Determining the Resistance Profile of Conocurvone, an Early-Stage Inhibitor of HIV-1 Replication
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Date
2001-03
Department
Hood College Biology
Program
Biomedical and Environmental Science
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Abstract
A unique inhibitor of HIV-1, conocurvone, was isolated from extracts of the
Australian shrub Conospermum incurvum. This compound, a trimeric
napthoquinone, is a potent inhibitor of HIV-1-induced cell killing (EC50 < 20uM)
that is relatively strain specific (HIV-1 RF > HIV-1 NL4-3 >>> HIV-1 111B). In vitro
mechanism of action studies revealed that conocurvone may possess dual
inhibitory activity against both HIV-1 integrase and HIV cell-mediated fusion. To
determine whether either of these mechanisms is a biologically relevant viral
target for conocurvone, a drug-resistant HIV-1 RF virus was generated by
passaging under drug selection in H9 cells. This virus, denoted cono-R, was
used as a template to identify conocurvone resistance-engendering mutations
and to determine the mechanism of action of conocurvone. Genotypic analysis
of this cono-R virus revealed four amino acid changes in gp120, four in gp41,
and none in integrase, suggesting that conocurvone targets the HIV-1 envelope
protein. One of the gp41 mutations introduces a premature stop codon at
position W766 and, as a result, truncated more than 100 amino acids from the
cytoplasmic tail of the HIV envelope protein, similar to some CD4-independent
viruses. Interestingly, when an analogous proviral clone (HIV-1pNL4-3 W766*)
was constructed, the resultant virus was replication-defective yet able to fully
induce syncitia formation in CEM-SS cells. Three of the cono-R gp120
mutations, V64A, G145E, and N315K conferred conocurvone-resistance to NL4-
3 when introduced into the pNL4-3 provirus. The G145E and N315K mutations
are located within the V1/V2 and the V3 loop respectively. These variable loops
have been implicated in determining viral coreceptor usage. These resistance
selection data indicate that conocurvone targets gp120 of HIV-1 and inhibits HIV
fusion in T cells. This study suggests that HIV-1 may escape the activity of
fusion inhibitors by introducing specific mutations that alter envelope
conformation and receptor usage.