The Requirement for Cellular Transportin 3 (TNPO3 or TRN-SR2) during Infection Maps to Human Immunodeficiency Virus Type 1 Capsid and Not Integrase

ABSTRACT Recent genome-wide screens have highlighted an important role for transportin 3 in human immunodeficiency virus type 1 (HIV-1) infection and preintegration complex (PIC) nuclear import. Moreover, HIV-1 integrase interacted with recombinant transportin 3 protein under conditions whereby Moloney murine leukemia virus (MLV) integrase failed to do so, suggesting that integrase-transportin 3 interactions might underscore active retroviral PIC nuclear import. Here we correlate infectivity defects in transportin 3 knockdown cells with in vitro protein binding affinities for an expanded set of retroviruses that include simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and Rous sarcoma virus (RSV) to critically address the role of integrase-transportin 3 interactions in viral infection. Lentiviruses, with the exception of FIV, display a requirement for transportin 3 in comparison to MLV and RSV, yielding an infection-based dependency ranking of SIV > HIV-1 > BIV and EIAV > MLV, RSV, and FIV. In vitro pulldown and surface plasmon resonance assays, in contrast, define a notably different integrase-transportin 3 binding hierarchy: FIV, HIV-1, and BIV > SIV and MLV > EIAV. Our results therefore fail to support a critical role for integrase binding in dictating transportin 3 dependency during retrovirus infection. In addition to integrase, capsid has been highlighted as a retroviral nuclear import determinant. Accordingly, MLV/HIV-1 chimera viruses pinpoint the genetic determinant of sensitization to transportin 3 knockdown to the HIV-1 capsid protein. We therefore conclude that capsid, not integrase, is the dominant viral factor that dictates transportin 3 dependency during HIV-1 infection.

[1]  C. Woodward,et al.  Integrase Interacts with Nucleoporin NUP153 To Mediate the Nuclear Import of Human Immunodeficiency Virus Type 1 , 2009, Journal of Virology.

[2]  A. Fassati,et al.  HIV-1 exploits importin 7 to maximize nuclear import of its DNA genome , 2009, Retrovirology.

[3]  R. König,et al.  Global Analysis of Host-Pathogen Interactions that Regulate Early-Stage HIV-1 Replication , 2008, Cell.

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

[5]  A. Engelman,et al.  The Lentiviral Integrase Binding Protein LEDGF/p75 and HIV-1 Replication , 2008, PLoS pathogens.

[6]  E. Poeschla,et al.  Integrase, LEDGF/p75 and HIV replication , 2008, Cellular and Molecular Life Sciences.

[7]  J. Lieberman,et al.  Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen , 2007, Science.

[8]  M. Emerman,et al.  Evidence for Direct Involvement of the Capsid Protein in HIV Infection of Nondividing Cells , 2007, PLoS pathogens.

[9]  J. Sodroski,et al.  Complex determinants within the Moloney murine leukemia virus capsid modulate susceptibility of the virus to Fv1 and Ref1-mediated restriction. , 2007, Virology.

[10]  Xiaojian Yao,et al.  Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication* , 2007, Journal of Biological Chemistry.

[11]  Z. Debyser,et al.  Lentiviral nuclear import: a complex interplay between virus and host , 2007, BioEssays : news and reviews in molecular, cellular and developmental biology.

[12]  P. Cherepanov.  LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro , 2006, Nucleic acids research.

[13]  Ariberto Fassati,et al.  HIV infection of non-dividing cells: a divisive problem , 2006, Retrovirology.

[14]  A. Engelman,et al.  Wild-Type Levels of Human Immunodeficiency Virus Type 1 Infectivity in the Absence of Cellular Emerin Protein , 2006, Journal of Virology.

[15]  D. Jans,et al.  HIV-1 integrase is capable of targeting DNA to the nucleus via an Importin α/β-dependent mechanism , 2006 .

[16]  A. Engelman,et al.  Biochemical and genetic analyses of integrase-interacting proteins lens epithelium-derived growth factor (LEDGF)/p75 and hepatoma-derived growth factor related protein 2 (HRP2) in preintegration complex function and HIV-1 replication. , 2006, Virology.

[17]  Michael Emerman,et al.  Retroviral infection of non-dividing cells: old and new perspectives. , 2006, Virology.

[18]  M. Emerman,et al.  The Cell Cycle Independence of HIV Infections Is Not Determined by Known Karyophilic Viral Elements , 2005, PLoS pathogens.

[19]  R. Benarous,et al.  The Interaction of LEDGF/p75 with Integrase Is Lentivirus-specific and Promotes DNA Binding* , 2005, Journal of Biological Chemistry.

