The zinc finger of nucleocapsid protein of Friend murine leukemia virus is critical for proviral DNA synthesis in vivo.
Nucleocapsid protein NCp10 of murine leukemia virus (MuLV) is encoded by the 3' domain of gag and contains a zinc finger of the form Cys-X2-Cys-X4-His-X4-Cys flanked by basic amino acids. In the course of virus assembly, NCp10 is necessary for core formation, and the zinc finger flanked by the basic residues is required for the packaging of the genomic RNA dimer. In vitro, NCp10 exhibits strong nucleic acid binding and annealing activities that appear to be critical for virus infectivity since NCp10 promotes dimerization of the viral RNA containing the E/DLS packaging-dimerization signal and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (PBS). Recent in vitro studies have suggested that NCp10 may also play a role in proviral DNA synthesis. To investigate the function of NCp10 in proviral DNA synthesis in vivo, we developed a simple and convenient genetic packaging system consisting of two DNA constructs expressing the packaging components gag-pol and env of Friend MuLV and a Moloney MuLV-based lacZ vector with either the MuLV E+ or a rat VL30 E packaging signal. This system allowed us to examine the consequences of a set of mutations in NCp10 on a single round of recombinant virus replication. Most mutations in the N- or C-terminal domain of NCp10 do not significantly alter infectivity, while those in the zinc finger drastically impair infectivity. Analysis of the viral RNA content in virions showed that all mutations in the zinc finger decrease but do not abolish packaging of the recombinant genome. Interestingly enough, mutation of Y-28 to S (mutation Y28S) in the zinc finger results in RNA packaging at a level similar to that observed upon deletion of three prolines and three arginines in the C-terminal domain of NCp10 (mutant delta PR3). However, mutant Y28S is noninfectious while mutant delta PR3 is only threefold less infectious than the wild-type virus, which prompted us to examine the role of NCp10 protein in proviral DNA synthesis in vivo using these nucleocapsid mutants. PCR amplification was used to analyze viral DNA synthesized in newly infected cells, and results indicate that the Y28S zinc finger mutation impairs reverse transcription, thus suggesting that the nucleocapsid protein zinc finger plays a key role in proviral DNA synthesis in vivo.
Yu
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LaboRetro, Institut National de la Santé et de la Recherche Médicale U412, Ecole Normale Supérieure de Lyon, France.
Darlix
J L
JL
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002213:Capsid / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D005622:Friend murine leukemia virus / Q000235:genetics / Q000502:physiology*; D015752:Genes, env; D015750:Genes, gag; D015751:Genes, pol; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000502:physiology*; D012361:RNA, Transfer, Pro / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D051381:Rats; D017384:Sequence Deletion; D014162:Transfection; D014758:Viral Core Proteins / Q000378:metabolism*; D014779:Virus Replication; D016335:Zinc Fingers
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D012361:RNA, Transfer, Pro; D012367:RNA, Viral; D014758:Viral Core Proteins; D012194:RNA-Directed DNA Polymerase