Phosphorylation of the HIV-1 capsid by MELK triggers uncoating to promote viral cDNA synthesis

Regulation of capsid disassembly is crucial for efficient HIV-1 cDNA synthesis after entry, yet host factors involved in this process remain largely unknown. Here, we employ genetic screening of human T-cells to identify maternal embryonic leucine zipper kinase (MELK) as a host factor required for optimal uncoating of the HIV-1 core to promote viral cDNA synthesis. Depletion of MELK inhibited HIV-1 cDNA synthesis with a concomitant delay of capsid disassembly. MELK phosphorylated Ser-149 of the capsid in the multimerized HIV-1 core, and a mutant virus carrying a phosphorylation-mimetic amino-acid substitution of Ser-149 underwent premature capsid disassembly and earlier HIV-1 cDNA synthesis, and eventually failed to enter the nucleus. Moreover, a small-molecule MELK inhibitor reduced the efficiency of HIV-1 replication in peripheral blood mononuclear cells in a dose-dependent manner. These results reveal a previously unrecognized mechanism of HIV-1 capsid disassembly and implicate MELK as a potential target for anti-HIV therapy.

[1]  O. Cosnefroy,et al.  HIV-1 capsid uncoating initiates after the first strand transfer of reverse transcription , 2016, Retrovirology.

[2]  M. Philippe,et al.  Cell cycle regulation of pEg3, a new Xenopus protein kinase of the KIN1/PAR-1/MARK family. , 2002, Developmental biology.

[3]  P. Prevelige,et al.  Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry. , 2003, Journal of molecular biology.

[4]  Amos B. Smith,et al.  Human Cytosolic Extracts Stabilize the HIV-1 Core , 2013, Journal of Virology.

[5]  F. Jensen,et al.  Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles , 1997, Journal of virology.

[6]  S. Antonarakis,et al.  TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm , 2013, Retrovirology.

[7]  E. Freed.  HIV-1 and the host cell: an intimate association. , 2004, Trends in microbiology.

[8]  M. Matsuoka,et al.  Cooperation of NF-kappaB2/p100 activation and the PDZ domain binding motif signal in human T-cell leukemia virus type 1 (HTLV-1) Tax1 but not HTLV-2 Tax2 is crucial for interleukin-2-independent growth transformation of a T-cell line. , 2007, Journal of virology.

[9]  C. Desgranges,et al.  Identification of Three Major Phosphorylation Sites within HIV-1 Capsid , 1999, The Journal of Biological Chemistry.

[10]  W. Greene,et al.  A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes , 2002, Nature Biotechnology.

[11]  Wesley I. Sundquist,et al.  Formation of a Human Immunodeficiency Virus Type 1 Core of Optimal Stability Is Crucial for Viral Replication , 2002, Journal of Virology.

[12]  P. Auewarakul,et al.  The effect of capsid mutations on HIV-1 uncoating. , 2007, Virology.

[13]  J. Lineberger,et al.  Nef Does Not Affect the Efficiency of Human Immunodeficiency Virus Type 1 Fusion with Target Cells , 2003, Journal of Virology.

[14]  T. Nakano,et al.  Phosphorylation of human immunodeficiency virus type 1 capsid protein at serine 16, required for peptidyl-prolyl isomerase-dependent uncoating, is mediated by virion-incorporated extracellular signal-regulated kinase 2. , 2014, The Journal of general virology.

[15]  J. Kappes,et al.  Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy , 2002, Antimicrobial Agents and Chemotherapy.

[16]  Wesley I. Sundquist,et al.  Functional Surfaces of the Human Immunodeficiency Virus Type 1 Capsid Protein , 2003, Journal of Virology.

[17]  E. Lillehoj,et al.  The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors , 1988, Journal of virology.

[18]  G. Towers,et al.  A model for cofactor use during HIV-1 reverse transcription and nuclear entry☆ , 2014, Current opinion in virology.

[19]  C. Aiken,et al.  Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly , 2017, Journal of Virology.

[20]  Indira Hewlett,et al.  The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability. , 2011, Virology.

