Elimination of Retroviral Infectivity by N-Ethylmaleimide with Preservation of Functional Envelope Glycoproteins

ABSTRACT The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2′-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.

[1]  A. Gettie,et al.  Presentation of Exogenous Whole Inactivated Simian Immunodeficiency Virus by Mature Dendritic Cells Induces CD4+ and CD8+ T-Cell Responses , 2003, Journal of acquired immune deficiency syndromes.

[2]  L. Gritz,et al.  Production and of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins. , 1991, AIDS.

[3]  C. Ehresmann,et al.  The Human Immunodeficiency Virus Type 1 Gag Polyprotein Has Nucleic Acid Chaperone Activity: Possible Role in Dimerization of Genomic RNA and Placement of tRNA on the Primer Binding Site , 1999, Journal of Virology.

[4]  J. Lifson,et al.  Human Immunodeficiency Virus Type 1 Activates Plasmacytoid Dendritic Cells and Concomitantly Induces the Bystander Maturation of Myeloid Dendritic Cells , 2004, Journal of Virology.

[5]  K. Nagashima,et al.  Quantitation of HLA Class II Protein Incorporated into Human Immunodeficiency Type 1 Virions Purified by Anti-CD45 Immunoaffinity Depletion of Microvesicles , 2003, Journal of Virology.

[6]  J. Lifson,et al.  Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences. , 1999, Virology.

[7]  J. Modrof,et al.  Ebola Virus Transcription Activator VP30 Is a Zinc-Binding Protein , 2003, Journal of Virology.

[8]  H. Jin,et al.  Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability , 2001, Journal of Virology.

[9]  J. Lifson,et al.  Chemical inactivation of retroviral infectivity by targeting nucleocapsid protein zinc fingers: a candidate SIV vaccine. , 1998, AIDS research and human retroviruses.

[10]  B. Roques,et al.  Zn(2+) binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein: evidence of a critical role of cysteine 49 in Zn(2+) dissociation. , 2002, Biochemistry.

[11]  A. Gettie,et al.  Enhanced in vitro stimulation of rhesus macaque dendritic cells for activation of SIV-specific T cell responses. , 2002, Journal of immunological methods.

[12]  E. Chertova,et al.  Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein , 1999, Journal of Virology.

[13]  J. Berg,et al.  Potential metal-binding domains in nucleic acid binding proteins. , 1986, Science.

[14]  J. Lifson,et al.  Sites, mechanism of action and lack of reversibility of primate lentivirus inactivation by preferential covalent modification of virion internal proteins. , 2003, Current molecular medicine.

[15]  Kenneth A. Taylor,et al.  Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution‐state structure of the nucleocapsid protein from HIV‐1 , 1992, Protein science : a publication of the Protein Society.

[17]  T Friedmann,et al.  Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Desrosiers,et al.  Whole inactivated SIV virion vaccines with functional envelope glycoproteins: safety, immunogenicity, and activity against intrarectal challenge , 2002, Journal of medical primatology.

[19]  B. Roques,et al.  Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro , 1997, Journal of virology.

[20]  B. Roques,et al.  Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+. , 2001, Journal of molecular biology.

[21]  J. Casas-Finet,et al.  Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design , 2001, Protein science : a publication of the Protein Society.

[22]  B. Roques,et al.  Protein Nucleocapsid Protein Chaperoning of Nucleic Acids at the Heart of HIV Structure , Assembly and cDNA Synthesis , 2002 .

[23]  L. Sun,et al.  Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation. , 1996, Biochemistry.

[24]  P. Boyer,et al.  Replication of Phenotypically Mixed Human Immunodeficiency Virus Type 1 Virions Containing Catalytically Active and Catalytically Inactive Reverse Transcriptase , 2001, Journal of Virology.

[25]  P. Brown,et al.  An Essential Interaction between Distinct Domains of HIV-1 Integrase Mediates Assembly of the Active Multimer (*) , 1995, The Journal of Biological Chemistry.

[26]  J. Sodroski,et al.  Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes , 1986, Journal of virology.

[27]  L. Arthur,et al.  The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent , 1993, Journal of virology.

[28]  Vm Vogt.  Retroviral Virions and Genomes , 1997 .

[29]  M. Chance,et al.  Extended x-ray absorption fine structure studies of a retrovirus: equine infectious anemia virus cysteine arrays are coordinated to zinc. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Summers,et al.  Inhibition of HIV-1 infectivity by zinc-ejecting aromatic C-nitroso compounds , 1993, Nature.

[31]  J. Lifson,et al.  Infectious and Whole Inactivated Simian Immunodeficiency Viruses Interact Similarly with Primate Dendritic Cells (DCs): Differential Intracellular Fate of Virions in Mature and Immature DCs , 2002, Journal of Virology.

[32]  Q. Sattentau,et al.  Inactivation of Human Immunodeficiency Virus Type 1 Infectivity with Preservation of Conformational and Functional Integrity of Virion Surface Proteins , 1998, Journal of Virology.

[33]  R. Gorelick,et al.  Human Immunodeficiency Virus Type 1 Nucleocapsid Zn2+ Fingers Are Required for Efficient Reverse Transcription, Initial Integration Processes, and Protection of Newly Synthesized Viral DNA , 2003, Journal of Virology.

