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Background:  Repeated bottleneck passages of RNA viruses result in fitness losses due to accumulation of deleterious mutations. The decrease in fitness due to repeated bottlenecks has been interpreted as the result of an accentuation of Muller’s ratchet effect. In contrast, repeated transfers of large virus populations result in exponential fitness increases HIV-1 manifested a drastic fitness loss after a limited number of plaque-to-plaque transfers in MT-4 cells, losses were drastic and rapid when compared with the fitness losses experienced by other RNA viruses. We now show the increase in fitness of four debilitated HIV-1 clones by repeated large population passages.

Methods:  Large virus populations passages were carried out in 5 x 10⁶ MT-4 cells with a multiplicity of infection of 0.1 PFU/cell. Fitness determination of the viral populations were performed by growth competition experiments with a reference clone. Quantification of viruses was achieved by a Heteroduplex Tracking assay. Complete genome sequences were determined on the 2 cDNA strands from cultures supernatant. Viral RNA was extracted, and amplified using RT-PCR and a nested PCR.

Results:  Comparison of the entire genomic nucleotide sequences of initial and final viral populations showed that few mutations, from 2 to 7 per clone, mediated fitness recovery. Of the 20 mutations, 8 affected coding regions, mainly by the introduction of non-synonymous mutations (75%); 25% of the overall mutations observed were reversions. Strikingly most of the mutations accumulated during fitness recovery (12 of 20) were located in the 5’-untranslated leader region of the genome and more specifically in the primer-binding-site loop. Two of the viruses incorporated the same mutation in the primer activation signal in the primer-binding-site loop, which is critical for tRNA₃^Lys-mediated initiation of reverse transcription..

Conclusions:  Large population passages have promoted the recovery in fitness of 4 debilitated viral populations. Unusual distribution of mutations in the 5’-untranslated leader region indicates the importance of the binding of the tRNA to the primer-binding-site loop and the initiation of reverse transcription. In addition, reversions appeared in high proportion, this fact together with the prevalence of non-synonymous replacements could disclose the operation, during large population passages, of a strong positive selection for optimal HIV-1 replication.

Keywords: Evolution; Fitness Recovery; Reversion