viruses

Article
Nectin-2 Acts as a Viral Entry Mediated Molecule That Binds to
Human Herpesvirus 6B Glycoprotein B
Hirohito Ogawa 1, * , Daisuke Fujikura 2 , Hikaru Namba 1 , Nobuko Yamashita 1 , Tomoyuki Honda 1
and Masao Yamada 1, *

1 Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan; [email protected] (H.N.);
[email protected] (N.Y.); [email protected] (T.H.)
2 School of Veterinary Medicine, Kitasato University, Higashi 23-35-1, Towada 034-8628, Japan;
[email protected]
* Correspondence: [email protected] (H.O.); [email protected] (M.Y.)

Abstract: Human herpesvirus 6B (HHV-6B) is a T-lymphotropic virus and the etiological agent of
exanthem subitum. HHV-6B is present in a latent or persistent form after primary infection and is
produced in the salivary glands or transmitted to this organ. Infected individuals continue to secrete
the virus in their saliva, which is thus considered a source for virus transmission. HHV-6B primarily
propagates in T cells because its entry receptor, CD134, is mainly expressed by activated T cells. The
virus then spreads to the host’s organs, including the salivary glands, nervous system, and liver.
However, CD134 expression is not detected in these organs. Therefore, HHV-6B may be entering
cells via a currently unidentified cell surface molecule, but the mechanisms for this have not yet
been investigated. In this study, we investigated a CD134-independent virus entry mechanism in the
parotid-derived cell line HSY. First, we confirmed viral infection in CD134-membrane unanchored



HSY cells. We then determined that nectin cell adhesion molecule 2 (nectin-2) mediated virus entry

 and that HHV-6B-insensitive T-cells transduced with nectin-2 were transformed into virus-permissive
Citation: Ogawa, H.; Fujikura, D.; cells. We also found that virus entry was significantly reduced in nectin-2 knockout parotid-derived
Namba, H.; Yamashita, N.; Honda, T.; cells. Furthermore, we showed that HHV-6B glycoprotein B (gB) interacted with the nectin-2 V-set
Yamada, M. Nectin-2 Acts as a Viral
domain. The results suggest that nectin-2 acts as an HHV-6B entry-mediated protein.
Entry Mediated Molecule That Binds
to Human Herpesvirus 6B
Keywords: HHV-6B; nectin-2; CD112; CD134; virus entry; glycoprotein B
Glycoprotein B. Viruses 2022, 14, 160.
https://doi.org/10.3390/v14010160

Academic Editor: Craig McCormick

1. Introduction
Received: 11 December 2021
Accepted: 13 January 2022 Human herpesvirus 6 (HHV-6) is classified into two distinct species, HHV-6A and
Published: 16 January 2022 HHV-6B [1–4], and human herpesvirus 7 (HHV-7) [5]. It belongs to the genus Roseolovirus in
the betaherpesvirus subfamily, which is a group of T-lymphotropic herpesviruses and the
Publisher’s Note: MDPI stays neutral
causal agents of exanthem subitum (ES), also known as roseola infantum. Roseoloviruses
with regard to jurisdictional claims in
cause life-long infections, as older children and adults continue to intermittently shed the
published maps and institutional affil-
iations.
virus and virus DNA in their saliva [6].
Human CD134 (also known as OX40) has been identified as an entry receptor for
HHV-6B [7]. This molecule is a member of the tumor necrosis factor receptor superfamily
4 (TNFRSF4) and is induced on CD4+ and CD8+ T cells [8]. The use of CD134 as an
Copyright: © 2022 by the authors. entry receptor is consistent with evidence that HHV-6B is a T-lymphotropic virus that
Licensee MDPI, Basel, Switzerland. propagates in primary activated T cells [9]. However, this virus was detected in the
This article is an open access article saliva, salivary glands [2,10,11], and livers of pediatric patients [12,13]. Furthermore,
distributed under the terms and HHV-6B was also detected in brain autopsy tissues [14], and encephalopathy with ES
conditions of the Creative Commons has also been reported [15]. Notably, the expression of CD134 was not detected in the
Attribution (CC BY) license (https:// salivary glands, liver, or brain samples available from the Human Protein Atlas (http:
creativecommons.org/licenses/by/ //www.proteinatlas.org) [16]. The tropism of HHV-6B for non-T cells that do not express
4.0/).

Viruses 2022, 14, 160. https://doi.org/10.3390/v14010160 https://www.mdpi.com/journal/viruses

Viruses 2022, 14, 160 2 of 19

CD134 might be analogous to the multiple receptor usage of various herpesviruses that
target cells from different lineages [17,18].
During the entry of herpes simplex virus (HSV) type 1 (HSV-1), glycoprotein (g) D
(gD) can interact with any of three classes of receptors: nectins [19,20], a tumor necrosis
factor family member designated as herpesvirus entry mediator (HVEM) [21], or 3-O-
sulfated heparan sulfate [22]. gB is required for infection, especially for efficient mem-
brane fusion during virus entry. HSV-1 gB can interact with three distinct receptors:
paired immunoglobulin-like type 2 receptor α (PILRα) [23], myelin-associated glycoprotein
(MAG) [24], and non-muscle myosin II (NM-II) [17,25]. gD is a conserved glycoprotein
among several alphaherpesviruses [26]. In general, there are three glycoproteins (gB, gH,
and gL) which are thought to be essential for the entry of all herpesviruses [18]. For
HHV-6A and HHV-6B, eight virion enveloped glycoproteins (gH, gL, gM, gN, gB, gO, gQ1,
and gQ2) have been described [27–30]. The gH/gL/gQ1/gQ2 complex is a viral ligand
for two HHV-6A- and HHV-6B-specific receptors, CD46 [31] and CD134 [7], respectively.
Another trimetric complex (gH/gL/gO) [32] and gB are also candidate virus entry factors;
however, cellular receptors that bind to these glycoproteins have not yet been discovered.
One hypothesis is that a novel cellular receptor may mediate HHV-6B entry into cells.
The frequent detection of HHV-6 DNA, especially HHV-6B DNA in saliva and salivary
glands [2,11,33], suggests that the salivary glands are a potential site of HHV-6 persis-
tence [34]. Therefore, it is important to elucidate how HHV-6B infects salivary glands that
do not express CD134. To address this, we identified an unknown cell surface molecule
using a full-length cDNA library prepared from the parotid gland cell line, HSY [35]. We
found that nectin cell adhesion molecule 2 (nectin-2) [36], previously named HveB and also
known as CD112, mediated the entry of HHV-6B. gB acted as a ligand for nectin-2, and
there was a binding region for entry in the V-set domain. Our results suggest that nectin-2
acts as an HHV-6B entry-mediated molecule.

2. Materials and Methods

2.1. Cells and Virus Strains
The T-cell lines MT-4, Sup-T1, and CCRF-HSB-2 and their transfectants were cultured
in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 100 U/mL
penicillin, 100 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal
bovine serum (FBS). The parotid gland cell line (HSY and its transfectants) [35], 293T cells,
and Platinum-GP retroviral packaging (Plat-GP) cells used for the production of retrovirus
vectors (Cell Biolabs, San Diego, CA, USA) were all cultured in Dulbecco’s modified
Eagle’s minimal essential medium (DMEM) supplemented with 100 U/mL penicillin,
100 mg/mL streptomycin (Sigma-Aldrich), and 10% FBS. HHV-6B strains HST and Z29
were propagated on cord blood mononuclear cells, and virus stocks were prepared as
described previously [37].

