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×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 GPΔMLD
expression vector was produced via site-directed mutagenesis using
FLAG-tagged EBOV GPΔMLD as the backbone (29). The N-terminal FLAG-tagged
EBOV GPΔMLD 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-ΔCD (furin CD deletion)/Furin-ΔP (furin P
deletion) expression vectors, MARCH8-GFP/EBOV GP-ΔMLD-GFP expression
vectors,
Furin-VC/MARCH8-VN/GP-ΔMLD-VC
expression vectors, calnexin-mCherry /TGN46-mCherry expression vectors,
and pNL-Luc-ΔEnv proviral reporter vector were used as previously
described (29).