Nucleotide excision repair and template-independent addition by HIV-1 reverse transcriptase in the presence of nucleocapsid protein.
During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.
Bampi
Carole
C
LaboRetro, Unité de Virologie Humaine, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, Institut Fédératif de Recherche 128, 69364 Lyon Cedex 07, France.
Bibillo
Arkadiusz
A
Wendeler
Michaela
M
Divita
Gilles
G
Gorelick
Robert J
RJ
Le Grice
Stuart F J
SF
Darlix
Jean-Luc
JL
D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D016049:Didanosine / Q000494:pharmacology; D024882:Drug Resistance, Viral; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013698:Templates, Genetic; D014779:Virus Replication; D015215:Zidovudine / Q000494:pharmacology
D017931:DNA Primers; D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D016049:Didanosine