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\begin{document}

\title{Human MARCH1, 2, and 8 Block Ebola Virus Envelope Glycoprotein Cleavage
via Targeting Furin P Domain}



\author[1]{Chunfu  Zheng}%
\author[2]{Changqing Yu}%
\author[3]{Wenbo Tan}%
\author[4]{Yu Bai}%
\author[5]{Yulong Zhou}%
\author[6]{Jinbo Zhai}%
\author[7]{Mengzhou Xue}%
\author[8]{Qiang Liu}%
\affil[1]{Guangdong Academy of Agricultural Sciences}%
\affil[2]{Yibin Vocational and Technical College}%
\affil[3]{Heilongjiang University}%
\affil[4]{Wenzhou Vocational College of Science and Technology}%
\affil[5]{Heilongjiang Bayi Agricultural University}%
\affil[6]{Universities of Inner Mongolia Autonomous Region}%
\affil[7]{The Second Affiliated Hospital of Zhengzhou University}%
\affil[8]{Nanchong Vocational and Technical College}%


\vspace{-1em}



  
  \date{June 02, 2024}


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\selectlanguage{english}
\begin{abstract}
Membrane-Associated Ring-CH (MARCH) family proteins were recently
reported to inhibit viral replication through multiple modes of action.
Previous work proved that human MARCH8 blocked Ebola virus (EBOV)
glycoprotein (GP) maturation. Our study here demonstrates that human
MARCH1 and MARCH2 share a similar pattern to MARCH8 in restricting EBOV
GP-mediated viral replication. Human MARCH1 and MARCH2 retain EBOV GP in
the trans-Golgi network (TGN), reduce its surface display, and impair
EBOV GP-pseudotyped virions infectivity. Furthermore, we uncover that
the host proprotein convertase (PC) furin could interact with human
MARCH1/2 and EBOV GP intracellularly. Importantly, the furin P domain is
confirmed to be recognized by MARCH1/2/8, which is critical for their
blocking activities. Besides, bovine MARCH2 and murine MARCH1 also
impair EBOV GP proteolytic processing. Altogether, our findings confirm
that MARCH1/2 proteins of different species showed a relatively
conserved feature in blocking EBOV GP proteolytic processing, which
could provide a reference for subsequent antiviral studies and may
facilitate the development of novel strategies to antagonize enveloped
virus infection.%
\end{abstract}%



\sloppy


\textbf{Human MARCH1, 2, and 8 Block Ebola Virus Envelope Glycoprotein
Cleavage via Targeting Furin P Domain}

Changqing Yu \textsuperscript{1 \#}, Wenbo Tan \textsuperscript{4\#}, Yu
Bai \textsuperscript{5\#}, Yulong Zhou \textsuperscript{8}, Jingbo Zhai
\textsuperscript{6}, Mengzhou Xue \textsuperscript{7}, Chunfu Zheng
\textsuperscript{2,9*}, Qiang Liu \textsuperscript{3*}

\textsuperscript{1} Egineering Center of Agricultural Biosafety
Assessment and Biotechnology, School of Advanced Agricultural Sciences,
Yibin Vocational and Technical College, Yibin, People's Republic of
China;

\textsuperscript{2} Institute of Animal Health, Guangdong Academy of
Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of
Guangdong Province, Scientific Observation and Experiment Station of
Veterinary Drugs and Diagnostic Techniques of Guangdong Province,
Ministry of Agriculture and Rural Affairs, Guangzhou, People's Republic
of China;

\textsuperscript{3} Nanchong Key Laboratory of Disease Prevention,
Control and Detection in Livestock and Poultry, Nanchong Vocational and
Technical College, Nanchong, People's Republic of China;

\textsuperscript{4} College of Advanced Agriculture and Ecological
Environment, Heilongjiang University, Harbin, People's Republic of
China;

\textsuperscript{5} College of Animal Science, Wenzhou Vocational
College of Science and Technology, Wenzhou, People's Republic of China;

\textsuperscript{6} Key Laboratory of Zoonose Prevention and Control at
Universities of Inner Mongolia Autonomous Region, Medical College, Inner
Mongolia Minzu University, Tongliao, People's Republic of China;

\textsuperscript{7} Department of Cerebrovascular Diseases, The Second
Affiliated Hospital of Zhengzhou University, 2 Jingba Road, Zhengzhou,
Henan, People's Republic of China;

\textsuperscript{8} College of Animal Science and Technology,
Heilongjiang Bayi Agricultural University, Daqing, People's Republic of
China;

\textsuperscript{9} Department of Microbiology, Immunology \& Infection
Diseases, University of Calgary, Calgary, Alberta, Canada.

\textsuperscript{\#} These authors contributed equally to this work.