[21]  T. Kigawa,et al.  RING Domain Mutations Uncouple TRIM5α Restriction of HIV-1 from Inhibition of Reverse Transcription and Acceleration of Uncoating , 2011, Journal of Virology.

[22]  A. Gartel,et al.  Thiazole Antibiotics Target FoxM1 and Induce Apoptosis in Human Cancer Cells , 2009, PloS one.

[23]  S. Shorte,et al.  Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration , 2012, PloS one.

[24]  A. Gronenborn,et al.  Rhesus TRIM5α Disrupts the HIV-1 Capsid at the InterHexamer Interfaces , 2011, PLoS pathogens.

[25]  T. Copeland,et al.  Biochemical and immunological analysis of human immunodeficiency virus gag gene products p17 and p24 , 1988, Journal of virology.

[26]  J. Luban,et al.  Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1 , 2004, Nature.

[27]  Mahdad Noursadeghi,et al.  HIV-1 Capsid-Cyclophilin Interactions Determine Nuclear Import Pathway, Integration Targeting and Replication Efficiency , 2011, PLoS pathogens.

[28]  J. Moore,et al.  AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor , 1998, Nature Medicine.

[29]  Yusuke Nakamura,et al.  Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family , 2007, Breast Cancer Research.

[30]  C. M. Owens,et al.  The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys , 2004, Nature.

[31]  P. Mischel,et al.  Siomycin A targets brain tumor stem cells partially through a MELK-mediated pathway. , 2011, Neuro-oncology.

[32]  Y. le Page,et al.  A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development , 2011, Journal of Cell Science.

[33]  A. Cimarelli,et al.  A Novel Entry/Uncoating Assay Reveals the Presence of at Least Two Species of Viral Capsids During Synchronized HIV-1 Infection , 2016, PLoS pathogens.

[34]  B. Gay,et al.  Active cAMP-dependent Protein Kinase Incorporated within Highly Purified HIV-1 Particles Is Required for Viral Infectivity and Interacts with Viral Capsid Protein* , 2003, Journal of Biological Chemistry.

[35]  M. Bollen,et al.  Substrate Specificity and Activity Regulation of Protein Kinase MELK* , 2005, Journal of Biological Chemistry.

[36]  J. Inoue,et al.  Selective Transmission of R5 HIV-1 over X4 HIV-1 at the Dendritic Cell–T Cell Infectious Synapse Is Determined by the T Cell Activation State , 2009, PLoS pathogens.

[37]  G. Melikyan,et al.  Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells , 2016, PLoS pathogens.

[38]  Y. Ikeda,et al.  Rhesus monkey TRIM5α restricts HIV-1 production through rapid degradation of viral Gag polyproteins , 2007, Nature Medicine.

[39]  Yusuke Nakamura,et al.  Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer , 2012, Oncotarget.

[40]  Zeger Debyser,et al.  Transportin-SR2 Imports HIV into the Nucleus , 2008, Current Biology.

[41]  W. Sundquist,et al.  Biological Crystallography Structures of the Hiv-1 Capsid Protein Dimerization Domain at 2.6 a Ê Resolution , 2022 .

[42]  T. Hope,et al.  KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection , 2016, PLoS pathogens.

[43]  M. Matsuoka,et al.  Cooperation of NF-κB2/p100 Activation and the PDZ Domain Binding Motif Signal in Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax1 but Not HTLV-2 Tax2 Is Crucial for Interleukin-2-Independent Growth Transformation of a T-Cell Line , 2007, Journal of Virology.

[44]  T. Masuda,et al.  Quantitative Analysis of Human Immunodeficiency Virus Type 1 DNA Dynamics by Real-Time PCR: Integration Efficiency in Stimulated and Unstimulated Peripheral Blood Mononuclear Cells , 2003, Virus Genes.

[45]  H. Fox,et al.  Trim5α Accelerates Degradation of Cytosolic Capsid Associated with Productive HIV-1 Entry* , 2006, Journal of Biological Chemistry.

[46]  H. Gendelman,et al.  Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone , 1986, Journal of virology.

