #7, #135 and #201 inhibit B19V replication in CD36+ EPCs.
CD36+ EPCs were infected with B19V followed by addition of each compound #7 (A),
compound #135 (B) and compound #201(C) at various concentrations as shown. At two days
post-infection, Hirt DNA samples were prepared from infected cells, and were subjected to
Southern blotting using the M20 probe. DMSO was used a vehicle control. CD36+ EPCs without
infection were set up as Mock. pM20 digest with Sal I was loaded as size marker. Mitochondrial
(mt) DNA was detected as loading control using a specific mt DNA probe (91). *mRF DNA,
monomer replicative form DNA, and **ssDNA, single-strand DNA.
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