2.2. Reverse Transcription-PCR (RT-PCR) and Quantitative RT-PCR

Total RNA was extracted using a NucleoSpin RNA Plus kit (Macherey-Nagel, Düren,
Germany) according to the manufacturer’s instructions. One-step RT-PCR was carried out
using a Qiagen OneStep RT-PCR kit (Qiagen, Hilden, Germany) according to the manufac-
turer’s instructions. HHV-6B U91 and U100 genes were amplified using the following primer
sets: 50 -GGCGCTGAAGCATGTAAGCA-30 and 50 -CAGAACAGTCAGGTATTTTCCCC-
30 for U91 [38] and HHV6B-U100-mRNA-F (50 -TGCACCATGATCGTCCTACG-30 ) and
HHV6B-U100-mRNA-R (50 -TCGCATTCCGATGGTTTCCA-30 ) for U100, which were de-
signed in this study. The β-actin gene (ACTB) was amplified using hACTB-mRNA71F (50 -
GCCGCCAGCTCACCAT-30 ) and hACTB-mRNA299R (50 -TCGATGGGGTACTTCAGGGT-
30 ), which were designed in this study.
For the quantitative RT-PCR of NECTIN2, total RNA was subjected to cDNA syn-
thesis using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan),
and 10 ng cDNA was amplified using a TB Green Premix Ex Taq II kit (Takara Bio) and
Viruses 2022, 14, 160 3 of 19

an Applied Biosystems StepOnePlus System (Thermo Fischer Scientific, Waltham, MA,

USA), all according to the manufacturer’s instructions. NECTIN2 isoforms were amplified
using the following primer sets: NEC2-alpha-qF (50 -TCTACGATCCGAAAGCTCAGGTGT-
30 ) and NEC2-alpha-qR (50 -CATCCTTGCCATCTGGTTCCATGG-30 ) for NECTIN2 alpha
and NEC2-delta-qF (50 -TCGGAGCACAGCCCACTCAAGAC-30 ) and NEC2-delta-qR (50 -
GTGGGCAGCTCATGGTATCGAGG-30 ) for NECTIN2 delta, which were designed in
this study.

2.3. Antibodies
APC anti-human CD134 monoclonal antibody (mAb) (clone: Ber-ACT35), APC mouse
immunoglobulin (Ig) G 1, κ isotype control mAb (clone: MOPC-21), PE anti-human CD112
(Nectin-2) mAb (clone: TX31), and PE mouse IgG1, κ isotype control mAb (clone: MOPC-21)
were purchased from BioLegend (San Diego, CA, USA). Anti-HHV-6A gQ1 mAb (clone:
119) was purchased from Cosmo Bio (Tokyo, Japan). Anti-nectin-2 mAb (clone: E-1),
anti-HHV-6 gB (clone: 6A5), and anti-HHV-6 p41 early antigen mAb (clone: 9A5D12)
were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse IgG 2B isotype
control mAb (clone: 20116) were purchased from Bio-techne (Minneapolis, MN, USA). Anti-
DDDDK-tag mAb (clone: FLA-1) and anti-hemagglutinin (HA)-tag mAb (clone: TANA2)
were purchased from MBL (Nagoya, Japan). Anti-β-tubulin mAb was purchased from
Fujifilm Wako (Osaka, Japan). Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L)
F(ab’)2 (A-11017) and Alexa Fluor 555 conjugated goat anti-mouse IgG (H+L) F(ab’)2
(A-21425) were purchased from Thermo Fisher Scientific. Horseradish peroxidase (HRP)-
conjugated goat anti-mouse IgG (H+L) was purchased from Jackson ImmunoResearch (115-
035-062; West Grove, PA, USA). HRP-conjugated anti-mouse IgG for immunoprecipitation
(IP) was purchased from Abcam (ab131368; Cambridge, UK).

2.4. Flow Cytometric Analysis

Cells were washed with cold fluorescence-activated cell sorting (FACS) buffer (phosphate-
buffered saline (PBS) containing 1% sodium azide and 2% FBS) and incubated with the
indicated mAbs at 4 ◦ C for 30 min, after blocking with purified mouse IgG (BioLegend,
clone MG1-45, 0.5 µg/mL, 4 ◦ C, 15 min). After several washes, the cells were stained with
7-AAD Viability Staining Solution (BioLegend) and analyzed using a BD FACSAria III Cell
Sorter (BD, Franklin Lakes, NJ, USA) and FlowJo software version 10 (BD).

2.5. Immunofluorescence Assay (IFA)

HSY cells and ex6 C7 cells were grown on poly L-lysine-coated micro slide glasses
(Matsunami Glass, Osaka, Japan). Cells were infected with HHV-6B and cultured for
2–4 days. T-cell lines (MT-4, CCRF-HSB-2, and its transformants) were infected with the
virus and cultured for 1–7 days in 24-well plates (AS ONE, Osaka, Japan). After centrifu-
gation, the cells were transferred to 14-well ring marked micro slide glasses (Matsunami
Glass) and air-dried. After fixation with cold acetone for 5 min, the cells were incubated
with primary mAb for 25 min at 35 ◦ C. After washing, the bound antibodies were detected
with secondary fluorescent dye-conjugated antibodies for 25 min at 35 ◦ C. After several
washes, cover slides were mounted using ProLong Diamond Antifade Mountant with DAPI
(Thermo Fisher Scientific). Fluorescent images were acquired using a BZ-X700 microscope
(Keyence, Osaka, Japan).

2.6. Establishment of a CD134-Membrane Unanchored HSY Cell Clone (Ex6 C7)

The transmembrane region of TNFRSF4 was mutated using the CRISPR/Cas9 system,
as described previously [39]. The 23-nucleotide guide sequence (50 -CCGTGCGGTTGCCGC
CATCCTGG-30 ) targeting the negative strand genomic DNA located on exon-6 of the
human TNFRSF4 gene was designed using a CRISPR direct software uploaded to http:
//crispr.dbcls.jp. The DNA fragment was cloned into pSpCas9(BB)-2A-Puro (PX459)
ver.2 [40]. The resulting plasmid was transfected into HSY cells using Lipofectamine 2000
Viruses 2022, 14, 160 4 of 19

(Thermo Fisher Scientific). Two days after puromycin selection at 0.125 µg/mL, single-cell
clones were isolated using a limiting dilution. Several cell clones were selected, and their
genomic DNA was sequenced to confirm gene mutations. The transmembrane helices were
predicted using TMHMM2.0 [41]. One clone (ex6 C7 cells) was established.

2.7. Establishment of Nectin-2 Knockout Ex6 C7 (Ex6 C7 Nec2-KO) Cell Clones

The NECTIN2 gene was mutated using the CRISPR/Cas9 system, as described above.
Briefly, the 23-nucleotide guide sequence (50 -CCTGCCGTCGAGATCGCCGCCGA-30 ) tar-
geting the negative strand genomic DNA of the human NECTIN2 gene was designed and
cloned into a plasmid vector. The resulting plasmid was transfected into ex6 C7 cells, and
the obtained cell clones were sequenced to confirm the gene mutations (Figure S1). Three
clones (ex6 C7 nec2-KO1, -KO2, and -KO3) were established.

2.8. Expression Screening

A full-length cDNA library was constructed from ex6 C7 cells using the In-Fusion
SMARTer Directional cDNA Library construction kit (Takara Bio) according to the manu-
facturer’s instructions and cloned into a Molony murine leukemia virus (MMLV)-based
retroviral vector, pMX (Cell Biolabs), as described previously [39,42]. The titer of the cDNA
library was more than 1 × 106 colony-forming units, and more than 80% of the individual
clones contained different cDNA fragments. Plat-GP cells were co-transfected with pMX
containing cDNA library genes and a VSV G-expressing plasmid, pCMV-VSV-G (Cell
Biolabs), using Lipofectamine 2000. Two days later, culture supernatants were collected and
cleared of cell debris by centrifugation at 300× g for 5 min and then passed through 0.45 µm
pore filters (Merck Millipore, Darmstadt, Germany). CCRF-HSB-2 cells (5 × 105 cells) were
infected with viruses at a multiplicity of infection of 0.1 with 0.8 µg/mL polybrene solution
(Nacalai Tesque, Kyoto, Japan). Cells (lib-CCRF-HSB-2 cells) were expanded for several
passages and stored at −80 ◦ C. The lib-CCRF-HSB-2 cells (1.2 × 105 cells) were infected
with HHV-6B and cultured for several passages to generate HHV-6B persistently infected
lib-CCRF-HSB-2 cells. The cells were incubated with anti-HHV-6A gQ1 mAb for 30 min at
4 ◦ C. After two washes with cold PBS, the cells were stained with anti-mouse IgG-Alexa
Fluor 488 and APC anti-human CD134 for 30 min at 4 ◦ C. After two washes, the cells
were separated on a BD FACSAria III by dot plots gating on Alexa Flour 488 (gQ1) versus
APC (CD134). Both Alexa Fluor 488-positive and APC-negative cells were considered
HHV-6B-infected cells (non CD134-dependent) and sorted. Then, the sorted cells were
expanded. After several rounds of sorting and culturing, genomic DNA was extracted
using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions, and
library genes inserted into the genomic DNA were amplified by PCR using pMXs-specific
primers and then sequenced.

