Mechanism of inhibition of HIV-1 infection in vitro by guanine-rich oligonucleotides modified at the 5' terminal by dimethoxytrityl residue.

Oligodeoxyribonucleotides (ODN) linked at their 5'-end with dimethoxytrityl (DmTr) residue were examined for antiviral activities against human immunodeficiency virus type 1 (HIV-1). We found that guanine-rich oligonucleotides exhibit anti-HIV activity upon 5'-end modification with DmTr. One oligonucleotide, DmTr-TGGGAGGTGGGTCTG (SA-1042), showed potent anti-HIV activity in vitro. A greater than 95% reduction of infectivity was observed if the cells were treated with 10 micrograms/ml of SA-1042 at the time of viral infection, PCR analysis confirmed that there was a significant reduction of provirus in the cells exposed to virus in the presence of SA-1042. By contrast, no inhibition was observed if the cells were treated with the oligomer 1 h after virus adsorption. SA-1042 prevented syncytium formation between chronically infected cells and CD4 positive uninfected cells. Furthermore, the oligomer interfered the interaction of purified gp120 to the CD4 receptor. By contrast, SA-1042 had no inhibitory effect on chronically HIV-infected cells. These results strongly suggest that the DMTr-ODNs with appropriate base sequences antagonize HIV-1 infection during the stage of virus-cell interaction.     

Effect of structural variations in cholesteryl-conjugated oligonucleotides on inhibitory activity toward HIV-1.

A number of oligonucleotide analogues containing internucleoside phosphorothioate linkages and a covalently attached cholesteryl residue was synthesized and tested for activity against HIV-1 in cultures of Molt3 cells. Structural features important for high antiviral activity are the presence of a cholesteryl moiety, a run of terminal phosphorothioate groups, and the presence of nucleoside residues. An increase in length of the tether between cholesteryl and phosphorus from six to 14 atoms has no significant effect on antiviral activity, and up to one-half of the internucleoside links in a cholesteryl-conjugated phosphorothioate oligomer and one-third of the internucleoside links in a nonconjugated phosphorothioate can be replaced with phosphodiester links without much change in antiviral activity. However, replacement of nucleoside units in the oligomers by a simple analogue (-OCH2CH2CH2O-) yields inactive or very weakly active compounds, even in the presence of a cholesteryl group. Dose-response patterns for assays in which cholesteryl-conjugated oligomers are added to test cells either simultaneously or subsequently to viral infection are similar for homooligomer derivatives and for oligomers containing "antisense" sequences, suggesting a similarity in mode of action for the two classes of oligomers in this system.     

Inhibition of human immunodeficiency virus type 1 activity in vitro by a new self-stabilized oligonucleotide with guanosine-thymidine quadruplex motifs

An oligonucleotide with a dimeric hairpin guanosine quadruplex (basket type structure) (dG3T4G3-s), containing phosphorothioate groups, was able to inhibit human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in peripheral blood mononuclear cells. This oligonucleotide lacks primary sequence homology with the complementary (antisense) sequences to the HIV-1 genome. Furthermore, this oligonucleotide may have increased nuclease resistance. The activity of this oligonucleotide was increased when the phosphodiester backbone was replaced with a phosphorothioate backbone. In vivo results showed that dG3T4G3-s was capable of blocking the interaction between gp120 and CD4. We also found that dG3T4G3-s specifically inhibits the entry of T-cell line-tropic HIV-1 into cells. This compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.     

A monoclonal antibody to CD4 domain 2 blocks soluble CD4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and HIV-1 infection of CD4+ cells.

The murine monoclonal antibody (MAb) 5A8, which is reactive with domain 2 of CD4, blocks human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ cells (L. C. Burkly, D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm, J. Immunol., in press). Here we show that, in contrast to the CD4 domain 1 MAb 6H10, 5A8 and its Fab fragment do not block soluble CD4 (sCD4) binding to virions, whereas they do inhibit sCD4-induced exposure of cryptic epitopes on gp41 and dissociation of gp120 from virions. Two other MAbs, OKT4 and L120, which are reactive with domains 3 and 4 of CD4, have little or no effect on HIV-1 infection, syncytium formation, or sCD4-induced conformational changes in the envelope glycoproteins. The mechanisms of action of 5A8 and 6H10 can be further distinguished in syncytium inhibition assays: 6H10 blocks competitively, while 5A8 does not. We opine that 5A8 blocks HIV-1 infection and fusion by interfering with conformational changes in gp120/gp41 and/or CD4 that are necessary for virus-cell fusion.     

The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T cells

mAb 60.3 and IB4 to CD18, the common beta-subunit of the human leukocytic cell adhesion molecule family, efficiently inhibit syncytium formation induced by the interaction of HIV type 1 (HIV-1)-infected monocytoid cells and CD4+ T cells. The antibodies also interfere with cellfree HIV-1 infection of U-937 clone 16 cells. Virus-induced aggregation of these cells and the subsequent syncytia formation leading to massive cell death are efficiently blocked, and the number of infected cells remains at a very low level, 2 to 5%, for the entire culture period. However, anti-CD18 mAb do not inhibit binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4. The results indicate participation of CD18, or of the protein complex CD11a-c/CD18, in addition to CD4, in the infection and cytopathic effect of HIV-1. They also suggest that intercellular adhesion contributes to virus transmission from cell to cell and may be an important mechanism for virus spreading.