\textsuperscript{*} Correspondence

Chunfu Zheng, zheng.alan@hotmail.com

Qiang Liu, liuqiang\_yyy@163.com

\textbf{Abstract:} Membrane-Associated Ring-CH (MARCH) family proteins
were recently reported to inhibit viral replication through multiple
modes of action. Previous work proved that human MARCH8 blocked Ebola
virus (EBOV) glycoprotein (GP) maturation. Our study here demonstrates
that human MARCH1 and MARCH2 share a similar pattern to MARCH8 in
restricting EBOV GP-mediated viral replication. Human MARCH1 and MARCH2
retain EBOV GP in the trans-Golgi network (TGN), reduce its surface
display, and impair EBOV GP-pseudotyped virions infectivity.
Furthermore, we uncover that the host proprotein convertase (PC) furin
could interact with human MARCH1/2 and EBOV GP intracellularly.
Importantly, the furin P domain is confirmed to be recognized by
MARCH1/2/8, which is critical for their blocking activities. Besides,
bovine MARCH2 and murine MARCH1 also impair EBOV GP proteolytic
processing. Altogether, our findings confirm that MARCH1/2 proteins of
different species showed a relatively conserved feature in blocking EBOV
GP proteolytic processing, which could provide a reference for
subsequent antiviral studies and may facilitate the development of novel
strategies to antagonize enveloped virus infection.

\textbf{KEYWORDS}

MARCH proteins, Ebola virus, furin, envelope glycoprotein, cleavage

\textbf{1 INTRODUCTION}

Currently, a group of host cell membrane-associated proteins has been
discovered to target viral envelope glycoproteins, including IFN-induced
transmembrane (IFITM) proteins, serine incorporator (SERINC) and MARCH
proteins (1). These cellular innate immunity factors could exert their
antiviral activities at multiple stages of viral replication.

Originally, MARCH proteins were identified to show homology with the E3
ubiquitin ligase of K3 and K5 of the Kaposi's sarcoma-associated
herpesvirus (KSHV) (2, 3). The MARCH family proteins now have been
extended to comprise at least 11 members, most of which share a similar
molecular structure, including a cytoplasmic N-terminal C4HC3
RING-finger (RING-CH finger) domain and two or more transmembrane
domains, except for MARCH7 and MARCH10, which contain no predicted
transmembrane domains. MARCH family proteins are widely involved in cell
surface protein regulation (3-9), innate immunity signal transduction
(10-12) and autophagy pathways regulation (13-15). MARCH1 and MARCH2
were originally found to downregulate the transferrin receptor (TfR) and
CD86 (B7-2) (3). MARCH1 was able to mediate MHC II ubiquitination and
thus promote dendritic cell selection of natural T regulatory cells
(16). In addition, MARCH1 could inhibit type I IFN signaling pathways
(17). MARCH2 recognized syntaxin-6 (18), regulated secretory proteins
trafficking (19), and negatively regulated cell autophagy (20) and NF-\selectlanguage{greek}κ\selectlanguage{english}B
essential modulator (NEMO) signaling (21).

Recently, MARCH proteins were found to show antiviral activities (22,
23). Tada et al. first reported that MARCH8 could inhibit HIV-1 and
VSV-G pseudotyped virus infection by targeting their envelope
glycoproteins (24). Subsequently, MARCH1 and MARCH2 were also found to
inhibit viral replication potentially (25-28). The viral proteins
targeted by MARCH proteins now are extended to envelope glycoproteins of
EBOV (27, 29, 30), severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) (27, 28, 30), murine leukemia virus (MLV) (24, 27),
influenza virus (IAV) (27, 29, 31), Nipah virus (NiV) (27), spring
viremia of carp virus (SVCV) (32) and rabies virus (RABV) (28), M2
protein of IAV (33), and N protein of porcine epidemic diarrhea virus
(PEDV) (15). Interestingly, in contrast to the negative role on viral
replication, MARCH8 was also critically supportive of hepatitis C virus
(HCV) replication (34).

Generally, two antiviral modes are employed by MARCH proteins to
restrict viral replication. On one side, MARCH proteins downregulated
viral envelope glycoproteins expression and induced their intracellular
degradation, which was the viral protein \emph{c} ytoplasmic \emph{t}
ail\emph{d} ependence (CTD), including envelope glycoproteins of VSV and
RABV, and M2 protein of IAV (24, 28, 33). On the other side, MARCH
proteins retained viral envelope glycoproteins at intracellular
compartments without degradation, which relied on their E3 ubiquitin
ligase activities but was the viral protein \emph{c} ytoplasmic\emph{t}
ail \emph{i} ndependence (CTI), including envelope glycoproteins of
HIV-1, EBOV, and SARS-CoV-2 (24, 29, 30, 35).

EBOV GP is critical for its target cell infection. EBOV
GP\textsubscript{0} precursor experiences initial N-glycosylation
modification in the endoplasmic reticulum (ER) and is then transported
to the trans-Golgi network (TGN), where it finishes its mature N- and
O-glycosylation (36). The mature N, O-glycosylated GP\textsubscript{0}
is cleaved into two subunits (GP\textsubscript{1} and
GP\textsubscript{2}) by furin protease at the highly conserved polybasic
sites (R-X-A/R-R) (36). The GP\textsubscript{1} and GP\textsubscript{2}
subunits are re-linked via disulfide bonds and form
GP\textsubscript{1,2} (37), assembled into self-trimers, and transported
to the cell membrane. GP\textsubscript{1}contains a mucin-like domain
(MLD), which is both N-glycosylated and highly O-glycosylated and thus
dramatically increases its molecular weight. Therefore, the molecular
weight of the mature glycosylated GP\textsubscript{1} is usually a
little larger than the full-length immature glycosylation form
GP\textsubscript{0}.