2.9. Generation of Nectin-2- and CD134-Expressing Cell Lines

Total RNA was extracted from HSY cells using the NucleoSpin RNA Plus kit (Macherey-
Nagel), and mRNA was reverse transcribed using a PrimeScript II 1st strand cDNA Synthe-
sis Kit (Takara Bio), all according to the manufacturer’s instructions. The NECTIN2 (isoform
δ) and TNFRSF4 genes were amplified from cDNA with KOD One (Toyobo, Osaka, Japan)
and inserted into pMXs-IRES-GFP (Cell Biolabs). After sequence confirmation, the plas-
mids were retrovirally transfected into CCRF-HSB-2 cells as described above. Transduced
GFP-positive cells were collected using a BD FACSAria III Cell Sorter (BD Biosciences) and
then used in the experiments (HSB2-nec2δ, HSB2-CD134, and HSB2-cont cells).

2.10. Generation of Nectin-2- and CD134-Expressing Cell Lines

Plasmids encoding FLAG-tagged nectin-2 (pCAGGS-nec2δ-FLAG) were prepared.
Briefly, the NECTIN2-δ gene was amplified from cDNA as prepared above using KOD
One (Toyobo) and inserted into pCAGGS [43] with in vivo Escherichia coli cloning (iVEC)
technology [44]. After sequence confirmation, the plasmids of the mutant nectin-2 were
Viruses 2022, 14, 160 5 of 19

constructed using a PrimeSTAR Mutagenesis Basal Kit (Takara Bio) and ECOS Competent
E. coli DH5α (Nippon Gene, Tokyo, Japan), all according to the manufacturer’s instruc-
tions. The resulting plasmids were designated pCAGGS-nec2δ-∆V-FLAG (mutant lacking
nectin-2δ V-set domain), pCAGGS-nec2δ-∆C2×2-FLAG (mutant lacking nectin-2δ C2-set
domains), pCAGGS-nec2δ-∆C2(55aa)-FLAG (mutant lacking nectin-2δ first C2-set domain),
and pCAGGS-nec2δ-∆C2(46aa)-FLAG (mutant lacking nectin-2δ second C2-set domain).
Plasmids encoding HA-tagged HHV-6B gB (pCAGGS-HST gB-HA and pCAGGS-Z29
gB-HA) were prepared as follows: Total RNA was extracted from HHV-6B HST and Z29
strains using the NucleoSpin RNA Plus kit (Macherey-Nagel) and mRNA was reverse
transcribed using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio), all according
to the manufacturer’s instructions. The gB genes were amplified from cDNA using KOD
One (Toyobo) and inserted into pCAGGS using an In-Fusion HD Cloning Kit (Takara Bio)
according to the manufacturer’s instructions. The plasmids were transformed into MAX
Efficiency Stbl2 competent cells (Thermo Fischer Scientific), and the transformants were
cultured at 30 ◦ C. After sequence confirmation, the mutant HST gB-HA plasmid lacking a
transmembrane domain was constructed using a PrimeSTAR Mutagenesis Basal Kit (Takara
Bio) and MAX Efficiency Stbl2 competent cells (Thermo Fischer Scientific), all according to
the manufacturer’s instructions.
A plasmid encoding HA-tagged HST gQ1 (pCAGGS-HST gQ1-HA) was prepared as
described above. Empty vector control plasmids (pCAGGS-FLAG and pCAGGS-HA) were
also constructed as controls.

2.11. Transfection, Immunoblotting, and Co-IP

The 293T cells were grown in 6-well plates 24 h before transfection at 37 ◦ C and 5%
CO2 . The plasmids were transfected into cells using polyethylenimine “Max” (1 mg/mL)
(Polysciences, Warrington, PA, USA). At 72 h post-transfection, cells were washed four
times with cold PBS, and then treated with 0.25 mL of cold lysis buffer (20 mM Tris-HCl (pH
7.4), 135 mM NaCl, 1% Triton X-100, 1% glycerol) containing a protease inhibitor cocktail
(25955-11; Nacalai Tesque) for 15 min at 4 ◦ C. Cell lysates were sonicated at 4 ◦ C for 10 s and
centrifuged at 21,000× g for 10 min at 4 ◦ C. Aliquots of the supernatants were collected and
stored at −80 ◦ C until the immunoblotting. The remaining supernatants were mixed with
anti-DDDDK-tag mAb-magnetic agarose (M185-10; MBL) or anti-HA-tag mAb-magnetic
agarose (M132-10; MBL) and incubated on a rotator at 4 ◦ C overnight. After washing four
times with cold lysis buffer, the beads were boiled with 30 µL of sodium dodecyl sulfate
(SDS) sample buffer and subjected to 10% SDS-polyacrylamide gel electrophoresis. The
proteins were transferred to polyvinylidene fluoride membranes (Immobilon-P; Merck
Millipore), which were blocked with 3% skim milk in PBS containing 0.05% Tween 20 for
1 h at room temperature, followed by incubation with the appropriate primary antibodies
for 1 h at room temperature. After washing, the bound antibodies were detected using HRP-
conjugated secondary antibodies. The bound antibodies were visualized with Immobilon
Western (Merck Millipore) and detected using a C-DiGit Blot Scanner (LI-COR Biosciences,
Lincoln, NE, USA). The experiment was performed at least thrice, and representative data
are shown.

2.12. Nectin-2 Binding Assay in HHV-6B-Infected Cells

MT-4 cells were infected with the virus (multiplicity of infection = 0.1) and cultured
for 6 d. Cells were washed with cold PBS and then treated with cold lysis buffer (0.5 mL)
containing a protease inhibitor cocktail (Nacalai Tesque) for 15 min at 4 ◦ C. Cell lysates were
sonicated at 4 ◦ C for 10 s and centrifuged at 21,000× g for 10 min at 4 ◦ C. The supernatants
were incubated on a rotator at 4 ◦ C for 4 h and mixed with anti-HHV-6 gB antibody (6A5)
binding Dynabeads Protein A (Thermo Fisher Scientific) on a rotator at 4 ◦ C overnight.
After washing four times with cold lysis buffer, the beads were boiled with 30 µL of SDS
sample buffer and subjected to immunoblotting. The experiment was performed at least
thrice, and representative data are shown.
Viruses 2022, 14, 160 6 of 19

2.13. Assay of Nectin-2 Binding to Purified HHV-6B gB

A plasmid expressing soluble HST gB-HA protein was transfected into 293T cells
grown in 6-well plates, as described above. At 72 h post-transfection, the collected soluble
gB-HA proteins were mixed with anti-HA-tag mAb-magnetic agarose (MBL) and incubated
on a rotator at 4 ◦ C for 1 h. The proteins were purified by washing four times with cold lysis
buffer and suspended in 0.4 mL of cold lysis buffer. The purified gB-HA binding magnetic
agarose was incubated with 0.4 µg of human CD112 Fc chimera recombinant protein
(A42495; Thermo Fisher Scientific) or recombinant human IgG1 Fc (778304; BioLegend)
on a rotator at 4 ◦ C for 1 h. After washing four times with cold lysis buffer, the magnetic
agaroses were boiled with 30 µL of SDS sample buffer and subjected to immunoblotting.
The experiment was performed twice, and representative data are shown.

2.14. Statistical Analysis

Statistical analyses were performed using GraphPad PRISM software version 8 (Graph-
Pad Software, San Diego, CA, USA).