[34]  F. Bushman,et al.  Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein , 1999, Journal of Virology.

[35]  J. Lifson,et al.  Activation of HIV-1 specific CD4 and CD8 T cells by human dendritic cells: roles for cross-presentation and non-infectious HIV-1 virus , 2002, AIDS.

[36]  D. McDonald,et al.  Visualization of the intracellular behavior of HIV in living cells , 2002, The Journal of cell biology.

[37]  R. Gorelick,et al.  Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[38]  D. Trono,et al.  Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses , 1992, Journal of virology.

[39]  L. Arthur,et al.  Probing the topography of HIV-1 nucleocapsid protein with the alkylating agent N-ethylmaleimide. , 1998, Biochemistry.

[40]  S. Osmanov,et al.  Serotyping of HIV type 1 infections: definition, relationship to viral genetic subtypes, and assay evaluation. UNAIDS Network for HIV-1 Isolation and Characterization. , 1998, AIDS research and human retroviruses.

[41]  R. Wagner,et al.  Effect of sulfhydryl reagents on the infectivity of vesicular stomatitis virus , 1980, Virology.

[42]  C. Supuran,et al.  Cysteine-modifying agents: a possible approach for effective anticancer and antiviral drugs. , 2002, Environmental health perspectives.

[43]  M. Emerman,et al.  Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene , 1992, Journal of virology.

[44]  Y. Sung,et al.  Generation of the replication-competent human immunodeficiency virus type 1 which expresses a jellyfish green fluorescent protein. , 1997, Biochemical and biophysical research communications.

[45]  B. Roques,et al.  Analysis of NCp7-dependent activation of HIV-1 cDNA integration and its conservation among retroviral nucleocapsid proteins. , 2003, Journal of molecular biology.

[46]  B. Roques,et al.  Zn2+ binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein: Evidence of a critical role of cysteine 49 in Zn2+ dissociation , 2002 .

[47]  J. Lifson,et al.  Nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo. , 1999, Virology.

[48]  L. Arthur,et al.  Genetic analysis of the zinc finger in the Moloney murine leukemia virus nucleocapsid domain: replacement of zinc-coordinating residues with other zinc-coordinating residues yields noninfectious particles containing genomic RNA , 1996, Journal of virology.

[49]  J. Lifson,et al.  MHC-I–restricted presentation of HIV-1 virion antigens without viral replication , 2001, Nature Medicine.

[50]  J. Lifson,et al.  Differential Incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and Major Histocompatibility Complex Class I and II Molecules into Human Immunodeficiency Virus Type 1 Virions and Microvesicles: Implications for Viral Pathogenesis and Immune Regulation , 2001, Journal of Virology.

[51]  A. Rein,et al.  Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication. , 1998, Trends in biochemical sciences.

[52]  L. Arthur,et al.  Inhibitors of HIV Nucleocapsid Protein Zinc Fingers as Candidates for the Treatment of AIDS , 1995, Science.

[53]  J. Lifson,et al.  Immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells. , 2004, Blood.

[54]  B. Korber,et al.  HIV sequence compendium 2002 , 2002 .

[55]  H. Issaq,et al.  Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses , 1992, Journal of virology.

[56]  M. Feinberg,et al.  Use of a green fluorescent protein as a marker for human immunodeficiency virus type 1 infection. , 1997, AIDS research and human retroviruses.

[57]  P. Cresswell,et al.  HLA class II on HIV particles is functional in superantigen presentation to human T cells: implications for HIV pathogenesis. , 1995, AIDS research and human retroviruses.

[58]  M. Shaharabany,et al.  Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. , 1991, The Journal of biological chemistry.

[59]  Darrin M York,et al.  Insights into the regioselectivity and RNA-binding affinity of HIV-1 nucleocapsid protein from linear-scaling quantum methods. , 2003, Journal of molecular biology.

[60]  F. Bushman,et al.  Cofactors for Human Immunodeficiency Virus Type 1 cDNA Integration In Vitro , 2003, Journal of Virology.

[61]  B. Vallee,et al.  [36] Reactions with N-ethylmaleimide and p-mercuribenzoate. , 1972, Methods in enzymology.

[62]  K. Nagashima,et al.  Characterization of clones of HIV-1 infected HuT 78 cells defective in gag gene processing and of SIV clones producing large amounts of envelope glycoprotein. , 1990, Journal of medical primatology.

[63]  Cc García,et al.  Antiviral and Virucidal Activities against Arenaviruses of Zinc-Finger Active Compounds , 2000, Antiviral chemistry & chemotherapy.

[64]  Frank Oellien,et al.  Enhanced CACTVS Browser of the Open NCI Database , 2002, J. Chem. Inf. Comput. Sci..

[65]  J. Groves,et al.  Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation. , 1996, Biochemistry.

[66]  L. Arthur,et al.  Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA , 1990, Journal of virology.

[67]  K. Goldenthal,et al.  Workshop Report: Traditional Approach Preventive HIV Vaccines: What Are the Cell Substrate and Inactivation Issues?1 , 1998 .

[68]  E. Damonte,et al.  Mode of inactivation of arenaviruses by disulfide-based compounds. , 2002, Antiviral research.

[69]  B. Roques,et al.  Determination of the pKa of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: Consequences on the binding of Zn2+ , 2001 .