Determinants of activity of the HIV-1 maturation inhibitor PA-457.

3-O-(3',3'-dimethylsuccinyl) betulinic acid, also termed PA-457 or DSB, is a novel HIV-1 inhibitor that blocks virus maturation by disrupting cleavage of the capsid precursor, CA-SP1. To better define the molecular target for PA-457, we prepared a panel of mutant viruses with point deletions spanning the CA-SP1 cleavage domain and characterized each of these viruses for PA-457 sensitivity. Our results indicate that amino acid residues in the N-terminal half of SP1 serve as determinants of PA-457 activity, while residues in the C-terminal half of SP1 were not involved in compound activity. These findings support and extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage and identify the CA-SP1 domain as the primary viral determinant for this novel inhibitor of HIV-1 replication.

[1]  M. Wainberg,et al.  Characterization of a Putative α-Helix across the Capsid-SP1 Boundary That Is Critical for the Multimerization of Human Immunodeficiency Virus Type 1 Gag , 2002, Journal of Virology.

[2]  J. Wills,et al.  Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins , 1995, Journal of virology.

[3]  Celia A. Schiffer,et al.  Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease , 2002, Journal of Virology.

[4]  A. Kaplan,et al.  Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles , 1993, Journal of virology.

[5]  M. Hammarskjöld,et al.  Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation , 1995, Journal of virology.

[6]  S. Goff,et al.  Mutations altering the Moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle , 1999, The EMBO journal.

[7]  Brian T. Foley,et al.  HIV Sequence Compendium 2018 , 2010 .

[8]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[9]  C. Cameron,et al.  Fine mapping and characterization of the Rous sarcoma virus Pr76gag late assembly domain , 1996, Journal of virology.

[10]  C. Aiken,et al.  The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid , 2004, Retrovirology.

[11]  P. Bieniasz,et al.  HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress , 2001, Nature Medicine.

[12]  M. Wainberg,et al.  Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication , 1998, Journal of Virology.

[13]  S. Goff,et al.  Infectivity of Moloney Murine Leukemia Virus Defective in Late Assembly Events Is Restored by Late Assembly Domains of Other Retroviruses , 2000, Journal of Virology.

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

[15]  S. Höglund,et al.  A Putative α-Helical Structure Which Overlaps the Capsid-p2 Boundary in the Human Immunodeficiency Virus Type 1 Gag Precursor Is Crucial for Viral Particle Assembly , 1998, Journal of Virology.

[16]  E. Freed,et al.  HIV-1 gag proteins: diverse functions in the virus life cycle. , 1998, Virology.

[17]  K. Jeang.  Retrovirology and young Turks... , 2004, Retrovirology.

[18]  B. Roques,et al.  Helical structure determined by NMR of the HIV‐1 (345–392)Gag sequence, surrounding p2: Implications for particle assembly and RNA packaging , 2005, Protein science : a publication of the Protein Society.

[19]  H. Zentgraf,et al.  The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity , 1995, Journal of virology.

[20]  A. Kaplan,et al.  The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions , 1994, Journal of virology.

[21]  E. Wimmer,et al.  Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli , 1988, Journal of virology.

[22]  David E. Martin,et al.  PA-457: A potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  C. Hutchison,et al.  Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. , 1989, AIDS research and human retroviruses.

[24]  C. Aiken,et al.  Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Replication by Specific Targeting of the Final Step of Virion Maturation , 2004, Journal of Virology.

[25]  J. Wills,et al.  Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein , 1997, Journal of virology.

[26]  L. Verplank,et al.  Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[27]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[28]  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.

[29]  S. Höglund,et al.  Functional domains of the capsid protein of human immunodeficiency virus type 1 , 1994, Journal of virology.

[30]  Curtis McMurtrey,et al.  3-O-(3′,3′-Dimethysuccinyl) Betulinic Acid Inhibits Maturation of the Human Immunodeficiency Virus Type 1 Gag Precursor Assembled In Vitro , 2006, Journal of Virology.

[31]  Chen Liang,et al.  Mutations within Four Distinct Gag Proteins Are Required To Restore Replication of Human Immunodeficiency Virus Type 1 after Deletion Mutagenesis within the Dimerization Initiation Site , 1999, Journal of Virology.

[32]  H. Kräusslich,et al.  Sequential Steps in Human Immunodeficiency Virus Particle Maturation Revealed by Alterations of Individual Gag Polyprotein Cleavage Sites , 1998, Journal of Virology.

[33]  E. Freed,et al.  The Late Domain of Human Immunodeficiency Virus Type 1 p6 Promotes Virus Release in a Cell Type-Dependent Manner , 2002, Journal of Virology.

[34]  J. Sodroski,et al.  Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[35]  E. Freed,et al.  p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease , 1995, Journal of virology.

[36]  H. Gelderblom,et al.  Efficient HIV‐1 replication can occur in the absence of the viral matrix protein , 1998, The EMBO journal.

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

[38]  D. Ott,et al.  Redundant Roles for Nucleocapsid and Matrix RNA-Binding Sequences in Human Immunodeficiency Virus Type 1 Assembly , 2005, Journal of Virology.

[39]  J. Wills,et al.  A large region within the Rous sarcoma virus matrix protein is dispensable for budding and infectivity , 1996, Journal of virology.

[40]  C. Aiken,et al.  Inhibition of HIV-1 Maturation via Drug Association with the Viral Gag Protein in Immature HIV-1 Particles* , 2005, Journal of Biological Chemistry.

[41]  E. Freed,et al.  Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[42]  R. Swanstrom,et al.  Synthesis, Assembly, and Processing of Viral Proteins , 1997 .