Role of the Carbohydrate-Binding Sites of Griffithsin in the Prevention of DC-SIGN-Mediated Capture and Transmission of HIV-1

Abstract

BackgroundThe glycan-targeting C-type DC-SIGN lectin receptor is implicated in the transmission of the human immunodeficiency virus (HIV) by binding the virus and transferring the captured HIV-1 to CD4+ T lymphocytes. Carbohydrate binding agents (CBAs) have been reported to block HIV-1 infection. We have now investigated the potent mannose-specific anti-HIV CBA griffithsin (GRFT) on its ability to inhibit the capture of HIV-1 to DC-SIGN, its DC-SIGN-directed transmission to CD4+ T-lymphocytes and the role of the three carbohydrate-binding sites (CBS) of GRFT in these processes.FindingsGRFT inhibited HIV-1(IIIB) infection of CEM and HIV-1(NL4.3) infection of C8166 CD4+ T-lymphocytes at an EC50 of 0.059 and 0.444 nM, respectively. The single mutant CBS variants of GRFT (in which a key Asp in one of the CBS was mutated to Ala) were about ∼20 to 60-fold less potent to prevent HIV-1 infection and ∼20 to 90-fold less potent to inhibit syncytia formation in co-cultures of persistently HIV-1 infected HuT-78 and uninfected C8166 CD4+ T-lymphocytes. GRFT prevents DC-SIGN-mediated virus capture and HIV-1 transmission to CD4+ T-lymphocytes at an EC50 of 1.5 nM and 0.012 nM, respectively. Surface plasmon resonance (SPR) studies revealed that wild-type GRFT efficiently blocked the binding between DC-SIGN and immobilized gp120, whereas the point mutant CBS variants of GRFT were ∼10- to 15-fold less efficient. SPR-analysis also demonstrated that wild-type GRFT and its single mutant CBS variants have the capacity to expel bound gp120 from the gp120-DC-SIGN complex in a dose dependent manner, a property that was not observed for HHA, another mannose-specific potent anti-HIV-1 CBA.ConclusionGRFT is inhibitory against HIV gp120 binding to DC-SIGN, efficiently prevents DC-SIGN-mediated transfer of HIV-1 to CD4+ T-lymphocytes and is able to expel gp120 from the gp120-DC-SIGN complex. Functionally intact CBS of GRFT are important for the optimal action of GRFT.

Griffithsin: An Antiviral Lectin with Outstanding Therapeutic Potential

“…Single mutations of any one of the Asp residues to Ala slightly weaken the affinity of binding to HIV-1 envelope glycoprotein gp120. However, the single mutations significantly decrease the potency in inhibition in single round HIV infection and cytopathicity assays as well as cell-to-cell transmission assays (cocultivation assays) [18,20]. To further study the importance of having three binding sites on each monomer, Xue et al [21] designed a series of mutants where two monomers connected by an amino acid linker were expressed to form an obligate dimer.…”

“…The importance of intermolecular crosslinking by GRFT has been suggested in several publications [18,19,20,21,26]. Evidence has been obtained from the protein perspective as well as from the glycan perspective.…”

“…GRFT has been shown to inhibit both processes [37,41]. In addition, single-site mutations on the mannose-binding site of GRFT have been shown to significantly diminish DC-SIGN associated cell-to-cell transmission [20]. Competition assays have shown that GRFT inhibits the binding of DC-SIGN to gp120 [20,40].…”

“…In addition, single-site mutations on the mannose-binding site of GRFT have been shown to significantly diminish DC-SIGN associated cell-to-cell transmission [20]. Competition assays have shown that GRFT inhibits the binding of DC-SIGN to gp120 [20,40]. GRFT is not cytotoxic to DCs and does not trigger DC apoptosis or maturation [42].…”

Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

“…The known dual roles of DC-SIGN as a facilitator of adaptive immune response as well as promoter of HIV-1 infection prompted us to examine if innate immune soluble factors such as C1q and gC1qR can potentially modulate viral transmission via DC-SIGN (47), similar to reports involving CD4 + T cells (48) and a lectin drug GRFT ( Griffithsia) isolated from the red algae (49). We also included DC-SIGNR (DC-SIGN-related), a homolog of DC-SIGN, in our study.…”