It was previously reported that human MARCH8 retained EBOV GP at TGN and
blocked its proteolytic cleavage via targeting furin under the CTI
antiviral pattern (29). Here, we extend to investigate the anti-EBOV GP
activities of human MARCH1/2, bovine MARCH1/2, and murine MARCH1/2. Our
results show that human MARCH1 and MARCH2, bovine MARCH2, and murine
MARCH1 could inhibit EBOV GP intracellular cleavage. Critically, human
MARCH1 and MARCH2 are also found to hijack furin, as MARCH8 did, to
block EBOV GP activities, demonstrating a conserved CTI antiviral mode
among MARCH molecules and thus shedding light on subsequent antiviral
studies.

\textbf{2 METHODOLOGY}

\textbf{2.1 Cell lines}

Human embryonic kidney 293T cells, cervical cancer HeLa cells, human
liver Huh-7 cells, and African green monkey kidney Vero cells were
cultured at 37\selectlanguage{ngerman}°C, and 5\% CO\textsubscript{2} in DMEM (Gibco)
supplemented with 8\% fetal bovine serum (FBS) (Gibco), streptomycin,
and penicillin (100 \selectlanguage{greek}µ\selectlanguage{english}g/ml). All cell lines were obtained from the
American Type Culture Collection (ATCC).

\textbf{2.2 DNA constructs}

The bovine MARCH1/2 and murine MARCH1/2 genes were obtained from
bovine/murine peripheral blood mononuclear cells (PBMC) through RT-PCR
amplification and cloned into the pCAGGS vector with the C-terminal HA
tag by EcoRI/XhoI restriction sites. The human MARCH8 was provided by
professor Kenzo Tokunaga (pCAGGS-3\selectlanguage{ngerman}×HA-MARCH8) or cloned into the pCAGGS
vector with a C-terminus HA tag (pCAGGS-MARCH8-HA). The human MARCH1 and
MARCH2 were synthesized and cloned into the pCAGGS vector
(pCAGGS-MARCH1/2-HA) according to the GenBank deposited sequences
(GenBank accession \#NM\_001166373 and \#NM\_016496).
Human/bovine/murine MARCH1-GFP and MARCH2-GFP expression vectors were
constructed by inserting MARCH1 or MARCH2 into the pEGFP-N1 vector
(GenBank accession \#U55762) via EcoRI/AgeI restriction sites. Human
MARCH1/MARCH2 was cloned into the pcDNA3.1-VN vector by EcoRI/AgeI
restriction sites, generating the pcDNA3.1-MARCH1/MARCH2-VN. The
N-terminal HA-tagged EBOV GP\selectlanguage{greek}Δ\selectlanguage{english}MLD expression vector was produced via
site-directed mutagenesis using FLAG-tagged EBOV GP\selectlanguage{greek}Δ\selectlanguage{english}MLD as the backbone
(29). The N-terminal FLAG-tagged EBOV GP\selectlanguage{greek}Δ\selectlanguage{english}MLD and full-length EBOV GP
were cloned into pcDNA3.1(+) expression vector based on their parental
vectors (29). The VSV-G expression vector, Furin/Furin-\selectlanguage{greek}Δ\selectlanguage{english}CD (furin CD
deletion)/Furin-\selectlanguage{greek}Δ\selectlanguage{english}P (furin P deletion) expression vectors,
MARCH8-GFP/EBOV GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-GFP expression vectors,
Furin-VC/MARCH8-VN/GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-VC expression vectors, calnexin-mCherry
/TGN46-mCherry expression vectors, and pNL-Luc-\selectlanguage{greek}Δ\selectlanguage{english}Env proviral reporter
vector were used as previously described (29).

\textbf{2.3 Confocal microscopy assay}

HeLa cells (2\selectlanguage{ngerman}×10\textsuperscript{5}) were seeded on glass slides 10 h
before transfection. The HeLa cells were transfected with Lipofectamine
3000 reagent (Thermo Fisher). Twenty-four hours later, the cells were
washed, fixed with 4\% paraformaldehyde, and permeabilized with 0.1\%
Triton X-100. After washing, the cells were blocked with 5\% bovine
serum albumin (BSA). Cells were incubated with an anti-HA primary
antibody for 1 h at RT for the immunofluorescence assay. After 3 washes,
cells were stained with an Alexa Fluor 647-conjugated secondary antibody
for 1 h at RT. Following 3 washes with PBS, cells were stained with DAPI
for 30 min, washed for 1 h at RT, and used for confocal microscopy assay
(ZEISS LSM700). At least 100 random cells per slide were analyzed, and
the most representative images from each slide were selected for
presentation.

