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15844388238
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note
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5 of each cell type per well) and incubated 3 hours at 37°C. β-Gal was detected by in situ staining with X-Gal. Because more stained cells were consistently observed with the total library compared to the single plasmid control, the library was subdivided into 1000 fractions (-4000 plasmids each), and pools of the fractions were screened in a similar fashion until a single positive fraction was identified. This fraction was further subdivided into 1000 subfractions (∼4 plasmids each) and screened in the same way. A positive subfraction was identified and plated onto agar. and individual colonies were picked and assayed. A single positive clone was identified (designated pcDNA3-fusin).
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16
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15844385009
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note
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There are five potential open reading frames in the cDNA sequence; the longest one encoding fusin starts with the first ATG, which has a surrounding sequence consistent with efficient translation initiation in vertebrate cells. To rule out the possibility that the functional activities might be encoded by the other open reading frames, we subcloned into pcDNAS a cDNA fragment containing only the remaining four downstream open reading frames. Unlike the full-length cDNA, the truncated fragment did not allow CD4-expressing NIH 3T3 cells to undergo fusion.
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17
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0027217002
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H. Herzog, Y. J. Hort, J. Shine, L. A. Selbie, DNA Cell Biol. 12, 465 (1993).
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22
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15844382571
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note
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Compared to the nucleotide sequence reported for the cDNA (21), there is one difference in the 3′ untranslated region: whereas that sequence has eight consecutive T residues beginning at nucleotide 1076. the fusin sequence has seven.
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23
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15844369573
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note
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Plasmid pcDNAS-fusin was digested with Xho I and blunt-ended with the Klenow fragment of E. coli DNA polymerase. The fusin cDNA insert was excised by digestion with Eco Rl and ligated into Stu I-Eco Rl-digested pSC59 (24). The resulting plasmid, designated pYF1-fusin, was used to generate vaccinia recombinant vCBYF1-fusin.
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24
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15844419939
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National Institute of Allergy and Infectious Diseases, personal communication
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S. Chakrabarti and B. Moss, National Institute of Allergy and Infectious Diseases, personal communication.
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Chakrabarti, S.1
Moss, B.2
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25
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0000498346
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F. M. Ausubel et al., Eds. Wiley, New York
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Earl, P.L.1
Cooper, N.2
Moss, B.3
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26
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15844398801
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note
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2-terminus of fusin (MEGISIYTSDNY-TEEMGSGDYDSMKEPCFREENANFNK; abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr) was synthesized by standard FMOC chemistry, purified by reversed-phase high-performance liquid chromatography (HPLC), and analyzed by analytical HPLC, mass spectrometry, and amino acid sequencing. The purified peptide was conjugated to keyhole limpet hemocyanin by means of the Cys residue with the use of m-maleimidobenzoyl-N-hydroxysuccinimide ester (108 peptides per carrier molecule). New Zealand White rabbits were immunized with 200 μg of conjugate emulsified in RiBi adjuvant (RiBi, Hamilton, MT) three times at 28-day intervals For preparation of purified immunoglobulin G, the serum was subjected to affinity chromatography on protein G sepharose.
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28
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15844423647
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note
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+ transformant of Mv 1 Lu (also containing the neomycin resistance gene) (3) was transfected with pZeoSV-fusin (with the use of DOTAP; Boeringer Mannheim); as a negative control, a monolayer of the same cells was transfected with pZeoSV-LacZ (Invitrogen) containing the lacZ gene linked to the SV40 promoter plus the Zeocin resistance gene. After 24 hours, the cells were washed and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum plus antibiotics (during the first 2 days with 1 mg/ml of G418 alone; subsequently, with G418 plus 1 mg/ml of zeocin). Colonies resistant to both antibiotics were picked, amplified, and screened for Env-dependent fusion permissiveness in the vaccinia assay system (with the use of luciferase as the reporter).
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29
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15844414355
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Y. Feng et al., data not shown
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Y. Feng et al., data not shown.
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B. R. Cullen, Ed, Oxford Univ. Press, New York
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50
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15844369572
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National Center for Human Genome Research, personal communication
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R. A. Morgan, National Center for Human Genome Research, personal communication.
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Morgan, R.A.1
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52
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15844362857
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note
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+ transformant of Mv 1 Lu; to M. Garfield, J. Lukszo, and J. Coligan, Laboratory of Molecular Structure, NIAID, for synthetic peptide synthesis, purification, and coupling; and to J. Sisler, Laboratory of Viral Diseases, NIAID, for oligonucleotide synthesis and assistance with DNA sequencing. We thank P. Murphy, NIAID, and B. Moss, NIAID, for helpful discussions and comments on the manuscript.
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