Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity.
The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.
Dib-Hajj
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Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
Khan
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Giedroc
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D001483:Base Sequence; D001667:Binding, Competitive; D002213:Capsid / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D036022:Capsid Proteins; D004247:DNA / Q000378:metabolism*; D015683:Gene Products, gag; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D016093:Mason-Pfizer monkey virus / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009695:Nucleic Acid Renaturation / Q000187:drug effects*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012190:Retroviridae / Q000737:chemistry* / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C080114:NC71 protein, Human immunodeficiency virus 1; C080115:NC96 protein, Mason-Pfizer monkey virus; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D004247:DNA