Showing papers by "Jun Komano published in 2006"

Mutations of Conserved Glycine Residues within the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 gp41 Can Inhibit Membrane Fusion and Incorporation of Env onto Virions

Abstract: SUMMARY: The membrane-spanning domain (MSD) of HIV-1 envelope protein (Env) has an additional glycine residue within a well-conserved putative transmembrane helix-helix interaction motif, GXXXG, and forms a G 690 G 691 XXG 694 sequence (G, glycine; X, any residues; the numbering indicates the position within the Env of an infectious molecular clone, HXB2). Different from vesicular stomatitis virus G (VSV-G), the glycine residues of the GXXXG motif of HIV-1 showed higher tolerance against mutations, and a simultaneous substitution of G690 and G694 with leucine residues only modestly decreased fusion activity and replication capacity of HIV-1. When G691 was further substituted with alanine, phenylalanine or leucine residue while G690 and G694 were substituted with leucine residues, the efficiency of membrane fusion decreased, with the decrease greatest occurring with the leucine substitution, a less severe decrease with phenylalanine, and the least severe decrease with alanine. Substitution with leucine residue also decreased the incorporation of Env onto virions, and the mutant showed the most delayed replication profile. Thus the presence of the extra glycine residue, G691, may increase the tolerance of the other two glycine residues against mutations than VSV-G. The fact that a more severe defect was observed for the leucine residue than the phenylalanine residue suggested that the function of Env depended on the steric nature rather than on the simple volume of the side chain of the amino acid residue at position 691. Based on this result, we propose a hypothetical model of the association among MSDs of gp41, in which G 691 locates itself near the helix-helix interface.

Rapid propagation of low-fitness drug-resistant mutants of human immunodeficiency virus type 1 by a streptococcal metabolite sparsomycin.

Abstract: Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.