title: Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.
abstract: Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form.
  
  
  OBJECTIVE
  Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.
authors: Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors: Tatsuro
authors: T
authors: Takahata
authors: Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
authors: Eri
authors: E
authors: Takeda
authors: Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Minoru
authors: M
authors: Tobiume
authors: Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Kenzo
authors: K
authors: Tokunaga
authors: Laboratory of Viral Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Masaru
authors: M
authors: Yokoyama
authors: Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors: Yu-Lun
authors: YL
authors: Huang
authors: Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors: Atsuhiko
authors: A
authors: Hasegawa
authors: Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
authors: Tatsuo
authors: T
authors: Shioda
authors: Laboratory of Viral Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors: Hironori
authors: H
authors: Sato
authors: Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors: Mari
authors: M
authors: Kannagi
authors: Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan tmasu.impt@tmd.ac.jp.
authors: Takao
authors: T
authors: Masuda
mesh_terms: D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D015870:Gene Expression; D017930:Genes, Reporter; D057809:HEK293 Cells; D019427:HIV Integrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D014443:Tyrosine; D008156:Luciferases; D019427:HIV Integrase; D054303:HIV Reverse Transcriptase; C514801:p31 integrase protein, Human immunodeficiency virus 1
keywords: HIV-1; integrase; reverse transcriptase; viral ribonucleoprotein complex