AEGiS-11IAC: Role of the HIV-A nucleocapsid protein (NCp7) and the A-rich loop in cell-free reverse transcriptase and infectivity assays.

11th International AIDS Conference


Vancouver, British Columbia — July 7-12, 1996

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Role of the HIV-A nucleocapsid protein (NCp7) and the A-rich loop in cell-free reverse transcriptase and infectivity assays.

Int Conf AIDS 1996 Jul 7-12; 11:227 (abstract no. Tu.A.491)
Wainberg M, Li X, Kleiman L, Parniak MA; McGill University AIDS Centre-Jewish General Hospital, Montreal, Canada. Fax: 514-340-7537.

OBJECTIVE: To determine the physiological relevance of NCp and the A-rich loop 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 NCp7: RNA ration 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 self-priming 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 7nt 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.

Keywords: AEGIS, RNA-Directed DNA Polymerase, Nucleocapsid Proteins, RNA, Transfer, Lys, HIV, DNA, Viral, Templates, Genetic, HIV-1, RNA, DNA Primers, Transcription, Genetic, RNA, Transfer, strong-stop DNA, pathogenicity, analysis, genetics, ICA11KWDaegis,rna-directeddnapolymerase,nucleocapsidproteins,rna,transfer,lys,hiv,dna,viral,templates,genetic,hiv-1,rna,dnaprimers,transcription,genetic,rna,transfer,strong-stopdna,pathogenicity,analysis,genetics,ica11

960707
TuA491

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