3. Results
3.1. HHV-6B Infects the HSY Parotid Gland Cell Line
To determine whether HHV-6B infects salivary gland derivative cells, we infected
HSY cells with an HHV-6B HST strain and detected the viral RNA using RT-PCR. U91
(immediate early) and U100 (late) mRNAs were detected in the HSY cells (Figure 1A). The
viral antigen was also detected in HSY cells by IFA using an anti-HHV-6 p41 early antigen
antibody (Figure 1B). We then compared the expression levels of the CD134 in the HSY
and T-lymphocyte cell lines, which have a different HHV-6B-permissive range, using FACS
analysis. The CD134 was highly and slightly expressed in the HHV-6B-permissive T-cell
line (MT-4) and HHV-6B-poorly permissive T-cell line (Sup-T1), respectively (Figure 1C).
CD134 was not detected in the HHV-6B-insensitive T-cell line (CCRF-HSB-2). CD134 was
detected only slightly in the HSY cells (Figure 1C).

3.2. HHV-6B Infects Ex6 C7 Cells (CD134-Membrane Unanchored HSY Cells)

To determine the requirement of CD134 for viral entry into the HSY cells, a TNFRSF4
gene-specific mutation was performed using the CRISPR-Cas9 system [40]. Because the
TNFRSF4 gene has several transcript variants, it was difficult to establish a complete CD134
knockout cell clone. Instead, the transmembrane region of the TNFRSF4 gene was deleted
so that CD134 was expressed in the HSY cells without its transmembrane anchor sequence.
Examination of the resulting amino acid sequence showed that a native transmembrane
segment had been lost and replaced with amino acids that were not predicted to form a
hydrophobic transmembrane anchor (Figure 2A). As expected, we obtained ex6 C7 cells
lacking CD134 expression on their surfaces (Figure 2B). The mutant CD134 did not appear
to be present inside the cells (Figure S2). Interestingly, we detected U91 and U100 mRNA
by RT-PCR as well as p41 early antigen by IFA in the HST strain-inoculated ex6 C7 cells,
although CD134 was not detected on the cell surface (Figure 2C,D). These data suggest that
HHV-6B enters ex6 C7 cells using an unknown cell surface molecule that is not CD134.

3.3. Nectin-2 Is Required for HHV-6B Infection

To identify the potential receptor molecules required for HHV-6B entry, we constructed
a cDNA library obtained from ex6 C7 cells in a retroviral vector, pMX, and retrovirally
transfected the cDNA library into an HHV-6B insensitive T-cell line, CCRF-HSB-2 (lib-
CCRF-HSB-2). We inoculated HHV-6B into lib-CCRF-HSB-2 cells, and then isolated the
transfectants detected as gQ1-positive via cell sorting. After several rounds, a NECTIN2
fragment was identified. Nectin-2 is a Ca2+ -independent Ig-like cell adhesion molecule
(CAM) belonging to the Ig superfamily and is ubiquitously expressed in a variety of
cells [36,45]. Nectin-2 is an entry receptor for alphaherpesviruses, HSV-1 mutant strains,
HSV-2, and pseudorabies virus (PRV) and interacts with gD [20,46,47].
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Figure 1.
Figure 1. HHV-6B
HHV-6B infectivity
infectivity in
in HSY
HSY cells
cells and
and relative
relative CD134
CD134 expression.
expression. (A)
(A) Viral
Viral RNA
RNA detection
detection
in HSY cells using RT-PCR.
in RT-PCR. Cells
Cells were
wereinfected
infectedwith
withHHV-6B
HHV-6BHST HSTandandtotal
totalRNA
RNAwas wasextracted
extractedat
4848
at h post-infection.
h post-infection.U91U91
(immediate
(immediateearly) and U100
early) (late) (late)
and U100 transcripts were detected.
transcripts β-actin β-actin
were detected. (ACTB)
was used
(ACTB) wasasused
an internal control.control.
as an internal M, DNA M, ladder; -, mock-infected
DNA ladder; HSY cells;
-, mock-infected +, HST-infected
HSY cells; +, HST-infectedHSY
cells; Temp-, No template control; N.C., negative control (HST-infected CCRF-HSB-2 cells); P.C.,
HSY cells; Temp-, No template control; N.C., negative control (HST-infected CCRF-HSB-2 cells);
positive control (HST-infected MT-4 cells). (B) Viral antigen detection in HSY cells using an immu-
P.C., positive control (HST-infected MT-4 cells). (B) Viral antigen detection in HSY cells using an
nofluorescence assay. Cells were infected with HHV-6B HST and treated with cold acetone at 48 h
immunofluorescence assay.HHV-6
post-infection (left panel). Cells were infected(red)
p41 protein withwas
HHV-6B HST Nuclei
detected. and treated with
of cells cold
were acetone
stained at
with
48 h post-infection (left panel). HHV-6 p41 protein (red) was detected. Nuclei of cells
DAPI (blue). Scale bars represent 50 μm. Virus-infected cells (red) were counted (right panel). Error were stained
with
bars DAPI (blue).
represent Scale bars
standard represent
deviations (n =50 Virus-infected
µm.cell).
3 per cells (red)
Asterisks indicate were counted
statistical (right(****
significance panel).
p<
0.0001,
Error Bonferroni’s
bars representmultiple
standardcomparison
deviations test).
(n = 3(C) CD134
per cell). expression on T-lymphocyte
Asterisks indicate statistical cell lines and
significance
HSYpcells
(**** was analyzed
< 0.0001, using
Bonferroni’s fluorescence-activated
multiple comparison test). cell sorting.
(C) CD134Cells were stained
expression with APC-con-
on T-lymphocyte cell
jugated anti-CD134 or isotype control antibodies. The antibody specific signals
lines and HSY cells was analyzed using fluorescence-activated cell sorting. Cells were stained of the cells are shown
with
in the histogram.anti-CD134 or isotype control antibodies. The antibody specific signals of the cells
APC-conjugated
are shown in the histogram.
3.2. HHV-6B Infects Ex6 C7 Cells (CD134-Membrane Unanchored HSY Cells)
To
Toassess the expression
determine level of
the requirement ofnectin-2,
CD134 for weviral
performed FACS
entry into the analyses
HSY cells,inaHSY cells,
TNFRSF4
which demonstrated that they had a greater intensity of nectin-2 staining
gene-specific mutation was performed using the CRISPR-Cas9 system [40]. Because the than other T-cell
lines (Figure
TNFRSF4 3A).has
gene This molecule
several has twovariants,
transcript splicing it variants, nectin-2α
was difficult (short form)
to establish and -2δ
a complete
(long form), which share an ectodomain and differ in the transmembrane
CD134 knockout cell clone. Instead, the transmembrane region of the TNFRSF4 gene was and cytoplasmic
regions
deleted [36]. Nectin-2δ,
so that CD134 was butexpressed
not nectin-2α,
in theisHSY
tyrosine
cellsphosphorylated in response anchor
without its transmembrane to cell-
to-cell adhesion
sequence. [48]. Toofconfirm
Examination whichamino
the resulting isoform was
acid dominant
sequence in HSY
showed cells,
that total trans-
a native RNA
was extracted from the cells, and nectin-2α and-2δ mRNA levels were
membrane segment had been lost and replaced with amino acids that were not predicted determined using
quantitative RT-PCR. As
to form a hydrophobic shown in Figure
transmembrane 3B, larger
anchor (Figureamounts of nectin-2δ
2A). As expected, wemRNA
obtained were
ex6
detected than nectin-2α mRNA, indicating that nectin-2δ was the dominant
C7 cells lacking CD134 expression on their surfaces (Figure 2B). The mutant CD134 did type. Therefore,
the
not subsequent
appear to be results areinside
present mainlytherelated
cells to nectin-2δ.
(Figure S2). Interestingly, we detected U91 and
U100 mRNA by RT-PCR as well as p41 early antigen by IFA in the HST strain-inoculated
ex6 C7 cells, although CD134 was not detected on the cell surface (Figure 2C,D). These
Viruses 2022, 13, x FOR PEER REVIEW 8 of 21

Viruses 2022, 14, 160 data suggest that HHV-6B enters ex6 C7 cells using an unknown cell surface molecule8that
of 19
is not CD134.