\textbf{2.4 Flow cytometry assay}

To detect BiFC, the pcDNA3.1-MARCH1-VN/MARCH2-VN/MARCH8-VN, and Furin-VC
expression vectors were co-transfected into 293T cells. After 36 h, the
293T cells were collected and used for flow cytometry analysis.

\textbf{2.5 Western blot (WB) assay}

293T cells (5×10\textsuperscript{5} or 2×10\textsuperscript{6}) were
seeded in 6-well plates or 10 cm dishes 12 h before transfection.
Thirty-six hours post-transfection, the cells were lysed with
radioimmunoprecipitation assay (RIPA) buffer (Sigma) and cleared by
centrifugation. Then, the supernatants were collected, mixed gently with
loading dye, and boiled in a water bath for 5 min. Subsequently, the
samples were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to a polyvinylidene
difluoride (PVDF) membrane, which was blocked with 4\% nonfat milk for 1
h at RT. After washing, the membrane was incubated with the primary
antibody and horseradish peroxidase (HRP)-conjugated secondary antibody.
Alternatively, the membrane was directly incubated with an
HRP-conjugated primary antibody. Finally, the PVDF membrane was exposed
to enhanced chemiluminescence (ECL).

\textbf{2.6 Antibodies}

Mouse anti-actin, mouse anti-HA, and mouse anti-FLAG monoclonal
antibodies were purchased from Sigma. HRP-conjugated anti-mouse
immunoglobulin G secondary antibodies were purchased from Pierce. Alexa
Fluor 647-conjugated secondary antibodies were purchased from Thermo
Fisher. HRP-conjugated anti-HA and anti-FLAG primary antibodies were
purchased from Roche and Sigma. The ECL detection kit was purchased from
Thermo Fisher.

\textbf{2.7 Co-immunoprecipitation (Co-IP) assay}

2×10\textsuperscript{6} 293T cells were seeded in 10 cm culture dishes
and transfected 12 h later. Forty-eight hours post-transfection, the
cells were lysed with RIPA buffer and cleared by centrifugation. The
supernatants were collected and incubated with anti-FLAG
antibody-conjugated magnetic beads (Sigma) overnight at 4°C. Then, the
beads were collected with a magnetic rack, repeatedly washed, and used
for the WB assay.

\textbf{2.8 Viral infectivity assay}

5×10\textsuperscript{5} 293T cells were seeded in 6-well plates 12 h
before transfection. 293T cells were co-transfected with
pcDNA3.1-FLAG-GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD/VSV-G, pNL-Luc-\selectlanguage{greek}Δ\selectlanguage{english}Env, and MARCH expression vectors.
Forty-eight hours post-transfection, virion-containing supernatants were
collected to infect the indicator Vero cells. Before infection,
supernatants were subjected to ultracentrifugation at 40000 \selectlanguage{ngerman}× g for 1 h,
and pellets were used for WB or P24\textsuperscript{Gag} ELISA quantity
assay. Thirty-six hours post-infection, Vero cells were lysed, and viral
infectivity was assessed via luciferase activity assay.

\textbf{2.9 Luciferase activity assay}

One day before viral infection, 1×10\textsuperscript{4} Vero cells were
seeded in 96-well plates. Virion-containing supernatants were then added
to Vero cells. After 48 h, the cell culture was terminated. Cells were
washed with PBS 3 times and lysed with 100 \selectlanguage{greek}μ\selectlanguage{english}L of RIPA buffer. 100 \selectlanguage{greek}μ\selectlanguage{english}L of
cell lysate was taken, mixed with an equal amount of substrate
(Bright-Glo luciferase assay system, Promega) for 10 min at RT, and used
for luminescence activity measurement.

\textbf{3 RESULTS}

\textbf{3.1 Human MARCH1 and MARCH2 inhibited EBOV GP proteolytic
processing}

Human MARCH1 and MARCH2 showed antiviral activities (26-28), and human
MARCH8 could suppress EBOV GP proteolytic processing (29, 30). Thus, in
this study, we aim to test whether mammalian MARCH1 and MARCH2 could
block EBOV GP cleavage. Sequence alignment indicated MARCH1 shared a
high, whereas MARCH2 was a low homology to MRAH8 (Fig. 1). However, they
all shared a similar membrane-associated structure (23). We cloned
MARCH1 and MARCH2 from humans, cattle, and mice. We then trace these
mammalian MARCH proteins' intracellular distribution. We inserted a
green fluorescent protein (GFP) tag in these MARCH proteins' C-terminus.
Confocal analysis indicated that these MARCH proteins showed
membrane-associated and scattered punctate distribution (Fig. 2A),
similar to the intracellular localization of MARCH8 as previously
reported (29). Next, we detected the anti-EBOV GP activity of these
MARCH proteins. As shown in Fig. 2B, similar to humans MARCH8, MARCH1
and MARCH2 also suppressed GP processing. Similarly, bovine MARCH2 and
murine MARCH1 could block GP cleavage (Fig. 2B). Intruguingly, murine
MARCH2 nearly lost such inhibition effects. Notably, in the presence of
bovine MARCH1, both GP\textsubscript{0} and
GP\textsubscript{1}expression was significantly impaired (Fig. 2B),
which differed from the other MARCH proteins' antiviral pattern. We then
applied the GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-mediated pseudovirus infection system to check
whether these MARCH proteins could block viral replication (29).
Compared to the vector control group, viral infectivity was
significantly suppressed in the presence of the MARCH proteins except
the murine MARCH2 (Fig. 2C), which matched their blocking effects on GP
cleavage. Therefore, these results demonstrated that human/murine MARCH1
and human/bovine MARCH2 blocked EBOV GP proteolytic processing. In
addition, human MARCH1 and MARCH2 also inhibited VSV-G pseudotyped
virion infectivity (Fig. 2D) and suppressed EBOV GP cleavage in human
liver Huh-7 cells (Fig. 2E).

