Abstract

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 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 has 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-1(JR-FL) proviral DNA. The constructed HIV-1 library contained 6.6 x 10(6) 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 to 0.30 microM in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated 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.

Highlights

  • Entry of R5 human immunodeficiency virus type 1 (HIV-1)1 into target cells requires sequential interaction of the envelope glycoprotein GP120 with CD4 and the coreceptor CCR5 [1]

  • These results indicated 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

  • We chose 10 amino acid positions in the V3 loop (Fig. 1A), and we incorporated the amino acid substitutions in 31 R5 HIV-1 strains obtained from the Los Alamos HIV data base (//hiv-web.lanl.gov)

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Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Molecular Clones—PM1 [37] and HeLa-CD4-LTR␤-gal (MAGI) cells [38] were provided by the National Institutes of Health AIDS Research and Preference Reagent Program. The ligation mixture was purified by Microcon-30 (Millipore Corp., Bedford, MA) and used to transform Escherichia coli strain DH5␣ by electroporation with an ECM 600 (BTX, San Diego) at 2.5 kV, generating pCR-SX-V3Lib containing 7.1 ϫ 106 independent clones with an AflII-NheI fragment. After purification of the pCR-SX-V3Lib DNA, the StuI-XhoI fragment from 15 ␮g of the plasmid was cloned into the StuI and XhoI sites of pJR-FL⌬SX. The HIV-1 library, designated pJR-FL-V3Lib, contained 6.6 ϫ 106 independent clones containing a 2077-bp (StuI-XhoI) env DNA fragment (ligation efficiency, 77%) (Table I). Viral stocks for analysis of the replication of the virus library HIV-1V3Lib were generated by transient transfection of 293T cells with pJR-FLV3Lib. 5 ϫ 105 of PM1-CCR5 or MT-2 cells were infected with HIV-1JR-FLan or HIV-1V3Lib stocks containing 600 ng of p24 Gag antigen/ ml. The cloned sequences were sequenced using an ABI Prism 310 (Applied Biosystems Inc.)

RESULTS
No independent clonesa
DISCUSSION

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