Figure 2.
Figure Establishmentof
2. Establishment TNFRSF4gene-specific
of TNFRSF4 gene-specificmutated
mutatedHSY HSYcells
cells(ex6
(ex6C7 C7 cells)
cells) and
and HHV-6B
HHV-6B
permissivity. (A) Comparison of amino acid sequences and predicted
permissivity. (A) Comparison of amino acid sequences and predicted transmembrane helices transmembrane helices around
around
the targettheregion
targetinregion in TMHMM2.0
TMHMM2.0 [41]. In ex6[41].
C7In ex6 aC7
cells, cells, a frameshift
frameshift mutation near mutation near the N-
the N-terminus of
terminus
the CD134oftransmembrane
the CD134 transmembrane region
region (left panel) led(left panel) ledofto
to translation antranslation
amino acidof an amino
sequence notacid se-
capable
quence notas
of serving capable of serving as
a transmembrane a transmembrane
region (right panels).region (rightexpression
(B) CD134 panels). (B) byCD134 expression
HSY cells and ex6 byC7
HSY cells and ex6 C7 cells was analyzed by means of fluorescence-activated
cells was analyzed by means of fluorescence-activated cell sorting analysis. (C) Viral RNA detection cell sorting analysis.
(C) Viral RNA detection in ex6 C7 cells by RT-PCR. Cells were infected with HHV-6B HST and total
in ex6 C7 cells by RT-PCR. Cells were infected with HHV-6B HST and total RNA was extracted at
RNA was extracted at 48 h post-infection. U91 (immediate early) and U100 (late) transcripts were
48 h post-infection. U91 (immediate early) and U100 (late) transcripts were detected. β-actin (ACTB)
detected. β-actin (ACTB) was used as an internal control. M, DNA ladder; -, mock-infected cells; +,
was used as ancells;
HST-infected internal
N.C.,control. M, DNA
no template ladder;
control. -, mock-infected
(D) cells; +, HST-infected
Viral antigen detection cells;and
in ex6 C7 cells N.C., no
HSY
template control. (D) Viral antigen detection in ex6 C7 cells and HSY cells using
cells using immunofluorescence assay. Cells were infected with HHV-6B HST and treated with cold immunofluorescence
assay. Cells
acetone at 48were infected with
h post-infection HHV-6B
(left panel). HST
HHV-6 andp41treated
proteinwith cold
(red) wasacetone
detectedat 48
in hboth
post-infection
cell types.
Nuclei of cells were stained with DAPI (blue). Scale bars represent 50 μm.
(left panel). HHV-6 p41 protein (red) was detected in both cell types. Nuclei of cells were Virus-infected cells (red)
stained
were counted (right panel). Error bars represent standard deviations (n = 3 per
with DAPI (blue). Scale bars represent 50 µm. Virus-infected cells (red) were counted (right panel). cell). Asterisks indi-
cate statistical significance (**** p < 0.0001, Bonferroni’s multiple comparison test).
Error bars represent standard deviations (n = 3 per cell). Asterisks indicate statistical significance
(**** p < 0.0001, Bonferroni’s multiple comparison test).
Viruses 2022, 13, x FOR PEER REVIEW 10 of 21
Viruses 2022, 14, 160 9 of 19

Figure 3.
Figure 3. Nectin-2
Nectin-2 is is expressed
expressedon onHSY
HSYcells,
cells,and
andHHV-6B-infected
HHV-6B-infected cells express
cells express different
differentlevels of of
levels
nectin-2. (A)
nectin-2. (A)Median
Medianfluorescence
fluorescenceintensity
intensity(MFI)
(MFI)ofofnectin-2
nectin-2expressed
expressedon onthe
thecell
cellsurface.
surface. HSY
HSY cells
cellsahad
had a greater
greater intensity
intensity of nectin-2
of nectin-2 staining
staining when when compared
compared with
with thethe T-cell
T-cell lines,
lines, MT-4,
MT-4, Sup-T1,
Sup-T1, and
and CCRF-HSB-2
CCRF-HSB-2 cells.cells.
ErrorError bars represent
bars represent standard
standard deviations
deviations (n = 3(n = cell).
per 3 per(B) cell).
Mean (B) copy
Meannumbers
copy
numbers
of nectin-2ofisoforms-α
nectin-2 isoforms-α and -δIsoform
and -δ mRNA. mRNA. δIsoform δ was dominantly
was dominantly detected detected in HSY
in HSY cells. cells.
Error bars
represent
Error barsstandard
representdeviations (n = 3 per (n
standard deviations cell).
= 3 (C)
perNectin-2
cell). (C) and CD134
Nectin-2 and expression levels in levels
CD134 expression MMLV-
transduced cells and cells
in MMLV-transduced parent
andCCRF-HSB-2 cells. (D)cells.
parent CCRF-HSB-2 Cells(D)
were infected
Cells with HHV-6B
were infected HST and
with HHV-6B
treated with cold acetone at 96 h post-infection (left panel). HHV-6 p41 protein
HST and treated with cold acetone at 96 h post-infection (left panel). HHV-6 p41 protein (red) (red) was detected
was
in
detected in HSB2-nec2δ cells and HSB2-CD134 cells with an immunofluorescence assay. Nuclei were
HSB2-nec2δ cells and HSB2-CD134 cells with an immunofluorescence assay. Nuclei of cells of
stained with
cells were DAPIwith
stained (blue). Scale(blue).
DAPI bars represent
Scale bars50represent
μm. Virus-infected cells (red) were
50 µm. Virus-infected cellscounted (right
(red) were
panel). Error bars represent standard deviations (n = 3 per cell). Asterisks indicate statistical signif-
counted (right panel). Error bars represent standard deviations (n = 3 per cell). Asterisks indicate
icance (**** p < 0.001, Bonferroni’s multiple comparison test). (E) Nectin-2 and CD134 expression
statistical significance (**** p < 0.001, Bonferroni’s multiple comparison test). (E) Nectin-2 and CD134
levels in HSY cells, ex6 C7 cells, and three lines of NECTIN2 gene-specific mutated ex6 C7 cells (ex6
C7-nec2-KO cells) were detected using FACS. (F) HSY cells, ex6 C7 cells, and ex6 C7-nec2-KO cells
were inoculated with the HHV-6B HST strain. HHV-6 p41 protein (red) was detected at 48 h post-
Viruses 2022, 14, 160 10 of 19

expression levels in HSY cells, ex6 C7 cells, and three lines of NECTIN2 gene-specific mutated ex6
C7 cells (ex6 C7-nec2-KO cells) were detected using FACS. (F) HSY cells, ex6 C7 cells, and ex6 C7-
nec2-KO cells were inoculated with the HHV-6B HST strain. HHV-6 p41 protein (red) was detected
at 48 h post-infection by IFA. Positive cells were determined by counting the number of red signals
in a microscopic field. Error bars represent standard deviations (n = 5 per cell). Asterisks indicate
statistical significance when compared with the parent ex6 C7 cells (*** p < 0.001, **** p < 0.0001,
Dunnett’s multiple comparison test).

Next, we generated CCRF-HSB-2 cells that overexpressed nectin-2δ (HSB2-nec2δ cells)

(Figure 3C). We also generated CCRF-HSB-2 cells overexpressing CD134 (HSB2-CD134
cells) and CCRF-HSB-2 cells into which the empty plasmid was retrovirally transfected
(HSB2-cont cells), and then analyzed their susceptibility to HHV-6B infection (Figure 3C,D).
Interestingly, HSB2-nec2δ cells were susceptible to viral entry; but the rate was much lower
than that of the HSB2-CD134 cells. While the HSB2-cont cells expressed nectin-2 and it was
detectable by FACS, they did not permit viral replication.
To evaluate the importance of endogenous nectin-2 in HSY cells for HHV-6B, we
generated nectin-2-knockout ex6 C7 (ex6 C7-nec2-KO) cells using the CRISPR-Cas9 sys-
tem. Although HHV-6B infection inhibition assays using soluble nectin-2 or anti-nectin-2
antibodies are standard, the baseline situation is that the efficiency of HHV-6B is low in
HSY cells (Figures 1–3); this makes it difficult to show the difference between the presence
and absence of soluble nectin-2 and the antibody. Alternatively, we selected a method
to generate ex6 C7-nec2-KO cells and examined viral infection. We obtained three cell
clones (ex6 C7-nec2-KO1, -KO2, and -KO3) that were confirmed to lack nectin-2 expression
by FACS (Figure 3E). These cells were inoculated with the HHV-6B HST strain, and their
susceptibility to HHV-6B infection was analyzed by IFA. Interestingly, viral antigen-positive
cells were significantly reduced in all nectin-2-knockout ex6 C7 cells when compared with
the parent ex6 C7 cells (Figure 3F).
These data suggest that nectin-2 is a viral entry-mediated molecule involved in the
infection of salivary gland cells by HHV-6B.