\textbf{3.2 Human MARCH1 and MARCH2 retained EBOV GP in the TGN
apparatus.}

Generally, mature GP\textsubscript{1,2} is presented on the cell
surface, enveloped into virions, and responsible for binding target
cells' receptors in EBOV next round of infection. Human MARCH1 and
MARCH2 reduced both GP\textsubscript{1} expression and
GP\textsubscript{1,2}-mediated viral infectivity. We thus deduced that
human MARCH1 and MARCH2 downregulated GP\textsubscript{1} cell surface
display. To confirm the hypothesis, the GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-GFP was co-transfected
with human MARCH1 or MARCH2. GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-GFP displayed a plasma membrane
(PM) localization, and the human MARCH1 and MARCH2 proteins showed
scattered punctate distribution (Fig. 3A). In the presence of human
MARCH1 or MARCH2, the GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-GFP protein was downregulated from PM and
was retained intracellularly (Fig. 3A). This evidence demonstrated
MARCH1 and MARCH2 trapped GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-GFP at an intracellular compartment,
where it was unable to be transported to PM. To further verify this, we
introduced the bimolecular fluorescence complementation (BiFC) system to
determine where the GP was retained. MARCH proteins were fused with the
Venus N-terminus (VN, residues 2-173) in their C-terminus, while the
GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD was fused with the Venus C-terminus (VC, residues 154-238) in
its C-terminus. Simultaneously, we applied the calnexin-mCherry
(CNX-mCherry) and TGN46-mCherry (TGN-mCherry) as the ER and TGN
localization markers, respectively (29). MARCH1-VN/MARCH2-VN/MARCH8-VN
and GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-VC were co-transfected with CNX-mCherry or TGN-mCherry to
trace their intracellular localization. Individual
MARCH1-VN/MARCH2-VN/MARCH8-VN/GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-VC was negative for any
fluorescent signals (data not shown). In the presence of MARCH1/2/8,
EBOV GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD was trapped intracellularly, consistent with the former
results (Fig. 3B). Further detection indicated
MARCH1-VN/MARCH2-VN/MARCH8-VN and GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD-VC pair groups showed minimal
overlap with CNX-mCherry but displayed a strong co-localization with
TGN-mCherry (Fig. 3B). Therefore, we concluded MARCH1 and MARCH2
retained GP in TGN, as MARCH8 did.

\textbf{3.3 MARCH1 and MARCH2 inhibited furin-mediated cleavage of EBOV
GP}

MARCH8 suppressed furin-mediated cleavage of EBOV GP (29). Therefore,
based on the above results (Fig. 2B), we further verified whether MARCH1
and MARCH2 could block furin-mediated GP cleavage. We first applied the
BiFC system to detect MARCH1/MARCH2/MARCH8 and furin intracellular
interaction. MARCH1-VN/MARCH2-VN/MARCH8-VN and furin-VC (FR-VC) were
co-transfected into 293T cells. Flow cytometry analysis showed
MARCH1-VN/MARCH2-VN/MARCH8-VN, and FR-VC had strong intracellular
interaction signals (Fig. 4A). Then, MARCH1-VN/MARCH2-VN/MARCH8-VN and
FR-VC were co-transfected into HeLa cells. As indicated in Fig. 4B,
these MARCH proteins and furin had a clear intracellular
co-localization. Next, to confirm their intracellular interactions, the
Co-IP assay was performed. The furin expression vector was
co-transfected with MARCH1/MARCH2 and EBOV GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD expression vectors to
further verify furin's binding to MARCH proteins and EBOV GP. As shown
in Fig. 4C, MARCH1/2, and GP-\selectlanguage{greek}Δ\selectlanguage{english}MLD could be co-immunoprecipitated by
furin, indicating they formed an intracellular complex, similar to the
results obtained in MARCH8, as previously identified (29). Relying on
their intracellular interactions, we identify whether these MARCH
proteins blocked furin-mediated GP cleavage. Generally, mature
GP\textsubscript{0} was cleaved into
GP\textsubscript{1}(\textasciitilde{}130 kDa) and GP\textsubscript{2}
(\textasciitilde{}26 kDa) by endogenous furin, whereas in the presence
of exogenous furin overexpression, mature GP\textsubscript{0} was
cleaved into three parts, i.e., the GP\textsubscript{1},
GP\textsubscript{1}\textsuperscript{*}and GP\textsubscript{2} (Fig. 4D,
lane 2), as previously demonstrated (29). Full-length GP was
co-transfected with furin in the absence or presence of these MARCH
proteins. In the presence of MARCH1, MARCH2, or MARCH8, furin-mediated
GP cleavage was inhibited because very few GP\textsubscript{1},
GP\textsubscript{1}\textsuperscript{*}, and GP\textsubscript{2} were
produced (Fig. 4D, lane 3/4/5). Thus, these results demonstrated MARCH1,
MARCH2, and MARCH8 could inhibit furin-mediated GP cleavage.

