Jpn. J. Infect. Dis., 54, 2001

Laboratory and Epidemiology Communications

The Optimal Molar Ratio between Binding‑DeflCient andFusion‑Deficient Murine Leukemia Virus Envelope Proteinsfor Complementation to Induce Syncytla
Tetsuro Matano *AIDS Research Center; National Institute ofInfectious Diseases,
Gakuen 4‑ 711, Musashi‑murayama, TobJo 208‑0011
Communicated by Hiroshi Yoshikura(Accepted March 14, 2001)

The mprine leukemia virus (MLV) envelope protein (Env)viral entry and cell‑to‑cellfusion. The ecotropic Friend MLV

We previously obtained a chimeric FMLV Env, CD4‑Env,
the RBD of which was completely replaced with a surface
domain of human CD4 (9). The CD4‑Env chimera was(FMLV) Env is oligomirized into a trimer in the endoplasmic

processed, expressed on the cell surface, and incorporated

plays an Important role in inducing membrane fusion during

reticulum, cleaved into the surface protein (SU, gp70) and theinto the virion. When coexpressed, CD4‑Env bound to thetransmembrane protein (TM, p 1 5E) in the Golgi apparatus,

wild‑type FMLV Env to formheter0‑0ligomers. Further, CD4‑
Env contributed the postbindingfusion function and comple‑
mented afusion‑deficient TM mutant to induce membranetransported to the cell surface, and incorporated into the virion(1). At the time of virus budding or shortly thereafter, pl5E
is cleaved into p12E and the C‑teminal 1 6‑amino‑acid longfusion (10). Because the CD4‑Env and the TM mutant are

良‑peptide (2). Expression of the wild‑type Env does not

required to form hetero101igomers in the complementation,induce syncytia in NIH 3T3 cells, whereas expression of thethe fusion efrlCiency may be dependent on the molar ratio ofthe fbmer to the latter. In our previous study, however, We

良‑deleted Env does (3,4). No syncytia are induced by the R‑

deleted FMLV Env in HeLa cells lacking the FMLV receptor.
Env‑mediated membrane fusion is initiated by the binding

examined the complementation between two molecules onlyof SU to the receptor followed by postbinding fusion (I).
The receptor‑binding domain (RBD) resides in the N‑teminal

the complementation to induce syncytia was detemined.

at the molar ratio of 1:I. In this study, the optlmal ratio forhalf of SU (5,6). Receptor‑binding and postbinding fusion

pCXN2 plasmids expressing FE (a wild‑type ecotropic
FMLV Env), FE.T470H (afusion‑deficient FMLV Env with afunctions can be contributed by two different molecules

point mutation in the extracellular domain ofTM), R‑deleted

respectively (7,8)･ ln a previousreport (7), a binding‑dencient

FE.D86K (a binding‑de丘cient FMLV Env with a point muta‑

Env with a polnt mutatlOn in RBD and afusion‑deficient Env
with a point mutation in TM functioned in a complementarytion in RBD),and R‑deleted CD4‑Env, respectively, wereusedfasion to induce membrane fusion.

syncytia by itself, but did by co‑expression with FE in NIH(10,1 1). Expression of 良‑deleted FE.D86K did not induce

3T3 cells. Similar complementation in inducing syncytla Was*Corresponding author: Tel: +8142‑561‑0771 ext. 335, Fax: +81‑

obseⅣed in experiments uslng R‑deleted CD4‑Env instead

42‑565‑33 1 5, E‑mail: matano@nih.go.jp

ofR‑deleted FE.D86K. Also, complementation was obseⅣed32

Jpn. J. Infect. Dig., 54, 2001

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Fig. 1. Symcytia assay on the DNA‑transfected HcLa cells cocultured withNIH 3T3 cells. HeLa cells in each well of6‑wellplates were cotransfected withdifferent amount ratios of DNAs (2.日Lg in totalin each transfection) expressing FE and R‑deleted CD4‑Env (A), FE.T470H and R‑deleted CD4‑Env (B), FE and R‑deleted FE.D86K (C), or FE.T470H and R‑deletedFEID86K (D). Twenty‑four hours aRer the transfection, the DNA‑transfected HeLa cells were diluted serially by two‑foldin quadruplicate, and 1 x 105 NIH 3T3 cells were added to 1 x 105 0fHeLa cells containlng the diluted DNA‑transfectedHeLa cells per well in 121Well plates･ After 241h coculture, 12‑well plates were examinedmicroscopically for syncytia. The
number of syn.cytia produced bythe undiluted transfected HeLa cells in each well (in 61Well plates) was calculated as

described prev10uSly ( I 2). The symcytia level is shown as a value relative to that obtained withthe cells transfected withFEand R(‑)‑CD4‑Env‑expression plasmids at a 1 : 1 ratio, which is rated as 100. Each value obtained丘･om each experiment isshown by open triangle. The mean values obtained舟om three sets of experiments are shown by +.

between FE.T470H and R‑deleted FE.D86K or 良‑deleteddeleted CD4‑Env or R‑deleted FE.D86K, the syncytia level

CD4‑Env. In those complementations, the effect of the molarwas greatly reduced and the optlmal ratio was shi鮎d to theratio of the two molecules on induction or syncytla Was

higher p12E/SU ratio (Fig. lB and lD).

examined.

First, NIH 3T3 cells were co‑transfected with different
molar ratios of R‑deleted CD4‑Env‑ and FE‑expression

from the Ministry of Education, Culture, Sports, Science andplasmids, pCXN2R(‑)CD4‑Env and pCXN2FE, and syncytia
induction was compared. Because the nucleotide length of

Ministry of Health, Labour and Welfare in Japan.

This work was supported by a grant for Scientific ResearchTechnology, and by a grant for AIDS Research from thepCXN2R(〕CD4‑Env is 8.3 kb and that orpCXN2FE is 8. 1kb, the DNA weight ratio approximates the DNA molar ra‑REFERENCES

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