[20]  Pamela A. Silver,et al.  Identification of an Evolutionarily Conserved Domain in Human Lens Epithelium-derived Growth Factor/Transcriptional Co-activator p75 (LEDGF/p75) That Binds HIV-1 Integrase* , 2004, Journal of Biological Chemistry.

[21]  J. Sodroski,et al.  TRIM5alpha mediates the postentry block to N-tropic murine leukemia viruses in human cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Emerman,et al.  Capsid Is a Dominant Determinant of Retrovirus Infectivity in Nondividing Cells , 2004, Journal of Virology.

[23]  Ian F. Harrison,et al.  Nuclear import of HIV‐1 intracellular reverse transcription complexes is mediated by importin 7 , 2003, The EMBO journal.

[24]  J. Olsen,et al.  Comparison of Gene Transfer Efficiencies and Gene Expression Levels Achieved with Equine Infectious Anemia Virus- and Human Immunodeficiency Virus Type 1-Derived Lentivirus Vectors , 2002, Journal of Virology.

[25]  S. Goff,et al.  Characterization of Intracellular Reverse Transcription Complexes of Human Immunodeficiency Virus Type 1 , 2001, Journal of Virology.

[26]  I. Plavec,et al.  Construction and Molecular Analysis of Gene Transfer Systems Derived from Bovine Immunodeficiency Virus , 2001, Journal of Virology.

[27]  S. Goff,et al.  Characterization of Intracellular Reverse Transcription Complexes of Moloney Murine Leukemia Virus , 1999, Journal of Virology.

[28]  C. Tabin,et al.  Production and design of more effective avian replication-incompetent retroviral vectors. , 1999, Developmental biology.

[29]  Jennifer L. Bachorik,et al.  Transportin-SR, a Nuclear Import Receptor for SR Proteins , 1999, The Journal of cell biology.

[30]  T. Hope,et al.  Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors , 1999, Journal of Virology.

[31]  J. Olsen.  Gene transfer vectors derived from equine infectious anemia virus , 1998, Gene Therapy.

[32]  T. Hope,et al.  HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[33]  F. Bushman,et al.  Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition , 1997, Journal of virology.

[34]  S. A. Chow,et al.  Characterization of feline immunodeficiency virus integrase and analysis of functional domains. , 1997, Virology.

[35]  J. Sodroski,et al.  CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia , 1997, Nature.

[36]  A. Engelman.  Biochemical characterization of recombinant equine infectious anemia virus integrase. , 1996, Protein expression and purification.

[37]  F. Gage,et al.  In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector , 1996, Science.

[38]  A. Kingsman,et al.  Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication , 1996, Journal of virology.

[39]  M. Emerman,et al.  Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus , 1994, Journal of virology.

[40]  P. Brown,et al.  Integration of murine leukemia virus DNA depends on mitosis. , 1993, The EMBO journal.

[41]  F. Bushman,et al.  Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[42]  M. Roth,et al.  Characterization of the forward and reverse integration reactions of the Moloney murine leukemia virus integrase protein purified from Escherichia coli. , 1993, The Journal of biological chemistry.

[43]  A. Engelman,et al.  Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro , 1992, Journal of virology.

[44]  M. Emerman,et al.  Human immunodeficiency virus infection of cells arrested in the cell cycle. , 1992, The EMBO journal.

[45]  M. Bukrinsky,et al.  Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[46]  M. Malim,et al.  Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes , 1991, The Journal of experimental medicine.

[47]  A. Miller,et al.  Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection , 1990, Molecular and cellular biology.

[48]  P. Brown,et al.  A nucleoprotein complex mediates the integration of retroviral DNA. , 1989, Genes & development.

[49]  R. Katz,et al.  Restriction endonuclease and nucleotide sequence analyses of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions , 1982, Journal of virology.

[50]  E. Poeschla,et al.  Durable, safe, multi-gene lentiviral vector expression in feline trabecular meshwork. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[51]  D. Jans,et al.  HIV-1 integrase is capable of targeting DNA to the nucleus via an importin alpha/beta-dependent mechanism. , 2006, The Biochemical journal.

[52]  E. Poeschla,et al.  FIV Vectors. , 2003, Methods in molecular biology.

[53]  R. Desrosiers,et al.  Recombinant simian immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys. , 1999, AIDS research and human retroviruses.

[54]  I. Mattaj,et al.  Nucleocytoplasmic transport: the soluble phase. , 1998, Annual review of biochemistry.

[55]  M. O. Dayhoff,et al.  Atlas of protein sequence and structure , 1965 .