Abstract:
OBJECTIVE: To ascertain the sequence changes that account for the enhanced replicative capacity of the ELI strain of HIV-1 passaged in H9 cells. METHODS: Virus stocks prepared from HeLa cells transfected with molecular clones of HIV-1ELI were used to infect peripheral blood mononuclear cells (PBMC) and CD4-positive cell lines. DNA isolated from infected cells was analyzed by PCR-SSCP (Single Strand Conformation Polymorphism) to locate sequence changes in the HIV-1 genome. These identified mutations were introduced into the parent genome, and the growth properties of the resulting viruses were characterized. RESULTS: Virus derived from a molecular clone of the ELI strain of HIV-1 exhibits delayed replication kinetics on H9 cells and is unable to infect productively U937 cells. When the virus that emerged from H9 cells was used to infect different CD4-positive cell lines, it was found to have acquired altered growth characteristics, viz., it exhibited accelerated growth kinetics on H9 cells and was able to infect U937 cells. Sequence changes in the genomes of the passaged virus were mapped to the env gene using PCR-SSCP. Clones were found with sequence changes in the N-terminus of gp41, a region involved with fusion, and with changes in both the N-terminus of gp41 and in the CD4-binding region of gp120. No changes were found in the immunodominant V3 loop region, a known determinant of viral tropism. Mutations in gp41 alone conferred upon ELI an enhanced replication capacity in H9 cells, since the mutant viruses infected these cells without the delay in virus production seen with the parent virus. The additional change in the CD4-binding site of gp120 was necessary before ELI was capable of infecting U937 cells. None of these mutations affected the ability of ELI to replicate in PBMC. CONCLUSIONS: These results demonstrate that HIV-1 can adapt to growth in certain tissue culture cell lines by selecting for variants with mutations in the env gene. These data also show that the CD4-binding region of gp120 and the fusogenic region of gp41 are determinants of HIV-1 tropism. While the mechanism of the enhanced replicative capacity remains to be elucidated, the locations of the mutations strongly suggest that increased virus uptake is involved.
Keywords: Antigenic Variation/GENETICS Cells, Cultured Comparative Study CD4-Positive T-Lymphocytes/MICROBIOLOGY *Genes, env Hela Cells/MICROBIOLOGY Human HIV Envelope Protein gp120/*GENETICS/PHYSIOLOGY HIV Envelope Protein gp41/*GENETICS/PHYSIOLOGY HIV-1/*GENETICS/PHYSIOLOGY Leukocytes, Mononuclear/MICROBIOLOGY Monocytes/MICROBIOLOGY Mutation Tumor Cells, Cultured/MICROBIOLOGY Virus Replication/*GENETICS ABSTRACT 921230
M92C5317
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