| 1) Harada, S., Yusa, K., Monde, K., Akaike, T.
and Maeda, Y.: Influence of membrane fluidity on human immunodeficiency
virus type 1 entry. Biochem. Biophys. Res. Commun. 329, 480-486, 2005. |
| For penetration of human immunodeficiency virus type 1
(HIV-1), formation of fusion-pores might be required for accumulating critical
numbers of fusion-activated gp41, followed by multiple-site binding of gp120
with receptors, with the help of fluidization of the plasma membrane and
viral envelope. Correlation between HIV-1 infectivity and fluidity was observed
by treatment of fluidity-modulators, indicating that infectivity was dependent
on fluidity. A 5% decrease in the fluidity suppressed the HIV-1 infectivity
by 56%. Contrarily, a 5% increase in fluidity augmented the infectivity
by 2.4-fold. An increased temperature of 40oC or treatment of 0.2% xylocaine
after viral adsorption at room temperature enhanced the infectivity by 2.6
and 1.5?fold, respectively. These were inhibited by anti-CXCR4 peptide,
implying that multiple-site binding was accelerated at 40oC or by xylocaine.
Thus, fluidity of both the plasma membrane and viral envelope was required
to form the fusion-pore and to complete the entry of HIV-1. |
| HIV-1感染が細胞膜とウイルスエンベロープの流動性に大きく影響されることを示した。5％の流動性の抑制でHIV-1の感染は56％阻害され、5％の流動性亢進で2.4倍感染が促進された。 |
| 2) Yusa, K., Maeda, Y., Fujioka,
A., Monde, K, and Harada, S.: Isolation of TAK-779-resistant HIV-1 from
an R5 HIV-1 gp120 V3 loop library. J. Biol. Chem. (in press) 2005. |
| The human immunodeficiency virus type 1 (HIV-1) envelope
glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor for viral
entry. The V3 loop in gp120 is a crucial region for determining coreceptor
usage during viral entry, and a variety of amino acid substitutions have
been observed in clinical isolates. To construct an HIV-1 V3 loop library,
we chose 10 amino acid positions in the V3 loop and incorporated random
combinations (27,648 possibilities) of the amino acid substitutions derived
from 31 R5 viruses into the V3 loop of HIV-1JR-FL proviral DNA. The constructed
HIV-1 library contained 6.6 x 106 independent clones containing a set of
0-10 amino acid substitutions in the V3 loop. To address whether restricted
steric alteration in the V3 loop could confer resistance to an entry inhibitor,
TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration
of TAK-779 from 0.10 μM to 0.30 μM in PM1-CCR5 cells with high expression
of CCR5. The selected viruses at passage 8 contained 5 amino acid substitutions
in the V3 loop without any other mutations in gp120 and showed 15-fold resistance
compared to the parental virus. These results indicate that a certain structure
of the V3 loop containing amino acid substitutions derived from 31 R5 viruses
can contribute to the acquisition of resistance to entry inhibitors binding
to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for
isolating and analyzing the specific biological features of HIV-1 with respect
to alterations of the V3 loop structure. |
| V3ループの変異ライブラリーを作成し、それぞれのTAK-779の感受性を比較した。今後、同様の戦略で中和抗体とV3変異との関係を調べる。 |