Identification of novel host-oriented targets for Human Immunodeficiency Virus type 1 using Random Homozygous Gene Perturbation

BackgroundHuman Immunodeficiency Virus (HIV) is a global threat to public health. Current therapies that directly target the virus often are rendered ineffective due to the emergence of drug-resistant viral variants. An emerging concept to combat drug resistance is the idea of targeting host mechanisms that are essential for the propagation of the virus, but have a minimal cellular effect.ResultsHerein, using Random Homozygous Gene Perturbation (RHGP), we have identified cellular targets that allow human MT4 cells to survive otherwise lethal infection by a wild type HIV-1NL4-3. These gene targets were validated by the reversibility of the RHGP technology, which confirmed that the RHGP itself was responsible for the resistance to HIV-1 infection. We further confirmed by siRNA knockdowns that the RHGP-identified cellular pathways are responsible for resistance to infection by either CXCR4 or CCR5 tropic HIV-1 variants. We also demonstrated that cell clones with these gene targets disrupted by RHGP were not permissible to the replication of a drug resistant HIV-1 mutant.ConclusionThese studies demonstrate the power of RHGP to identify novel host targets that are essential for the viral life cycle but which can be safely perturbed without overt cytotoxicity. These findings suggest opportunities for the future development of host-oriented therapeutics with the broad spectrum potential for safe and effective inhibition of HIV infection.

[1]  Wesley I. Sundquist,et al.  Tsg101 and the Vacuolar Protein Sorting Pathway Are Essential for HIV-1 Budding , 2001, Cell.

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

[3]  Amy S. Espeseth,et al.  Host Cell Factors in HIV Replication: Meta-Analysis of Genome-Wide Studies , 2009, PLoS pathogens.

[4]  J. Kahn,et al.  Time trends in primary HIV-1 drug resistance among recently infected persons. , 2002, JAMA.

[5]  J. Condra,et al.  In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors , 1995, Nature.

[6]  E. Hohenester.  Structural insight into Slit-Robo signalling. , 2008, Biochemical Society transactions.

[7]  Terri Wrin,et al.  The prevalence of antiretroviral drug resistance in the United States , 2004, AIDS.

[8]  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.

[9]  Richard A Koup,et al.  Homozygous Defect in HIV-1 Coreceptor Accounts for Resistance of Some Multiply-Exposed Individuals to HIV-1 Infection , 1996, Cell.

[10]  A. Nakagawara,et al.  A novel HECT-type E3 ubiquitin ligase, NEDL2, stabilizes p73 and enhances its transcriptional activity. , 2003, Biochemical and biophysical research communications.

[11]  F. Bushman,et al.  Retroviral DNA Integration: ASLV, HIV, and MLV Show Distinct Target Site Preferences , 2004, PLoS biology.

[12]  M. Kinch,et al.  The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza. , 2009, Virology.

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

[14]  J. Moore.  Coreceptors--Implications for HIV Pathogenesis and Therapy , 1997, Science.

[15]  Stanley N Cohen,et al.  tsg101: A Novel Tumor Susceptibility Gene Isolated by Controlled Homozygous Functional Knockout of Allelic Loci in Mammalian Cells , 1996, Cell.

[16]  Amy S. Espeseth,et al.  Genome-scale RNAi screen for host factors required for HIV replication. , 2008, Cell host & microbe.

[17]  P. Ahlquist,et al.  The Host Cell Sulfonation Pathway Contributes to Retroviral Infection at a Step Coincident with Provirus Establishment , 2008, PLoS pathogens.

[18]  T. Brümmendorf,et al.  Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters and alternative splicing regulate adhesion. , 2001, Current opinion in cell biology.

[19]  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.

[20]  R. Varshney,et al.  Cereal Genomics: An Overview , 2004 .

[21]  R. Lamb,et al.  Mechanisms for enveloped virus budding: can some viruses do without an ESCRT? , 2008, Virology.

[22]  J. Wheeler,et al.  Assessing Theoretical Risk and Benefit suggested by Genetic Association Studies of CCR5: Experience in a Drug Development Programme for Maraviroc , 2007, Antiviral therapy.

[23]  Stanley N Cohen,et al.  EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Hiroaki Kitano,et al.  The PANTHER database of protein families, subfamilies, functions and pathways , 2004, Nucleic Acids Res..

[25]  M. Chen,et al.  Isolation and characterization of two divergent infectious molecular clones of HIV type 1 longitudinally obtained from a seropositive patient by a progressive amplification procedure. , 1997, AIDS research and human retroviruses.

[26]  Kuan-Teh Jeang,et al.  A Genome-wide Short Hairpin RNA Screening of Jurkat T-cells for Human Proteins Contributing to Productive HIV-1 Replication* , 2009, The Journal of Biological Chemistry.

[27]  Marc Parmentier,et al.  Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene , 1996, Nature.

[28]  J. Lippincott-Schwartz,et al.  Rab9 GTPase Is Required for Replication of Human Immunodeficiency Virus Type 1, Filoviruses, and Measles Virus , 2005, Journal of Virology.

[29]  Justine R. Smith,et al.  Sequence- and target-independent angiogenesis suppression by siRNA via TLR3 , 2008, Nature.

[30]  Jane Y. Wu,et al.  Slit‐2/Robo‐1 modulates the CXCL12/CXCR4‐induced chemotaxis of T cells , 2007, Journal of leukocyte biology.

[31]  P. Bieniasz,et al.  Late budding domains and host proteins in enveloped virus release. , 2006, Virology.

[32]  Y. Rao,et al.  The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors , 2001, Nature.

[33]  M. Reitz,et al.  Growth of macrophage-tropic and primary human immunodeficiency virus type 1 (HIV-1) isolates in a unique CD4+ T-cell clone (PM1): failure to downregulate CD4 and to interfere with cell-line-tropic HIV-1 , 1995, Journal of virology.