[47]  J. Clements,et al.  LuSIV cells: a reporter cell line for the detection and quantitation of a single cycle of HIV and SIV replication. , 2000, Virology.

[48]  HIV-1-associated PKA acts as a cofactor for genome reverse transcription , 2013, Retrovirology.

[49]  Naoki Kishimoto,et al.  Uncoating of Human Immunodeficiency Virus Type 1 Requires Prolyl Isomerase Pin1* , 2010, The Journal of Biological Chemistry.

[50]  S. Yamaoka,et al.  A Carboxy-Terminally Truncated Human CPSF6 Lacking Residues Encoded by Exon 6 Inhibits HIV-1 cDNA Synthesis and Promotes Capsid Disassembly , 2013, Journal of Virology.

[51]  M. Bollen,et al.  Inhibition of Spliceosome Assembly by the Cell Cycle-regulated Protein Kinase MELK and Involvement of Splicing Factor NIPP1* , 2004, Journal of Biological Chemistry.

[52]  S. Gygi,et al.  Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis , 2007, The EMBO journal.

[53]  J. Sodroski,et al.  A B-Box 2 Surface Patch Important for TRIM5α Self-Association, Capsid Binding Avidity, and Retrovirus Restriction , 2009, Journal of Virology.

[54]  M. Malim,et al.  Human Immunodeficiency Virus Type 1 Spinoculation Enhances Infection through Virus Binding , 2000, Journal of Virology.

[55]  R. Gorelick,et al.  The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6 , 2013, Retrovirology.

[56]  E. Campbell,et al.  HIV-1 Uncoating Is Facilitated by Dynein and Kinesin 1 , 2014, Journal of Virology.

[57]  Ariberto Fassati,et al.  Multiple roles of the capsid protein in the early steps of HIV-1 infection. , 2012, Virus research.

[58]  C. Lionne,et al.  Electrostatic repulsion between HIV‐1 capsid proteins modulates hexamer plasticity and in vitro assembly , 2010, Proteins.

[59]  K. Mollier,et al.  Nuclear Import Defect of Human Immunodeficiency Virus Type 1 DNA Flap Mutants Is Not Dependent on the Viral Strain or Target Cell Type , 2006, Journal of Virology.

[60]  L. Chaloin,et al.  VSV-G pseudotyping rescues HIV-1 CA mutations that impair core assembly or stability , 2008, Retrovirology.

[61]  Mark Yeager,et al.  Atomic-level modelling of the HIV capsid , 2011 .

[62]  E. Kinoshita,et al.  Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE , 2009, Nature Protocols.

[63]  S. Goff,et al.  Host factors exploited by retroviruses , 2007, Nature Reviews Microbiology.

[64]  M. Goedert,et al.  Phosphorylation of microtubule‐associated protein tau by AMPK‐related kinases , 2012, Journal of neurochemistry.

[65]  T. Hope,et al.  Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription , 2011, Proceedings of the National Academy of Sciences.

[66]  D. Solter,et al.  New member of the Snf1/AMPK kinase family, Melk, is expressed in the mouse egg and preimplantation embryo , 1997, Molecular reproduction and development.

[67]  C. Aiken,et al.  HIV-1 uncoating: connection to nuclear entry and regulation by host proteins. , 2014, Virology.

[68]  Benjamin A. Himes,et al.  Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site , 2016, Nature Communications.

[69]  A. Gartel,et al.  Identification of a chemical inhibitor of the oncogenic transcription factor forkhead box M1. , 2006, Cancer research.

[70]  S. Okada,et al.  Expansion of Activated Memory CD4+ T Cells Affects Infectivity of CCR5-Tropic HIV-1 in Humanized NOD/SCID/JAK3null Mice , 2013, PloS one.

[71]  F. Diaz-Griffero,et al.  Inhibition of Reverse Transcriptase Activity Increases Stability of the HIV-1 Core , 2012, Journal of Virology.

[72]  J. Inazawa,et al.  Overexpressed NF-kappaB-inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells. , 2008, Blood.

[73]  Joseph Sodroski,et al.  Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. , 2006, Proceedings of the National Academy of Sciences of the United States of America.