title: Generation and characterization of a SIVmac239 clone corrected at four suboptimal nucleotides.
abstract: 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.
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. cmfennessey@gmail.com.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Fennessey
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. reidcm@mail.nih.gov.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Reid
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. leslie.lipkey@nih.gov.
authors.forename: Leslie
authors.initials: L
authors.lastname: Lipkey
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. laura.newman@nih.gov.
authors.forename: Laura
authors.initials: L
authors.lastname: Newman
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. oswaldk@mail.nih.gov.
authors.forename: Kelli
authors.initials: K
authors.lastname: Oswald
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. michael.piatak@nih.gov.
authors.forename: Michael
authors.initials: M
authors.lastname: Piatak
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. roserj@mail.nih.gov.
authors.forename: James D
authors.initials: JD
authors.lastname: Roser
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. Elena.Chertova@nih.gov.
authors.forename: Elena
authors.initials: E
authors.lastname: Chertova
authors.affiliation: Laboratory Animal Sciences Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA. jeremy.smedley@wanprc.org.|Washington National Primate Research Center, University of Washington, Seattle, WA, USA. jeremy.smedley@wanprc.org.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Smedley
authors.affiliation: Statistical Consulting, Data Management Services, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA. Greg.Alvord@nih.gov.
authors.forename: W
authors.initials: W
authors.lastname: Gregory Alvord
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. gregory.delprete@nih.gov.
authors.forename: Gregory Q
authors.initials: GQ
authors.lastname: Del Prete
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. estesj@mail.nih.gov.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Estes
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. lifsonj@mail.nih.gov.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Lifson
authors.affiliation: Retroviral Evolution Section, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Building 535, Rm. 408, Frederick, MD, 21702-1201, USA. keelebf@mail.nih.gov.
authors.forename: Brandon F
authors.initials: BF
authors.lastname: Keele
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D008253:Macaca mulatta; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000235:genetics*; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000235:genetics* / Q000502:physiology*; D019562:Viral Load; D014766:Viremia; D014774:Virulence; D014779:Virus Replication
chemical_list: D009711:Nucleotides