Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.
During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3' polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5'-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.
Post
Klara
K
Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Kankia
Besik
B
Gopalakrishnan
Swathi
S
Yang
Victoria
V
Cramer
Elizabeth
E
Saladores
Pilar
P
Gorelick
Robert J
RJ
Guo
Jianhui
J
Musier-Forsyth
Karin
K
Levin
Judith G
JG
D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D011687:Purines / Q000032:analysis; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D048348:Reverse Transcription; D016914:Ribonuclease H / Q000378:metabolism; D015032:Zinc / Q000737:chemistry; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D018832:Molecular Chaperones; C071884:NCP7 protein, Human immunodeficiency virus 1; D011687:Purines; C034477:RNA primers; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus; D012313:RNA; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D015032:Zinc