AEGiS-11IAC: Populations of defective HIV-1 genomes in PBMC cells infected in vivo.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Populations of defective HIV-1 genomes in PBMC cells infected in vivo.

Int Conf AIDS 1996 Jul 7-12; 11:229 (abstract no. Tu.A.513)
Sanchez G, Xu X, Chenine AL, Chermann JC, Hirsch I; INSERM, Merseille, France. Fax: (33) 91 41 92 50.

OBJECTIVE: To study the presence of complete and defective HIV genome in peripheral blood mononuclear cells (PBMC) from people with progressive and nonprogressive HIV infection. In spite of enormous variability of HIV genome, occurence of extensive deletions or other alterations in HIV DNA in vivo is poorly documented.

METHODS: Long distance polymerase chain reaction (Ld-PCR) was used to amplify the HIV DNA localized between long terminal repeats (LTR) present in the Ficoll-purified PBMC from HIV-infected persons. Ld-PCR followed by Southern blott hybridization with [32P]-oligonucleotide probes derived from six distant regions spread all over the HIV genome permitted to identify complete as well as largely deleted HIV genomes.

RESULTS: Different populations of distinct HIV-1 DNA fragments of highly variable size ranging from 600 bp to full length provirus were present in PBMC from HIV-infected persons. Defective genomes were observed in all but one of 10 studied patients. Full-length HIV DNA was missing from the Ld-PCR products of two nonprogressors. Most of defective fragments reacted with probes specific for both extremities of HIV genome. The frequency of deletions was proportional to their proximity to the central part of HIV genome. A comparison of electrophoretic mobility of defective fragments with the expected sizes of DNA fragments based on analysis of the hybridization pattern suggests the occurrence of one hit deletion events and does not provide an evidence for involvement of other extensive DNA aberrations in the formation of defective genomes. Defective genomes tended to gradually disappear after activation of PBMC with phytohemaglutinin. Only full-length Ld-PCR product was obtained in PBMC infectedin vitro. This suggests that, in contrast to situation in vivo, defective genomes are negatively selected in vitro.

CONCLUSIONS: We demonstrate for the first time the presence of a high proportion of defective genomes involving large deletions in PBMC infected in vivo, and provide an easy approach to study distribution of defective and complete genomes in HIV-infected persons.

Keywords: AEGIS, HIV-1, Proviruses, HIV Infections, Polymerase Chain Reaction, Genome, Viral, Genome, In Vitro, Human, genetics, ICA11

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TuA513