title: Functional reconstitution of lost activity of chemically cross-linked or mutant moloney murine leukemia virus nucleocapsid proteins by trans-complementation.
abstract: Intermolecular crosslinking of the retroviral gag structural proteins, capsid (CA) and nucleocapsid (NC), by oxidation of cysteine thiols interferes with virus assembly and infectivity. PD156202 is a dithiobisbenzamide with broad antiviral activity against retroviruses. Treatment of cell free Moloney murine leukemia virus (MuLV) particles with PD156202 induced crosslinking of gag structural proteins and inhibited reverse transcription in mellitin permeabilized virions. Site directed mutagenesis of NC cysteines in the zinc finger of MuLV proviral DNA resulted in virus particles that were noninfectious. The NC mutant virus did not display any intermolecular crosslinking following treatment with PD156202 under nonreducing conditions, which supported that NC cysteine residues participated in PD156202 mediated crosslinking. Replication of MuLV NC mutant virus could be restored by independent expression of wild type NC, making it susceptible to PD156202. These results demonstrate that oxidation of NC cysteines are detrimental for virus assembly and that complementation with wild type NC restored the nucleocapsid protein activity.
authors.affiliation: Department of Infectious Diseases, Pfizer Global Research and Development, Ann Arbor Laboratories, Michigan 48105, USA. andrea.heldsinger@pfizer.com
authors.forename: A
authors.initials: A
authors.lastname: Heldsinger
authors.forename: L
authors.initials: L
authors.lastname: Sharmeen
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000494:pharmacology; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012192:Retroviridae Infections / Q000821:virology; D014412:Tumor Virus Infections / Q000821:virology; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D001549:Benzamides; D003432:Cross-Linking Reagents; D004220:Disulfides; D015683:Gene Products, gag; D019590:Nucleocapsid Proteins; C098073:PD 156202; D003545:Cysteine