3.4. Nectin-2 Is a Functional Entry-Mediated Molecule for HHV-6B HST and Z29 Strains
We examined whether the susceptibility of nectin-2 as a viral entry-mediated molecule
was different between the HST and Z29 strains. Viral antigens were detected in both
HST- and Z29-infected-HSB2-nec2δ cells without a cytopathic effect (CPE), whereas some
virus-infected HSB2-CD134 cells showed typical CPE (balloon-like cells) (Figure 4A). There
was no difference in the infectivity of the HSB2-CD134 and HSB2-nec2δ cells in the HST and
Z29 strains (Figure 4B). Viral infectivity using nectin-2 as a viral entry-mediated molecule
was approximately 29 and 27 times lower than that of CD134 for HST and Z29 infection
(p < 0.0001), respectively (Figure 4B).

3.5. Nectin-2 Enables HHV-6B Replication in CCRF-HSB-2 Cells

Viral replication was examined using HSB2-cont, HSB2-nec2δ, HSB2-CD134, and MT-4
cells for 7 days. Viral proteins were detected in HSB2-nec2δ, HSB2-CD134, and MT-4 cells
(Figure 5A). The number of HHV-6B-infected HSB2-nec2δ cells increased gradually and
reached a peak of 10% (Figure 5B). HHV-6B activity was low in HSB2-nec2δ cells until the
fourth day and then increased, indicating that the infectious virus had been produced. The
number of HHV-6B-infected HSB2-CD134 cells increased rapidly for 3 days and then these
cells died via the lytic cycle 4 days post-infection (d.p.i.), whereas 100% of the MT-4 cells
were infected by multi-step growth at 4 d.p.i. MT-4 cells are human T-cell lymphotropic
virus type 1 (HTLV-1) which transform and express HTLV-1 Tax, which is a transcriptional
activator of the viral long terminal repeat [49,50]. Tax might protect HTLV-1-infected T-cells
from apoptosis [50]. Unlike HSB2-CD134 cells, MT-4 cells that are highly infected with
HHV-6B might remain alive because of Tax.
 HST- and Z29-infected-HSB2-nec2δ cells without a cytopathic effect (CPE), whereas some
virus-infected HSB2-CD134 cells showed typical CPE (balloon-like cells) (Figure 4A).
There was no difference in the infectivity of the HSB2-CD134 and HSB2-nec2δ cells in the
HST and Z29 strains (Figure 4B). Viral infectivity using nectin-2 as a viral entry-mediated
Viruses 2022, 14, 160 molecule was approximately 29 and 27 times lower than that of CD134 for HST and Z29
11 of 19
infection (p < 0.0001), respectively (Figure 4B).

Figure 4. Infectivity of HST and Z29 in HSB2-nec2δ and HSB2-CD134 cells. (A) Viral antigen detection
Figure 4. Infectivity of HST and Z29 in HSB2-nec2δ and HSB2-CD134 cells. (A) Viral antigen detec-
in HSB2-nec2δ, HSB2-CD134, and HSB2-cont cells determined using an immunofluorescence assay.
tion in HSB2-nec2δ, HSB2-CD134, and HSB2-cont cells determined using an immunofluorescence
Cells were
assay. Cellsinfected with HHV-6B
were infected with HST
HHV-6Band Z29
HSTstrains
and Z29(multiplicity of infection of
strains (multiplicity = 0.1) and then
infection treated
= 0.1) and
with cold acetone at 48 h post-infection. HHV-6 p41 protein (red) was detected in
then treated with cold acetone at 48 h post-infection. HHV-6 p41 protein (red) was detected in virus-virus-infected
HSB2-nec2δ and HSB2-CD134
infected HSB2-nec2δ cells. HSB2-cont
and HSB2-CD134 cells were
cells. HSB2-cont used
cells as aused
were negative control. control.
as a negative Nuclei of cells
Nuclei
of cells
were were with
stained stained with
DAPI DAPIArrowhead
(blue). (blue). Arrowhead
indicatesindicates balloon-like
balloon-like cells.
cells. Scale Scale
bars bars represent
represent 50 µm.
50 μm.
(B) (B) Virus-infected
Virus-infected cells
cells (red) were(red) were counted.
counted. Error barsError bars represent
represent standard standard
deviations deviations
(n = 3 per(n =3
cell).
per cell). Asterisks
Asterisks and nsstatistical
and ns indicate indicate statistical significance
significance (**** p <Tukey’s
(**** p < 0.0001, 0.0001, multiple
Tukey’s multiple
comparisoncompar-
test)
isonno
and test) and no significance,
significance, respectively.
respectively.

3.6.
3.5. Nectin-2
Nectin-2 Interacts with HHV-6B
Enables HHV-6B gB in CCRF-HSB-2 Cells
Replication
To determine
Viral the ligand
replication for nectin-2,
was examined we investigated
using HSB2-cont, the HHV-6B glycoproteins.
HSB2-nec2δ, HSB2-CD134,Four and
envelope
MT-4 cellsglycoproteins,
for 7 days. Viral gB,proteins
gD, gH,were
anddetected
gL, are in essential for viral
HSB2-nec2δ, entry [18], and
HSB2-CD134, and MT-
the
gD of some
4 cells (Figurealphaherpesviruses
5A). The number ofbinds to nectin-2 [20,46,47].
HHV-6B-infected HSB2-nec2δ HHV-6B contains
cells increased at least
gradually
eight envelope
and reached glycoproteins;
a peak however,
of 10% (Figure there isactivity
5B). HHV-6B no gD that is conserved
was low among
in HSB2-nec2δ several
cells until
alphaherpesviruses
the fourth day and then [26].increased,
We focused on gB, that
indicating as it the
is ainfectious
fusion glycoprotein
virus had beenthatproduced.
is highly
conserved
The number among herpesviruses [51],
of HHV-6B-infected and its cellcells
HSB2-CD134 surface receptors
increased have for
rapidly not3been
daysidentified
and then
in
these cells died via the lytic cycle 4 days post-infection (d.p.i.), whereas 100% of thenectin-
HHV-6B. We constructed HA-tagged HST gB and gQ1 as well as FLAG-tagged MT-4
2cells
expression plasmids
were infected to evaluategrowth
by multi-step the interactions
at 4 d.p.i. between
MT-4 cells HHV-6B
are humanglycoproteins and
T-cell lympho-
nectin-2. Then,
tropic virus type293T cells overexpressing
1 (HTLV-1) glycoproteins
which transform and express andHTLV-1
nectin-2Tax,
were examined
which by
is a tran-
co-IP. As a result,
scriptional gB-HA
activator and
of the nectin-2-FLAG
viral long terminal were immunoprecipitated
repeat [49,50]. Tax mightusing
protectanti-FLAG
HTLV-1-
and anti-HA antibodies (Figure 6A). Next, to confirm the direct binding of nectin-2 to
HST gB under cell-free conditions, soluble HST gB-HA lacking a transmembrane domain
was generated and examined by IP. Recombinant nectin-2 ectodomain tagged with the Fc
portion of human IgG1 (nectin-2-FC) was immunoprecipitated with soluble HST gB-HA
(Figure 6B). The bands at which the magnetic agarose bound to the Fc proteins were not
detected. We also confirmed that nectin-2 interacts with gB in HHV-6B-infected cells. Co-IP
was performed in HHV-6B-infected MT-4 cells, and nectin-2 was immunoprecipitated using
gB (Figure 6C). To further address the nectin-2 interactions with gB, we constructed HA-
tagged Z29 gB expression plasmids and evaluated the interactions between strain-specific
gB and nectin-2. Both strains of gB-HA and nectin-2-FLAG were immunoprecipitated
with each other (Figure 6D). This result is supported by the viral infectivity data for the
HSB2-nec2δ cells between the HST and Z29 strains (Figure 4B).
Together, these findings indicate that nectin-2 interacts with HHV-6B gB.
 Viruses 2022, 13, x FOR PEER REVIEW 12 of 21

Viruses 2022, 14, 160 12 of 19

infected T-cells from apoptosis [50]. Unlike HSB2-CD134 cells, MT-4 cells that are highly
infected with HHV-6B might remain alive because of Tax.