\textbf{3.4 Human MARCH1, MARCH2, and MARCH8 recognized Furin P domain}

As the Co-IP assay and flow cytometry analysis indicated (Fig. 4A and
C), MARCH1 , MARCH2, and MARCH8 interacted with furin. Furin CD and P
domains are critically important for substrate cleavage. To identify
which domain was necessary for recognition by human MARCH1, MARCH2, and
MARCH8, we utilized two previously created furin mutants (29), i.e., the
furin CD deletion mutant (FR\selectlanguage{greek}Δ\selectlanguage{english}CD) and the furin P deletion mutant (FR\selectlanguage{greek}Δ\selectlanguage{english}P),
to perform Co-IP assay with these MARCH proteins. As shown in Fig. 5,
MARCH1, MARCH2, and MARCH8 could bind to both full-length furin and the
FR\selectlanguage{greek}Δ\selectlanguage{english}CD but rarely recognize the FR\selectlanguage{greek}Δ\selectlanguage{english}P, implying these MARCH proteins
recognize furin P domain.

\textbf{4 DISCUSSION}

MARCH proteins have recently been shown to inhibit viral infections by
targeting their envelope glycoproteins. Generally, MARCH proteins
exerted their antiviral functions through two different modes, i.e., the
pattern of CTD or CTI. Human MARCH8 blocked EBOV GP and SARS-CoV-2 S
glycoprotein processing and glycosylation maturation through the CTI
antiviral mode (29, 30). Recent work also indiated human MARCH1, MARCH2,
and MARCH8 inhibited HIV-1, RABV, and many other viral infections
(26-28); mice MARCH1 and MARCH8 suppressed MLV and other viral infection
(27). This study further verified human MARCH1 and MARCH2, bovine
MARCH2, and murine MARCH1 suppressed EBOV GP proteolytic cleavage via
the CTI antiviral mode (Fig. 2B), demonstrating the MARCH protein
antiviral potency is conserved across many mammalian species. The
unprocessed mature glycosylation EBOV GP\textsubscript{0} is notably
larger than GP\textsubscript{1}, as previously demonstrated (29).
However, in the presence of human MARCH1/2, bovine MARCH2, and murine
MARCH1, the mature glycosylated EBOV GP\textsubscript{0} did not appear
(Fig. 2B), indicating these MARCHs also blocked EBOV GP glycosylation
maturation besides inhibiting its cleavage.

Like human MARCH8, human MARCH1 and MARCH2 retained EBOV GP at TGN. They
blocked its transportation to PM, thus reducing its cell surface
presentation (Fig. 3A). MARCH1 displayed a relatively high sequence
homology to MARCH8 but not MARCH2 (Fig. 1). However, they all inhibited
EBOV GP cleavage, implying protein conformation was critical to these
MARCH proteins' CTI antiviral activities. Interestingly, both EBOV
GP\textsubscript{0} and GP\textsubscript{1} were devoid of expression in
the presence of bovine MARCH1 (Fig. 2B). It is uncertain which step
bovine MARCH1 blocked EBOV GP expression, i.e., at the transcription or
expression process, and further work is needed to clarify the accurate
mechanism. In contrast to its human and bovine paralogue, murine MARCH2
lost its blocking on EBOV GP cleavage (Fig. 2B), similar to a previous
report of MARCH2 inactivation in inducing MLV envelope glycoprotein
degradation (27). Given the high sequence homology between human and
murine MARCH2, it would be expected to identify which domains are
critical for their gain or loss of function in the CTI antiviral mode.

