Abstract
The third hypervariable region, or V3 loop, represents the principal neutralizing domain of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequential viral isolates from a laboratory worker (LW) accidentally infected with HIV-1IIIB in 1985 were analyzed using type-specific neutralizing monoclonal antibodies directed to the V3 loop. A single amino acid substitution, Ala-->Thr at position 21 in the V3 loop of HIV-1LW isolated in 1987, was shown to determine the loss of the neutralizing epitope recognized by one of the monoclonal antibodies (M77). However, this antibody efficiently recognized linear V3 loop peptides containing either the Ala or Thr residue at position 21, indicating that a local change in conformation was responsible for the epitope loss in the native gp120. Molecular modeling studies, experimentally supported by different amino acid replacements at position 21, indicated that the Ala-->Thr substitution leads to a drastic change in the domain of the V3 loop, which contains the complementary surface for antibody binding. These results provide evidence for the first time that a conformation-dependent epitope within the V3 loop of HIV-1 is involved in the generation of neutralization escape mutants in vivo.
Highlights
Worker (LW)accidentally infected witHh N-lIIIBin 1985 Different approaches have beenfollowed in order toidentify wereanalyzedusingtype-specificneutralizingmono- amino acid changes which are critical for neutralization
A distinctive worker who became accidentally infectedwith HIV-1111~(Weiss characteristic of this loop is the extensive degree of genetic et al, 1988).This unique in vivo human model allowed US to variability found among individual isolates of HIV-1
Estimated by comparing its conformationalenergy with that of tions can be made: 1)the Ala 4 Ser substitution at position the extended V3 loop structure where GPGR forms a type I1 21 should not result in the loss of binding by M77, because Ala turn (LaRosa et al.,1990;Gupta and Myers, 1990),and there- and Ser have a similar size and thesmall side chains of maining amino acid residues adopt extended conformations, Ser can be accommodated in thespecific cavity
Summary
96-well plates by coculturing 1 x lo uninfected cells with 5 x lo virus-infected cells. 1 am~ino aci~d sequ~ence, f'orming a cas~sette in~to wh~icholig~onucleo tides ' representing V3 coding regions and containing an MluI 4-base overhang a t one end and a blunt end a t the other end can be ligated. SupTl cells infected with HIV-lIIIB (A) or LW87 ( B )and HeLat a t cells transfected with pHXB2 (enu LW85-2) (C) were labeled with [35Slcysteine.The clarified lysates from cells and virus containing supernatants were immunoprecipitated with an HN-1positive human serum (lane I), M77 (lane2 ) ,and 0.5p (lane). The amino acid sequence of the V3 loopwas converted into secondary structural stateusing a prediction algorithm (Deleage and Roux, 1989; Gupta and Myers, 1990) so that each amino acid was assigned one of the four secondary states, i.e. helix
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