Figure
Figure 5. HHV-6B
5. HHV-6B growth
growth in in HSB2-nec2δcells.
HSB2-nec2δ cells. (A)
(A) Viral
Viral antigen
antigendetection
detectionin in
HSB2-cont, HSB2-
HSB2-cont, HSB2-nec2δ,
nec2δ, HSB2-CD134, and MT-4 cells using an immunofluorescence assay. Cells were infected with
HSB2-CD134, and MT-4 cells using an immunofluorescence assay. Cells were infected with the
the HHV-6B HST strain (multiplicity of infection = 0.1) and then treated with cold acetone for 1–7
HHV-6B
days HST strain (multiplicity
post-infection (d.p.i.). HHV-6of infection
p41 = 0.1)
protein (red) wasand then in
detected treated with cold
virus-infected acetoneand
HSB2-nec2δ for 1–7 days
HSB2-CD134
post-infection cells. HSB2-cont
(d.p.i.). HHV-6cellsp41were used as
protein a negative
(red) control. HHV-6B-infected
was detected HSB2-CD134
in virus-infected HSB2-nec2δ and
cells died due to the lytic cycle at 4 d.p.i. Nuclei of cells were stained with DAPI (blue). Scale bars
HSB2-CD134 cells. HSB2-cont cells were used as a negative control. HHV-6B-infected HSB2-CD134
represent 50 μm. (B) Virus-infected cells (red) were counted 1–7 d.p.i. Error bars represent standard
cells died due to the lytic cycle at 4 d.p.i. Nuclei of cells were stained with DAPI (blue). Scale
bars represent 50 µm. (B) Virus-infected cells (red) were counted 1–7 d.p.i. Error bars represent
standard deviations (n = 3 per cell). Asterisks indicate statistical significances when compared with
the HSB2-cont cells (* p < 0.05, **** p < 0.0001, Dunnett’s multiple comparison test except for the data
of HSB2-CD134 because of missing values). N.A. indicates not applicable (uncountable due to lytic
infection progression).
 Viruses 2022, 13, x FOR PEER REVIEW 14 of 21
Viruses 2022, 14, 160 13 of 19

Figure 6. Interaction of HHV-6B gB with nectin-2. (A) The 293T cells were co-transfected with
pCAGGS-HST-gB-HA
Figure 6. Interaction of or HHV-6B
pCAGGS-HST-gQ1-HA
gB with nectin-2.and(A)
pCAGGS-nec2δ-FLAG
The 293T cells were or pCAGGS-FLAG.
co-transfected with
Cell lysates were subjected
pCAGGS-HST-gB-HA to immunoblotting (IB) and
or pCAGGS-HST-gQ1-HA andimmunoprecipitation
pCAGGS-nec2δ-FLAG (IP)orwith the indicated
pCAGGS-FLAG.
antibodies. HHV-6B
Cell lysates were gB consists
subjected of three polypeptides
to immunoblotting of 102 kDa, and its(IP)
(IB) and immunoprecipitation proteolytic
with the cleavage
indicated
antibodies.
products HHV-6B
of 59 and 50 gB
kDa consists of three polypeptides
[52]. C-terminal of 102 kDa, and
HA-tagged recombinant gB its proteolytic
proteins cleavage prod-
were expressed and
ucts of
used. (B)59Recombinant
and 50 kDa nectin-2-Fc
[52]. C-terminal HA-tagged recombinant
was immunoprecipitated withgB proteins
soluble were expressed
HST-gB-HA or HA. and
(C)
used. (B) Recombinant nectin-2-Fc was immunoprecipitated with soluble HST-gB-HA
HHV-6B-infected MT-4 cells were cultured for 6 d and lysates were subjected to co-IP with anti-HHV-6 or HA. (C)
HHV-6B-infected MT-4 cells were cultured for 6 d and lysates were subjected to
gB antibody (6A5). Mock-infected MT-4 cells were used as a negative control. (D) 293T cells wereco-IP with anti-
HHV-6 gB antibody (6A5). Mock-infected MT-4 cells were used as a negative control. (D) 293T cells
co-transfected with pCAGGS-gB (HST or Z29)-HA or pCAGGS-HA and pCAGGS-nec2δ-FLAG or
were co-transfected with pCAGGS-gB (HST or Z29)-HA or pCAGGS-HA and pCAGGS-nec2δ-
pCAGGS-FLAG. Cell lysates were subjected to IB with the indicated antibodies (left panel). Cell
FLAG or pCAGGS-FLAG. Cell lysates were subjected to IB with the indicated antibodies (left panel).
lysates werewere
Cell lysates subjected to IPtowith
subjected anti-FLAG
IP with or anti-HA
anti-FLAG or anti-HA antibodies (right
antibodies (rightpanel).
panel).WCE,
WCE, whole-
whole-
cell
cellextract.
extract.

3.7.
3.7. The
The V-set
V-set Domain
DomainofofNectin-2
Nectin-2IsIsImportant
Importantfor
forInteractions
Interactionswith
withHHV-6B
HHV-6BgB gB
To
Todetermine
determine which
which domain
domain of of nectin-2
nectin-2 was
was required
required for
for binding
binding to
to HHV-6B
HHV-6B gB,
gB,
several
severaldeletion
deletionmutants
mutantsof ofnectin-2δ
nectin-2δwere
wereconstructed
constructed(Figure
(Figure7A).
7A).These
Theseplasmids
plasmidswere
were
transfected into 293T cells together with the HHV-6B gB-expressing plasmids and examined
Viruses 2022, 13, x FOR PEER REVIEW 15 of 21

Viruses 2022, 14, 160 14 of 19

transfected into 293T cells together with the HHV-6B gB-expressing plasmids and exam-
ined by co-IP.
by co-IP. The full-length
The full-length domain,domain,
as wellasas well as the mutants
the mutants lackingdomains
lacking C2-set C2-set domains
(∆C2×2-
(ΔC2×2-FLAG), first C2-set domain (ΔC2(55aa)-FLAG), and
FLAG), first C2-set domain (∆C2(55aa)-FLAG), and second C2-set domain second C2-set domain
(∆C2(46aa)-
(ΔC2(46aa)-FLAG), but not a mutant lacking the V-set domain (ΔV-FLAG),
FLAG), but not a mutant lacking the V-set domain (∆V-FLAG), were immunoprecipitatedwere immuno-
precipitated
with HHV-6Bwith HSTHHV-6B
and Z29HST and Z297B).
gBs (Figure gBsThese
(Figure 7B).
data These data that
demonstrate demonstrate
the V-set that the
domain
V-set domain
of nectin-2 of nectin-2for
is important is important
interactionsforwith
interactions
HHV-6B with
gB. HHV-6B gB.