Furin cleaves a group of host intracellular proproteins, which regulate
many critical biological processes (38). It also could recognize many
viral envelope glycoproteins, including HIV-1 Env, EBOV GP, IAV H5N1 HA,
and SARS-CoV-2 S (38, 39). Viral glycoproteins cleavage by furin or
other PCs is important for their target cell infection. This study
demonstrated that human MARCH1, MARCH2, and MARCH8 specifically
interacted with furin, thus blocking EBOV GP cleavage (Fig. 4C and D).
Therefore, we deduce that other viral glycoproteins or intracellular
proproteins that critically rely on furin cleavage should also be
sensitive to human MARCH1, MARCH2, and MARCH8 mediated blocking. MARCH1,
MARCH2, and MARCH8 retained EBOV GP in TGN (Fig. 3B) and interacted with
furin and EBOV GP (Fig. 4C). These lines of evidence proved that MARCH1,
MARCH2, and MARCH8 locked the MARCH1/MARCH2/MARCH8-furin-EBOV GP complex
in TGN and thereby prevented their anterograde transport to PM, which
clarified a conserved CTI antiviral mode among these human MARCH
proteins (the antiviral mode is summarized in Fig. 6). Notably, furin is
an autocleavage protein, and mature furin is cleaved by itself at TGN or
the cell surface and then secreted outside the cells. Our results showed
that intracellular furin expression was enhanced in the presence of
human MARCH proteins (Fig. 4D, lane 3/4/5), probably because these human
MARCH proteins blocked furin autocleavage activity and retained it at
TGN, thus leading to its intracellular accumulation.

Besides MARCH8 (29), many host antiviral proteins also targeted furin to
block virus replication (40). In this report, we demonstrated that human
MARCH1 and MARCH2, and maybe bovine MARCH2 and murine MARCH1, also
inhibited furin-mediated cleavage of EBOV GP. Thus, targeting cellular
furin or other PCs to restrict enveloped viruses' infections may be a
conserved defense mode employed by host antiviral factors and deserves
further exploration.

Generally, in pursuit of survival, viruses evolve some viral proteins to
combat host cell antiviral factors. For instance, HIV-1 Vpu and Nef
could antagonize cellular BST-2 and SERINC5, respectively (41-43);
SARS-CoV-2 ORF7a counteracted SERINC5 antiviral effects (44). Though
MARCH proteins were proven to inhibit viral replication potently, less
viral antagonizing mechanisms have been discovered.

Only recently, Liu et al. reported that the M2 protein of IAV H1N1 could
evade MARCH8 induced degradation through its cytoplasmic tail K78/K79
variation (33). Senecavirus A (SVA) 2AB was discovered to induce MARCH8
autophagic degradation (45). MARCH1, MARCH2, and MARCH8 are highly
expressed in monocyte-derived macrophages (MDM) (24, 26), which are
critical targets for EBOV infection. Thus, to effectively replicate in
MDMs, whether EBOV needs to overcome these human MARCH proteins'
restriction deserves to be investigated.

\textbf{5 CONCLUSION}

In summary, we revealed a conserved CTI antiviral mode employed by
mammalian MARCH1/2 proteins to inhibit EBOV GP furin-mediated cleavage,
which provides new insights for current antiviral studies and may
facilitate the development of novel antiviral strategies.

\textbf{AUTHOR CONTRIBUTIONS}

Changqing Yu, Wenbo Tan, and Yu Bai contributed to the concept and
design of this work, acquisition, analysis, interpretation of data, and
manuscript preparation; Yulong Zhou, Jingbo Zhai, and Mengzhou Xue
contributed to analysis, discussion, or acquisition of data; Qiang Liu
and Chunfu Zheng contributed to the concept of this work, acquisition of
data, images acquisition, analysis and interpretation of data, and
manuscript revision. All authors contributed to this study and approved
the submitted version.

\textbf{ACKNOWLEDGMENTS}

We thank professor Kenzo Tokunaga, professor Shuanghui Yin, and Dr. Nan
Wang for the reagents, materials, and technical support and assistance.
This work was supported by Natural Science Funding Key Program of Yibin
Vocational and Technical College (Grant No. ZR22ZD-01), and the Science
and Technology Planning Project of Yibin (Grant No. YBZYSC22BK07) to
Changqing Yu; the Nanchong Vocational and Technical College for Basic
Scientific Research (Grant No. ZRA1904), and the Science and Technology
Planning Project of Nanchong (Grant No. 22XCZX0003) to Qiang Liu.

\textbf{CONFLICT OF INTEREST STATEMEN}

The authors declare that they have no conflict of interests.

\textbf{DATA AVAILABILITY STATEMENTS}

The datasets used during the study are included in this article, and
materials are available from the corresponding author on request.

\par\null

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\textbf{FIGURE LEGENDS}

\textbf{Figure 1. Amino acid (aa) sequence alignment of MARCH
proteins.}Human MARCH8 aa sequence was aligned with MARCH1 and MARCH2.
Dots represent identical aa, while dishes indicate the deletion. M1,
human MARCH1; M2, human MARCH2; M8, human MARCH8.

