title: Second site escape of a T20-dependent HIV-1 variant by a single amino acid change in the CD4 binding region of the envelope glycoprotein.
abstract: BACKGROUND
  We previously described the selection of a T20-dependent human immunodeficiency virus type-1 (HIV-1) variant in a patient on T20 therapy. The fusion inhibitor T20 targets the viral envelope (Env) protein by blocking a conformational switch that is critical for viral entry into the host cell. T20-dependent viral entry is the result of 2 mutations in Env (GIA-SKY), creating a protein that undergoes a premature conformational switch, and the presence of T20 prevents this premature switch and rescues viral entry. In the present study, we performed 6 independent evolution experiments with the T20-dependent HIV-1 variant in the absence of T20, with the aim to identify second site compensatory changes, which may provide new mechanistic insights into Env function and the T20-dependence mechanism.
  
  
  RESULTS
  Escape variants with improved replication capacity appeared within 42 days in 5 evolution cultures. Strikingly, 3 cultures revealed the same single amino acid change in the CD4 binding region of Env (glycine at position 431 substituted for arginine: G431R). This mutation was sufficient to abolish the T20-dependence phenotype and restore viral replication in the absence of T20. The GIA-SKY-G431R escape variant produces an Env protein that exhibits reduced syncytia formation and reduced cell-cell fusion activity. The escape variant was more sensitive to an antibody acting on an early gp41 intermediate, suggesting that the G431R mutation helps preserve a pre-fusion Env conformation, similar to T20 action. The escape variant was also less sensitive to soluble CD4, suggesting a reduced CD4 receptor affinity.
  
  
  CONCLUSIONS
  The forced evolution experiments indicate that the premature conformational switch of the T20-dependent HIV-1 Env variant (GIA-SKY) can be corrected by a second site mutation in Env (GIA-SKY-G431R) that affects the interaction with the CD4 receptor.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, The Netherlands. c.e.baldwin@amc.uva.nl
authors.forename: Chris E
authors.initials: CE
authors.lastname: Baldwin
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism*; D002460:Cell Line; D000077560:Enfuvirtide; D019143:Evolution, Molecular; D015686:Gene Products, env / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D015700:HIV Envelope Protein gp41 / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008561:Membrane Fusion; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D014779:Virus Replication
chemical_list: D019380:Anti-HIV Agents; D015704:CD4 Antigens; D015686:Gene Products, env; D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments; D000077560:Enfuvirtide