HIV-1 integrase (IN) is composed of three domains, the N-terminal domain (NTD, residues 1-50), the catalytic core domain (CCD, residues 51-212), and the C-terminal domain (CTD, residues 213-288). All the three domains are required for the two known integration reactions. CCD contains the catalytic triad and is believed to bind viral DNA specifically, and CTD binds viral DNA in a nonspecific manner. As no clear evidence has confirmed the involvement of NTD in DNA binding directly, NTD has not been seriously considered and less is known about its function in viral replication. In the current work, using a SPR technology-based assay, the HIV-1 viral DNA was determined to bind directly to NTD with a K(D) value of 8.8 microM, suggesting that the process of preintegrated complex formation for HIV-1 IN might involve the direct interaction of NTD with viral DNA in addition to binding of viral DNA to the catalytic core domain and C-terminal domain. Moreover, such viral DNA/IN binding could be inhibited by the marine product hyrtiosal from the marine sponge Hyrtios erectus with an IC(50) of 9.60+/-0.86 microM. Molecular dynamic analysis correlated with a site-directed mutagenesis approach further revealed that such hyrtiosal-induced viral DNA/IN binding inhibition was caused by the fact that hyrtiosal could bind HIV-1 NTD at Ser17, Trp19, and Lys34. As hyrtiosal was recently discovered by us as a protein tyrosine phosphatase 1B (PTP1B) inhibitor,1 this work might also supply multiple-target information for this marine product, and the verified HIV-NTD/HIV-1 IN interaction model could have further implications for new HIV-1 IN inhibitor design and evaluation.