\textbf{Figure 2. MARCH proteins blocked EBOV GP cleavage.} A.
human/bovine/murine MARCH1/2-GFP and human MARCH8-GFP vectors were
transfected into HeLa cells. Nuclei were stained with DAPI, and
fluorescent signals were detected by confocal microscopy. B. GP
(full-length) was co-expressed with human/bovine/murine MARCH1/2 or
human MARCH8 in 293T cells. GP was detected with anti-FLAG, and MARCH
proteins were detected with anti-HA. C. GP\selectlanguage{greek}Δ\selectlanguage{english}MLD, pNL-Luc-\selectlanguage{greek}Δ\selectlanguage{english}Env, and MARCH
expression vectors were co-transfected into 293T cells. Supernatants
containing the pseudovirions were collected to infect Vero cells.
Infectivity was indicated as luciferase values. D. VSV-G, pNL-Luc-\selectlanguage{greek}Δ\selectlanguage{english}Env,
and human MARCH1/2 expression vectors were co-transfected into 293T
cells. Supernatants containing the pseudovirions were collected to
infect Vero cells. E. GP (full-length) was co-expressed with human
MARCH1/2 in human Huh-7 cells. GP was detected with anti-FLAG, and MARCH
proteins were detected with anti-HA. HM, human MARCH; BM, bovine MARCH;
MM, murine MARCH; M1, human MARCH1; M2, human MARCH2.

\textbf{Figure 3. Intracellular co-localization assay for MARCH proteins
and EBOV GP.} A. MARCH1/2 was co-expressed with the GP\selectlanguage{greek}Δ\selectlanguage{english}MLD-GFP or empty
vector in HeLa cells. GP\selectlanguage{greek}Δ\selectlanguage{english}MLD-GFP was used as a control. MARCH1/2 was
detected by anti-HA, and nuclei were stained with DAPI. Fluorescent
signals were monitored by confocal microscopy. B. In HeLa cells, the
MARCH1/2/8-VN/GP\selectlanguage{greek}Δ\selectlanguage{english}MLD-VC BiFC pairs were co-expressed with CNX-mCherry or
TGN-mCherry, and nuclei were stained with DAPI. Fluorescent signals were
collected through confocal microscopy. M1, human MARCH1; M2, human
MARCH2; M8, human MARCH8; TGN-MC, TGN-mCherry; CNX-MC, CNX-mCherry.

\textbf{Figure 4. MARCH proteins suppressed EBOV GP intracellular
cleavage by furin.} A. MARCH1/2/8-VN/furin-VC BiFC pairs were
co-transfected into 293T cells. After 36 h, cell samples were collected
and pretreated for flow cytometry assay. MARCH8-VN was previously
indicated negative for any fluorescent signals (29). B.
MARCH1/2/8-VN/furin-VC BiFC pairs were co-transfected into HeLa cells.
The MARCH1/2/8-VN proteins were fused with a C-terminal HA tag. Cells
were stained with fluorescent anti-HA to detect MARCH proteins and with
DAPI to detect nuclei. Fluorescent signals were obtained under confocal
microscopy. C. FLAG-tagged furin was co-expressed with HA-tagged
MARCH1/2 and HA-tagged GP\selectlanguage{greek}Δ\selectlanguage{english}MLD proteins in 293T cells. Samples were
immunoprecipitated with anti-FLAG and used for WB assay. Furin was
detected with anti-FLAG, and MARCH1/2 and GP\selectlanguage{greek}Δ\selectlanguage{english}MLD were detected by
anti-HA. D. GP was co-expressed with furin and MARCH1/2/8. GP and furin
were detected by anti-FLAG, and MARCH1/2/8 were detected with anti-HA.
FR, furin; M1, human MARCH1; M2, human MARCH2; M8 human MARCH8; SP,
signal peptide.

\textbf{Figure 5. Critical domain identification of furin interacted
with human MARCH1/2/8.} Full-length or Flag-tagged \selectlanguage{greek}Δ\selectlanguage{english}CD/\selectlanguage{greek}Δ\selectlanguage{english}P furin was
co-immunoprecipitated with HA-tagged MARCH1/2/8. Furin was detected with
anti-FLAG, and MARCH1/2/8 were detected with anti-HA. M1, human MARCH1;
M2, human MARCH2; M8 human MARCH8; \selectlanguage{greek}Δ\selectlanguage{english}CD/\selectlanguage{greek}Δ\selectlanguage{english}P, furin \selectlanguage{greek}Δ\selectlanguage{english}CD/\selectlanguage{greek}Δ\selectlanguage{english}P mutant.

\textbf{Figure 6. Antiviral model of MARCH molecules in inhibiting EBOV
GP maturation.} MARCH proteins were localized in membrane-associated
compartments, including ER, TGN, endosomes, and PM. EBOV GP experiences
mature glycosylation modification at TGN and is cleaved by Furin into
the GP1 and GP2 subunits. The GP1 and GP2 are re-linked and transported
from TGN to PM. However, in the presence of MARCH proteins, furin
interacted with both MARCHs and EBOV GP and formed the MARCH-furin-EBOV
GP complex at TGN. Resultantly, EBOV GP cleavage/glycosylation
maturation was blocked and was trapped by MARCH proteins at TGN, which
blocked its translocation from TGN to PM. PM, plasma membrane; TGN,
trans-Golgi network; ER, endoplasmic reticulum.\selectlanguage{english}
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