AEGiS-11IAC: HIV-1 nucleocapsid protein (NCp7) specifically directs initiation of (-) DNA synthesis in cell-free reverse transcriptase assays.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

HIV-1 nucleocapsid protein (NCp7) specifically directs initiation of (-) DNA synthesis in cell-free reverse transcriptase assays.

Int Conf AIDS 1996 Jul 7-12; 11:438 (abstract no. Pub.A.1001)
Li X, Kleiman L, Parniak MA, Wainberg MA; McGill University AIDS Centre-Jewish General Hospital, Montreal, Canada. Fax: (514) 340-7537.

OBJECTIVE: To determine the physiological relevance of NCp in reverse transcription.

METHODS: Reactions were performed using recombinant HIV RT in the presence of NCp.

RESULTS: In the presence of tRNALys.3, NCp7 was found to stimulate synthesis of minus-strand strong-stop DNA [(-) ss DNA], consistent with previous reports. However, specific DNA synthesis was only observed at a NCp7: RNA ratio similar to that predicted to be present in virions. Moreover, at these concentrations, NCp7 inhibited synthesis of non-specific reverse transcribed DNA products, which are initiated due to selfpriming by RNA templates. In contrast to results obtained with tRNALys.3 as primer, NCp7 inhibited synthesis of (-) ss DNA products primed by a 18 nt ribonucleotide (rPR), complementary to the PBS, even though rPR can initiate synthesis of such material in the absence of pre-annealing with NCp7. Primer placement bandshift assays showed that NCp7 was necessary for efficient formation of the tRNA/RNA complex. In contrast, NCp7 was found to prevent formation of the rPR/RNA complex. We also investigated the roles of an A-rich loop upstream of the PBS, a 7 nt region immediately downstream of the PBS, and a 54 nt deletion further downstream of the PBS in interactions with tRNALys.3. Only deletions in the 54 nt region, that may prevent formation of the U5/leader stem, but not deletions in the A-rich loop or the 7 nt sequence, were found to prevent tRNALys.3 placement and priming and viral infectivity.

CONCLUSIONS: NCp may represent an important target for antiviral chemotherapy as may agents that target the 54 nt sequence downstream of the PBS.

Keywords: AEGIS, RNA-Directed DNA Polymerase, Nucleocapsid Proteins, HIV-1, RNA, Transfer, Lys, DNA, Viral, Templates, Genetic, RNA, Resin Cements, DNA Replication, Transcription, Genetic, RNA, Transfer, DNA Primers, strong-stop DNA, Direct, genetics, ICA11

960707
PubA1001