title: Short communication: Simultaneous substitutions of V38M and N43T-N44K in the gp41 heptad repeat 1 (HR1) disrupt HIV type 1 gPr160 endoproteolytic cleavage (*).
abstract: We cloned and sequenced gp41 HIV-1 from plasma of AIDS patients under HAART and T-20 (enfuvirtide, Fuzeon) therapy and revealed several T-20 resistance-associated mutations. Two mutations, a single V38A and a double N43T-N44K were the most frequent; however, they were not found together in one clone. We anticipated that simultaneous mutations of these three residues might play a vital role in the viral life cycle. To address this problem, we introduced N43T-N44K and V38M + N43T-N44K substitutions to a cloned gp41 and introduced modified gp41 into the pNL4-3 molecular clone. HEK293T cells were transfected with the obtained vectors and released viruses were examined for reverse transcriptase (RT) activity, infectivity on reporter TZM-bl cells, and in Western blotting. Nearly equal RT activity was demonstrated in viruses with and without mutations. However, viruses with the V38M + N43T-N44K mutations were not infectious and, as shown by Western blotting, gPr160 cleavage was impaired. These data suggest that V38M + N43T-N44K mutations perturbed the natural conformation of gPr160 in a way that access of furin to the cleavage site (REKR) was blocked. Therefore, the residues V38 + N43-N44 retain the gPr160 conformation in proximity to the furin cleavage site and, as a consequence, are critical for virus infectivity. These data may explain why viruses with V38M + N43T-N44K mutations were not previously detected in the plasma of T-20-experienced patients.
authors.affiliation: Institute of Human Virology, University of Maryland, Baltimore, Maryland 21201, USA. Morozovv@rki.de
authors.forename: V A
authors.initials: VA
authors.lastname: Morozov
authors.forename: A V
authors.initials: AV
authors.lastname: Morozov
authors.forename: S
authors.initials: S
authors.lastname: Lagaye
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D023241:Antiretroviral Therapy, Highly Active; D002460:Cell Line; D024882:Drug Resistance, Viral; D045683:Furin / Q000378:metabolism; D019302:HIV Envelope Protein gp160 / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000235:genetics*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011499:Protein Processing, Post-Translational
chemical_list: D019302:HIV Envelope Protein gp160; D015700:HIV Envelope Protein gp41; C514797:gp160 protein, Human immunodeficiency virus 1; C514787:gp41 protein, Human immunodeficiency virus 1; D045683:Furin