Analysis of sequence requirements for biological activity of cyanovirin-N, a potent HIV (human immunodeficiency virus)-inactivating protein.
Site-directed mutagenesis of DNA constructs coding for the novel, HIV-inactivating proteins cyanovirin-N (CV-N) and FLAG-cyanovirin-N (F-CV-N) was performed using mutagenic oligonucleotide primers in the polymerase chain reaction or by a restriction site elimination maneuver. The mutant constructs were expressed in Escherichia coli and the recombinant protein products were tested for binding to the HIV surface envelope glycoprotein gp 120 and for antiviral activity against infectious HIV. Results showed an overall very high correlation (r2 > 0.9) between the relative gp120 binding affinities and the anti-HIV activities of CV-N, F-CV-N, and the various mutants. An outlier, however, was a mutant which lacked one of the internal disulfide linkages normally present in CV-N and which showed modest gp120 binding but no antiviral activity against HIV. These findings are consistent with the view that gp120 binding is a necessary but not sufficient requirement for the HIV-inactivating activity of CV-N and related proteins; the sequence specificities for gp120 binding and anti-HIV activity are not identical.
Mori
T
T
Laboratory of Drug Discovery Research and Development, National Cancer Institute-FCRDC, Frederick, Maryland 21702-1201, USA.
Shoemaker
R H
RH
Gulakowski
R J
RJ
Krepps
B L
BL
McMahon
J B
JB
Gustafson
K R
KR
Pannell
L K
LK
Boyd
M R
MR
D000595:Amino Acid Sequence / Q000235:genetics / Q000502:physiology*; D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D017421:Sequence Analysis; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004220:Disulfides; D015699:HIV Envelope Protein gp120; C107733:cyanovirin N; D012694:Serine; D003545:Cysteine