Figure 7. Interactions between HHV-6B gB and nectin-2 deletion mutants. (A) Schematic representa-
Figure 7. Interactions between HHV-6B gB and nectin-2 deletion mutants. (A) Schematic represen-
tion of the constructed nectin-2δ deletion mutants. Deleted domains are represented by gray lines.
tation of the constructed nectin-2δ deletion mutants. Deleted domains are represented by gray lines.
(B) 293T cells were transfected with plasmids encoding HHV-6B gB-HA and nectin-2δ-FLAG mutants
(B) 293T cells were transfected with plasmids encoding HHV-6B gB-HA and nectin-2δ-FLAG mu-
and subjected
tants to immunoblotting
and subjected (IB) and
to immunoblotting immunoprecipitation
(IB) and (IP) (IP)
immunoprecipitation withwith
anti-FLAG or anti-HA
anti-FLAG or anti-
antibodies.
HA WCE,
antibodies. whole-cell
WCE, extract.
whole-cell extract.
Viruses 2022, 14, 160 15 of 19

4. Discussion
The HHV-6B entry receptor has previously been identified as CD134 [7], but the virus
has been detected in tissues in which CD134 is not expressed [10,12–14]. Single receptors
for the cell entry of HHV-6A, HHV-6B, and HHV-7 have been identified (CD46, CD134,
and CD4, respectively) [7,53,54]. While single receptors have been identified for the entry
of HHV-6A (CD46), HHV-6B (CD134), and HHV-7 (CD4) into T cells, other herpesviruses
use multiple receptors that enable entry into diverse cell types [18]. gB, gH, gL, gM, and
gN are conserved glycoproteins from the family Herpesviridae [55,56]. Among them, gB
and gH/gL play key roles in membrane fusion and herpesvirus infection [57], and the gB
and gH of HHV-6 also induce cell-to-cell fusion [58]. In HHV-6B, gH is a component of a
tetrameric complex (i.e., gH/gL/gQ1/gQ2) [59], which is a viral ligand for the receptor
CD134 [7]. The binding of a viral receptor to gB has not been reported for HHV-6B. In
this study, we demonstrated that the ectopic expression of nectin-2 conferred HHV-6B
susceptibility to cells normally insensitive to the virus and that the infectivity of HHV-6B
was reduced in nectin-2 knockout salivary gland cells. Furthermore, we demonstrated that
nectin-2 interacts with HHV-6B gB.
Nectins are a family of Ca2+ -independent Ig-like CAMs, and there are four members,
nectin-1 to nectin-4 [45]. All members, except for the nectin-1 splice variant (nectin-1γ),
have an extracellular region containing three Ig-like domains (a distal V-set domain and
two C-set domains), a single transmembrane region, and a cytoplasmic region. These
molecules homophilically and heterophilically interact in trans with each other to form cell–
cell adherens junctions (AJs) cooperatively with E-cadherin in a variety of cells [60]. Among
them, nectin-1 and nectin-2 have been identified as alphaherpesvirus (i.e., HSV-1, HSV-2,
and PRV) entry receptors that interact with their respective viral gDs [19,20]. Interestingly,
our data showed that HHV-6B gB, which has no known sequence or structural similarities
with alphaherpesvirus gDs, interacts with nectin-2 (Figures 6, 7 and S3).
Ig-like molecules are used as entry receptors for alphaherpesviruses, as well as other
viruses (e.g., nectin-4 and CD150 for measles virus, CD4 for human immunodeficiency
virus, and CD155 for poliovirus) [61–64]. Notably, the V domain of these molecules is
critical for virus entry into the cell [47,65–69]. In HSV-1, gD binds to nectin-1, and gD
contacts the β-sheet of the V-set domain [70,71]. Martinez et al. [47] reported that the V-set
domain of nectin-2 was critical for HSV entry via this molecule. Although the amino acid
identity in the V-set domain between nectin-1 and nectin-2 is approximately 67% [46],
the overall conformation of the nectin-2 V-set domain is similar to that of nectin-1 [72].
Similarly, the CD155 V-set domain has also been identified as an important binding site
for poliovirus [69,73]. It seems that the equivalent region of the V domain of different Ig
molecules can be utilized by several viruses for binding to the cell surface and for entry [47].
A similar region within the V-set domain was predicted to be the HHV-6B gB-binding site
in nectin-2.
Although HSB2-cont cells and parent CCRF-HSB-2 cells expressed nectin-2 that was
detectable using FACS (Figure 3), they did not permit viral replication, possibly because of
the low affinity of the interaction. Indeed, recombinant nectin-2-Fc was found to be weakly
bound to gB-HA in a nectin-2 direct binding assay (Figure 6B), and the efficiency of viral
infection was low, even in the ex6 C7 cells, in which the mean fluorescence intensity was
approximately three times higher than that in the HSB2-cont cells (Figure 3C,E). Therefore,
nectin-2 must be overexpressed to support effective viral entry.
HHV-6B has been detected in T-cells, salivary glands, liver cells, and neural cells,
which do not express CD134 [10,12,13,74]. In this study, we identified nectin-2 as an HHV-
6B entry-mediated molecule using a cDNA library derived from a parotid gland cell line,
HSY. Although nectin-2 might not be an entry receptor comparable to CD134, induction
of virus infection via the overexpression of nectin-2 suggests that nectin-2 is involved in
binding as well as the entry process. Nectin-2 is ubiquitously expressed and localized in AJs
in a variety of cells and salivary glands [45]. In polarized epithelial cells (apico-basal cell
polarity), intercellular adhesion is mediated through a junctional complex that comprises
Viruses 2022, 14, 160 16 of 19

tight junctions (TJs), AJs, and desmosomes (DSs), and TJs are formed at the apical side of
the AJs [75]. TJs provide a physical barrier that prevents the lateral movement of proteins
between the apical and basolateral membranes [76]. The functional significance of TJs in the
salivary gland epithelium is similar (e.g., polarized saliva secretion and barrier maintenance
between the extracellular environment and the glandular lumen) [76]. We hypothesize that
HHV-6B infection of salivary glands in vivo can be explained by two molecules, nectin-2
and CD134. HHV-6B infects T-lymphocytes via CD134-mediated entry, and infected T cells
transfer HHV-6B to epithelial cells via the nectin-2 expressed at AJs in the salivary glands
through the basolateral side. In the epithelia, viruses (transferred and/or propagated) are
released from the apical region to the luminal side of the salivary glands. This mechanism
is similar to that through which the measles virus infects the epithelia, which is mediated
by nectin-4, and virus particles released from the apical membrane enter the luminal side
of the respiratory tract [77]. It can be speculated that the number of HHV-6B-infected
cells might be decreased at the time of entry into the salivary gland cells via nectin-2, and
progeny viruses might be less productive in salivary glands. Further studies are required
to improve our understanding of why nectin-2 represents low-efficiency HHV-6B entry
and how nectin-2 mediates virus entry into the salivary glands.
In this study, we found that HHV-6B mediates nectin-2 and its entry into salivary gland
cells (intercalated duct cells of the parotid gland). As nectin-2 is ubiquitously expressed
in a variety of cells, including hepatocytes and astrocytes [16,75,78], this molecule may
play a key role in the pathogenesis of HHV-6B-associated diseases in the liver and central
nervous system. Further studies of these interactions will provide new insights into HHV-
6B infection.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/v14010160/s1, Figure S1: Target sequences in the nectin-2 knockout ex6 C7 cell clones,
Figure S2: CD134 without the transmembrane anchor sequence was not detected inside the ex6 C7
cells, Figure S3: Comparison of the gD and gB amino acid sequences.
Author Contributions: Conceptualization, H.O. and M.Y.; methodology, H.O., D.F., T.H. and M.Y.;
investigation, H.O. and D.F.; resources, H.O., D.F., H.N., N.Y. and M.Y.; data curation, H.O., D.F. and
M.Y.; writing—original draft preparation, H.O.; writing—review and editing, H.O., D.F., T.H. and
M.Y.; supervision, M.Y.; funding acquisition, H.O., N.Y., T.H. and M.Y. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported by JSPS KAKENHI [grants #JP19K07592, #JP20K08180, #JP18H02664,
and #JP21H02738] and the Takeda Science Foundation [grant no. not assigned], and Okayama Medical
Foundation [grant no. not assigned].
Acknowledgments: We thank Yukinari Isomoto and the staff at the Central Research Laboratory,
Okayama University Medical School for their technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.

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