Generation and characterization of a SIVmac239 clone corrected at four suboptimal nucleotides

SIVmac239 is a commonly used virus in non-human primate models of HIV transmission and pathogenesis. Previous studies identified four suboptimal nucleotides in the SIVmac239 genome, which putatively inhibit its replicative capacity. Since all four suboptimal changes revert to the optimal nucleotide...

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Bibliographic Details
Published in:Retrovirology 2015-06, Vol.12 (1), p.49, Article 49
Main Authors: Fennessey, Christine M, Reid, Carolyn, Lipkey, Leslie, Newman, Laura, Oswald, Kelli, Piatak, Jr, Michael, Roser, James D, Chertova, Elena, Smedley, Jeremy, Gregory Alvord, W, Del Prete, Gregory Q, Estes, Jacob D, Lifson, Jeffrey D, Keele, Brandon F
Format: Article
Language:English
Subjects:
Analysis
Animals
Care and treatment
Cells, Cultured
Cloning
Complications and side effects
Disease Models, Animal
Genomics
Health aspects
Macaca mulatta
Mutagenesis, Site-Directed
Nucleotides
Nucleotides - genetics
Primates
Risk factors
Simian Acquired Immunodeficiency Syndrome - virology
Simian Immunodeficiency Virus - genetics
Simian Immunodeficiency Virus - physiology
Viral Load
Viremia
Virulence
Virus diseases
Virus Replication
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Summary:SIVmac239 is a commonly used virus in non-human primate models of HIV transmission and pathogenesis. Previous studies identified four suboptimal nucleotides in the SIVmac239 genome, which putatively inhibit its replicative capacity. Since all four suboptimal changes revert to the optimal nucleotide consensus sequence during viral replication in vitro and in vivo, we sought to eliminate the variability of generating these mutations de novo and increase the overall consistency of viral replication by introducing the optimal nucleotides directly to the infectious molecular clone. Using site directed mutagenesis of the full-length/nef-open SIVmac239 clone, we reverted all four nucleotides to the consensus/optimal base to generate SIVmac239Opt and subsequently tested its infectivity and replicative capacity in vitro and in vivo. In primary and cell line cultures, we observed that the optimized virus displayed consistent modest but not statistically significant increases in replicative kinetics compared to wild type. In vivo, SIVmac239Opt replicated to high peak titers with an average of 1.2 × 10(8) viral RNA copies/ml at day 12 following intrarectal challenge, reaching set-point viremia of 1.2 × 10(6) viral RNA copies/ml by day 28. Although the peak and set point viremia means were not statistically different from the original "wild type" SIVmac239, viral load variation at set point was greater for SIVmac239WT compared to SIVmac239Opt (p = 0.0015) demonstrating a greater consistency of the optimized virus. Synonymous mutations were added to the integrase gene of SIVmac239Opt to generate a molecular tag consisting of ten genetically distinguishable viral variants referred to as SIVmac239OptX (Del Prete et al., J Virol. doi: 10.1128/JVI.01026-14 , 2014). Replication dynamics in vitro of these optimized clones were not statistically different from the parental clones. Interestingly, the consistently observed rapid reversion of the primer binding site suboptimal nucleotide is not due to viral RT error but is changed post-integration of a mismatched base via host proofreading mechanisms. Overall, our results demonstrate that SIVmac239Opt is a functional alternative to parental SIVmac239 with marginally faster replication dynamics and with increased replication uniformity providing a more consistent and reproducible infection model in nonhuman primates.
ISSN:1742-4690
1742-4690
DOI:10.1186/s12977-015-0175-3