Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition.
The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.
Barrientos
Laura G
LG
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD 20892, USA. lbarrientos1@cdc.gov
Matei
Elena
E
Lasala
Fátima
F
Delgado
Rafael
R
Gronenborn
Angela M
AM
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D015699:HIV Envelope Protein gp120 / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells; D008358:Mannose; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D008362:Mannosides / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D053586:Virus Internalization / Q000187:drug effects*
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D015699:HIV Envelope Protein gp120; D037241:Mannose-Binding Lectins; D008362:Mannosides; D011994:Recombinant Proteins; C107733:cyanovirin N; D008358:Mannose