12th International AIDS Conference Geneva, Switzerland - June 28-July 3, 1998

Int Conf AIDS 1998 Jun 28-Jul 3; 12:10-1 (abstract no. 11143)

Rhodes D, Solomon A, Deacon N
Macfarlane Burnet Centre for Medical Research, Fairfield, Vic., Australia.

BACKGROUND: The Sydney Bloodbank Cohort (SBBC) is a unique collection of individuals infected with an attenuated HIV-1 virus from a common donor. Molecular characterisation of viruses from one recipient, C98, shows the loss of sequence from the nef/LTR region over time indicating continued evolution of the cohort viruses. We have developed a sensitive replication fitness assay to examine the effects early and late deletions in the nef/LTR region have on virus replication.

METHODS: Proviral DNA was amplified from peripheral blood mononuclear cells (PBMC's) from cohort members and from virus cultured from PBMC's collected at different time points. Chimeric viruses were constructed to contain the cohort nef/LTR sequences in an NL4-3 background. The fitness of the chimeric C98 viruses was calculated relative to wildtype (NL4-3) following growth in a competition assay. Cells, MT-2 or PBMC's, were infected with a mixture of chimeric C98 and wild-type viruses at low multiplicities of infection, and passaged several times in culture. The nef/LTR region was PCR amplified from the proviral DNA in the infected cells using fluorescently labelled primers, and the change in the proportion of wild type to mutant nef/LTR's quantitated.

RESULTS: Evolution of the nef/LTR region has been observed in isolated and PBMC samples from the SBBC member C98. The evolved species lacks more sequence from the nef/LTR region and is now the predominate proviral species. The fitness of the chimeric virus containing the earlier, larger nef/LTR form was calculated to be 0.47 times as fit as that of wild type. The competition assays also show that viruses containing the newly evolved smaller nef/LTR sequences are also less fit than wild type, and subtle differences in replication are apparent between chimeras containing the larger and smaller nef/LTR sequences.

CONCLUSIONS: We have clearly demonstrated that the defective nef/LTR regions from the SBBC viruses led to decreased replication competency compared with wild type nef/LTR sequence. The competition assay described here will be widely applicable to the quantitation of the effects other subtle mutations have on virus replication.

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