Complex determinants in human immunodeficiency virus type 1 envelope gp120 mediate CXCR4-dependent infection of macrophages

Host cell range, or tropism, combined with coreceptor usage defines viral phenotypes as macrophage tropic using CCR5 (M-R5), T-cell-line tropic using CXCR4 (T-X4), or dually lymphocyte and macrophage tropic using CXCR4 alone or in combination with CCR5 (D-X4 or D-R5X4). Although envelope gp120 V3 is necessary and sufficient for M-R5 and T-X4 phenotypes, the clarity of V3 as a dominant phenotypic determinant diminishes in the case of dualtropic viruses. We evaluated D-X4 phenotype, pathogenesis, and emergence of D-X4 viruses in vivo and mapped genetic determinants in gp120 that mediate use of CXCR4 on macrophages ex vivo. Viral quasispecies with D-X4 phenotypes were associated significantly with advanced CD4+-T-cell attrition and commingled with M-R5 or T-X4 viruses in postmortem thymic tissue and peripheral blood. A D-X4 phenotype required complex discontinuous genetic determinants in gp120, including charged and uncharged amino acids in V3, the V5 hypervariable domain, and novel V1/V2 regions distinct from prototypic M-R5 or T-X4 viruses. The D-X4 phenotype was associated with efficient use of CXCR4 and CD4 for fusion and entry but unrelated to levels of virion-associated gp120, indicating that gp120 conformation contributes to cell-specific tropism. The D-X4 phenotype describes a complex and heterogeneous class of envelopes that accumulate multiple amino acid changes along an evolutionary continuum. Unique gp120 determinants required for the use of CXCR4 on macrophages, in contrast to cells of lymphocytic lineage, can provide targets for development of novel strategies to block emergence of X4 quasispecies of human immunodeficiency virus type 1.     

Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties.

The tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") are known to significantly enhance the binding affinity of oligonucleotides to their complementary target DNA or RNA strands. To investigate their effect on the nuclease resistance, they were incorporated into model oligomers with a natural phosphodiester backbone, and enzymatic degradation was monitored in an in vitro assay with snake venom phosphodiesterase as the hydrolytic enzyme. In both cases, a single incorporation at the 3'-terminus completely protected the oligonucleotides against 3'-exonuclease attack. Further investigations indicate that the observed high nuclease resistance is not due to the lack of binding affinity to the enzyme's active site, since these modified oligonucleotides were able to inhibit degradation of a natural DNA fragment by bovine intestinal mucosal phosphodiesterase in a dose-dependent manner.     

Use of [4,6-Di-14C]-5′-DMT-thymidine Phosphoramidite Reagent for the Radiolabeling of Synthetic Oligonucleotides

Abstract

A novel synthesis of 5′-radiolabeled oligonucleotides is described. The labeling is carried out by the phosphoramidite method with the aid of building block 1. The feasibility of the method is demonstrated by preparation of 5′-radiolabeled 3′-phosphorylated dodecathymidylate phosphorothioate.     

Positioning of Functionalities in a Heteroduplex Major Groove: Synthesis of 7-Deaza-2-Amino-2′-deoxyadenosines

Abstract

We have synthesized the 7-iodo-, 7-cyano-and 7-propynyl-7-deaza-2-ainino-2′-deoxyadenosines and incorporated each into several oligonucleotide (ODN) sequences. These oligonucleotides exhibit enhanced binding affinities to RNA complements relative to unmodified sequences.     

Synthesis of Chimerical Oligonucleotides Containing Internucleosidic Phosphodiester and S-Pivaloyl Mercaptoethyl Phosphotriester Linkages

Abstract

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages and their thioate analogs are described. Their synthesis involves new methodology for the deprotection of base-labile oligonucleotides.     

Synthesis of Fully Protected Nucleoside- Folic Acid Conjugated Phosphoramidites and Their Incorporation into Antisense Oligonuleotides

Abstract

A novel synthesis of the nucleoside-folic acid conjugates has been accomplished. This approach allowed us to synthesize several analogs, which were converted to phosphoramidites and successfully incorporated into therapeutically active antisense oligonucleotides.