TY - JOUR
T1 - Generation and characterization of a SIVmac239 clone corrected at four suboptimal nucleotides
AU - Fennessey, Christine M.
AU - Reid, Carolyn
AU - Lipkey, Leslie
AU - Newman, Laura
AU - Oswald, Kelli
AU - Piatak, Michael
AU - Roser, James D.
AU - Chertova, Elena
AU - Smedley, Jeremy
AU - Gregory Alvord, W.
AU - Del Prete, Gregory Q.
AU - Estes, Jacob D.
AU - Lifson, Jeffrey D.
AU - Keele, Brandon F.
N1 - Funding Information:
We acknowledge Drs. Rob Gorelick and David Ott from the AIDS and Cancer Virus Program for helpful discussions. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
PY - 2015/6/16
Y1 - 2015/6/16
N2 - BACKGROUND: 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.RESULTS: 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.CONCLUSIONS: 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.
AB - BACKGROUND: 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.RESULTS: 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.CONCLUSIONS: 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.
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U2 - 10.1186/s12977-015-0175-3
DO - 10.1186/s12977-015-0175-3
M3 - Article
C2 - 26076651
AN - SCOPUS:85047596234
SN - 1742-4690
VL - 12
SP - 49
JO - Retrovirology
JF - Retrovirology
ER -