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Impairment of viral replication capacity by nef alleles from HIV elite controllers
© Mwimanzi et al; licensee BioMed Central Ltd. 2011
Published: 3 October 2011
Elite controllers (EC) are HIV-1 infected persons who can spontaneously control viremia to extremely low levels. Because it is known that nef is required for HIV-1 replication and pathogenesis in vivo, we wish to clarify how HIV-1 nef plays a role in acquiring EC phenotype.
Materials and methods
Viral RNAs were extracted from plasma samples of 45 ECs and 48 chronic progressors (CP) and used for amplification of nef by RT-PCR. The amplified products were cloned into a plasmid and subsequently sequenced. A representative nef clone was selected for each patient and cloned into an NL43 proviral plasmid backbone. Infectious recombinant viruses were prepared by transfecting 293T cells with these resultant proviral clones and tested for their viral replication capacity in PBMCs obtained from multiple healthy donors. Viral replication was monitored by measuring the concentration of p24 Gag in the culture supernatant by p24 ELISA every 3 days. The same virus stocks were further tested for their infectivity using TZM-bl cells.
All nef alleles isolated from EC and CP had intact ORFs. Phylogenetic analysis revealed no specific lineage or clustering, suggesting the absence of common genetic defects in EC-derived nef alleles. All viruses expressing patient-derived nef showed a wide range of viral replication in PBMC, confirming the importance of nef alleles in the enhancement of viral replication capacity. The viruses harboring EC-derived nef replicated and peaked on Day 12 after infection or even later; whereas the viruses harboring CP-derived nef peaked on Day 9. Also, the peak level of p24 values was significantly lower in the EC-nef viruses than in the CP-nef viruses. Because Nef activity is highly dependent on PBMC donors, we tested 4 different donors' PBMC and compared the initial burst of their viral replication at Day 9. In all donors'PBMC, viruses carrying EC-nef displayed lower p24 values than those carrying CP-nef, suggesting that the viral replication capacity was much impaired by nef alleles in EC. Corroboratively, in viral infectivity assay in TZM-bl cells, the EC-nef viruses showed significantly lower infectivity than the CP-nef viruses.
Taken together, our data indicate that nef alleles present in circulating plasma viruses in EC are much impaired in boosting viral replication, suggesting that such phenotypic alterations in Nef function play a role, at least partly, in the sustained maintenance of low viral load in vivo.
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.