title: Proline residues in the HIV-1 NH2-terminal capsid domain: structure determinants for proper core assembly and subsequent steps of early replication.
abstract: Recent analyses suggest that the p24 capsid (p24(CA)) domain of the HIV-1 group-specific antigen (Gag) may be divided into two structurally and functionally distinct moieties: (i) an amino-terminal portion, previously shown to bind the cellular chaperone cyclophilin A, and (ii) a carboxy-terminal domain, known to contribute to the interaction of the Gag and Gag-Pol precursors during the early assembly process. In order to gain deeper insight into the role of the amino-terminal domain of the p24(CA) protein during viral replication, eight highly conserved proline residues known to promote turns and to terminate alpha-helices within the p24 tertiary structure were replaced by a leucine residue (P-position-L). Following transfection of the proviral constructs in COS7 cells, the majority of the mutants resembled wild-type viruses with respect to the assembly and release of virions. However, although the released particles contained wild-type levels of genomic viral RNA, the mature products of the Gag and Gag-Pol polyproteins as well as the Env glycoproteins-all of them, except mutant P225L-were either noninfectious or severely affected in their replicative capacity. Entry assays monitoring the process of viral DNA synthesis led to the classification of selected provirus mutants into four different phenotypes: (i) mutant P225L was infectious and allowed complete reverse transcription including formation of 2-LTR circles; (ii) mutants P149L, P170L, and P217L failed to form 2-LTR circles; (iii) mutant P222L displayed a severe defect in binding and incorporating cyclophilin A into virions, was delayed with respect to DNA polymerization, and failed to form a 2-LTR replication intermediate; and (iv) mutant P133L was unable even to synthesize a first-strand cDNA product. All replication-defective mutants were characterized by severe alterations in the stability of virion cores, which were in two cases reflected by visible changes in the core morphology. These results suggest that proline residues in the NH(2)-terminal capsid domain represent critical structure determinants for proper formation of functional virion cores and subsequent stages of early replication.
authors.affiliation: Institute of Medical Microbiology, Institute of Pathology, University of Regensburg, Franz-Josef-Strauss Allee 11, Regensburg, D-93053, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Fitzon
authors.forename: B
authors.initials: B
authors.lastname: Leschonsky
authors.forename: K
authors.initials: K
authors.lastname: Bieler
authors.forename: C
authors.initials: C
authors.lastname: Paulus
authors.forename: J
authors.initials: J
authors.lastname: Schröder
authors.forename: H
authors.initials: H
authors.lastname: Wolf
authors.forename: R
authors.initials: R
authors.lastname: Wagner
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015683:Gene Products, gag / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D011392:Proline / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014771:Virion / Q000235:genetics / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D019065:Virus Assembly / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
chemical_list: D015683:Gene Products, gag; D016655:HIV Core Protein p24; D015699:HIV Envelope Protein gp120; D010455:Peptides; D011498:Protein Precursors; D012367:RNA, Viral; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D011392:Proline; D019696:Peptidylprolyl Isomerase