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

Fennessey CM, Reid C, Lipkey L, Newman L, Oswald K, Piatak M, Roser JD, Chertova E, Smedley J, Gregory Alvord W, Del Prete GQ, Estes JD, Lifson JD, Keele BF - Retrovirology (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Model of tRNA-mediated mismatched integration of the PBS following viral reverse transcription. Proposed model of the method by which the mismatched pairing in the primer binding site is generated and integrated into the host genome. The tRNALys3 binds the viral PBS and functions as a primer for the initiation of RT but retains a mismatched base to wild type SIVmac239 with a guanine to uracil (G/U) pairing. Reverse transcription progresses with the tRNA-primed U5 region disassociating from the viral RNA PBS, and reannealing to its 3′ end and continuing transcription through the PBS. Following complete RNAseH digestion of the parental RNA (except the PPT, which subsequently acts as a reverse primer for RT), the nascent double stranded DNA circularizes and uses itself as template to complete transcription. Integration of the resulting double stranded viral DNA occurs with a mismatched base containing cytosine and adenine (C/A) at position 860 of the PBS. The base is generated from the primer is a C (green) and the suboptimal base is retained as an A (blue) on antisense strand which encodes a T in viral progeny. Length of viral genome not drawn to scale.
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Fig7: Model of tRNA-mediated mismatched integration of the PBS following viral reverse transcription. Proposed model of the method by which the mismatched pairing in the primer binding site is generated and integrated into the host genome. The tRNALys3 binds the viral PBS and functions as a primer for the initiation of RT but retains a mismatched base to wild type SIVmac239 with a guanine to uracil (G/U) pairing. Reverse transcription progresses with the tRNA-primed U5 region disassociating from the viral RNA PBS, and reannealing to its 3′ end and continuing transcription through the PBS. Following complete RNAseH digestion of the parental RNA (except the PPT, which subsequently acts as a reverse primer for RT), the nascent double stranded DNA circularizes and uses itself as template to complete transcription. Integration of the resulting double stranded viral DNA occurs with a mismatched base containing cytosine and adenine (C/A) at position 860 of the PBS. The base is generated from the primer is a C (green) and the suboptimal base is retained as an A (blue) on antisense strand which encodes a T in viral progeny. Length of viral genome not drawn to scale.

Mentions: In order to assess the relative fitness costs of each suboptimal nucleotide, wild type virus was grown in vitro on SupT1-R5 cells for 2 months and sampled at least weekly for sequence analysis of vRNA to identify mutants and selection of optimal nucleotides. By day 21, the PBS had completely changed to the optimal version but the Env and Pol mutations had not yet arisen. While the Env mutation occurred at approximately week 10, the Pol mutations were not seen within the 2 months of this experiment. Although it is clear from published research that all four are suboptimal clones that will mutate to optimal nucleotides given sufficient time and viral replication [1, 6], the mutation with the greatest impact on early replication appears to be the PBS mutation followed by the Env mutation. In fact, the PBS mutation accumulated so rapidly that in order to precisely document this turnover we repeated the experiment sampling at 12 and 24 h and then daily for 8 days. Single genome amplification (SGA) was performed on cellular DNA to determine the proportion of sequences encoding the wild type, suboptimal thymine/adenine (T/A) pairing or the optimal cytosine/guanidine (C/G) dimer for the PBS mutation (Figure 6a). Sequence analysis from samples obtained within the first 24 h revealed that ~50% of all sequences contained the suboptimal dimer (T/A), and ~10% contained the optimal pairing (C/G). The remaining ~40% of all sequences contained a mismatched pairing of C/A at position 860 of the PBS. Since these sequences were obtained from DNA by SGA, where only a single, double stranded template is amplified for any given reaction, we concluded that following reverse transcription and integration, the viral genome contains a mismatched pairing at the suboptimal PBS position. Careful examination of the complex reverse transcription process with a tRNA3Lys primer actually predicts a mixed base within the PBS (Figure 7). Interestingly, while the fraction of cells with mixed bases was constant over the 8-day culture, we found that the proportion of the suboptimal pairing (T/A) decreased from ~50% of the sequences down to less than 10% and the proportion of the optimal nucleotide bases (C/G) increased in the exact opposite amount from nearly 10% to over 50% (Figure 6a). The rate of loss of the suboptimal nucleotide and the reciprocal gain of the optimal nucleotide was ~5% per day.Figure 6


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

Fennessey CM, Reid C, Lipkey L, Newman L, Oswald K, Piatak M, Roser JD, Chertova E, Smedley J, Gregory Alvord W, Del Prete GQ, Estes JD, Lifson JD, Keele BF - Retrovirology (2015)

Model of tRNA-mediated mismatched integration of the PBS following viral reverse transcription. Proposed model of the method by which the mismatched pairing in the primer binding site is generated and integrated into the host genome. The tRNALys3 binds the viral PBS and functions as a primer for the initiation of RT but retains a mismatched base to wild type SIVmac239 with a guanine to uracil (G/U) pairing. Reverse transcription progresses with the tRNA-primed U5 region disassociating from the viral RNA PBS, and reannealing to its 3′ end and continuing transcription through the PBS. Following complete RNAseH digestion of the parental RNA (except the PPT, which subsequently acts as a reverse primer for RT), the nascent double stranded DNA circularizes and uses itself as template to complete transcription. Integration of the resulting double stranded viral DNA occurs with a mismatched base containing cytosine and adenine (C/A) at position 860 of the PBS. The base is generated from the primer is a C (green) and the suboptimal base is retained as an A (blue) on antisense strand which encodes a T in viral progeny. Length of viral genome not drawn to scale.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4469405&req=5

Fig7: Model of tRNA-mediated mismatched integration of the PBS following viral reverse transcription. Proposed model of the method by which the mismatched pairing in the primer binding site is generated and integrated into the host genome. The tRNALys3 binds the viral PBS and functions as a primer for the initiation of RT but retains a mismatched base to wild type SIVmac239 with a guanine to uracil (G/U) pairing. Reverse transcription progresses with the tRNA-primed U5 region disassociating from the viral RNA PBS, and reannealing to its 3′ end and continuing transcription through the PBS. Following complete RNAseH digestion of the parental RNA (except the PPT, which subsequently acts as a reverse primer for RT), the nascent double stranded DNA circularizes and uses itself as template to complete transcription. Integration of the resulting double stranded viral DNA occurs with a mismatched base containing cytosine and adenine (C/A) at position 860 of the PBS. The base is generated from the primer is a C (green) and the suboptimal base is retained as an A (blue) on antisense strand which encodes a T in viral progeny. Length of viral genome not drawn to scale.
Mentions: In order to assess the relative fitness costs of each suboptimal nucleotide, wild type virus was grown in vitro on SupT1-R5 cells for 2 months and sampled at least weekly for sequence analysis of vRNA to identify mutants and selection of optimal nucleotides. By day 21, the PBS had completely changed to the optimal version but the Env and Pol mutations had not yet arisen. While the Env mutation occurred at approximately week 10, the Pol mutations were not seen within the 2 months of this experiment. Although it is clear from published research that all four are suboptimal clones that will mutate to optimal nucleotides given sufficient time and viral replication [1, 6], the mutation with the greatest impact on early replication appears to be the PBS mutation followed by the Env mutation. In fact, the PBS mutation accumulated so rapidly that in order to precisely document this turnover we repeated the experiment sampling at 12 and 24 h and then daily for 8 days. Single genome amplification (SGA) was performed on cellular DNA to determine the proportion of sequences encoding the wild type, suboptimal thymine/adenine (T/A) pairing or the optimal cytosine/guanidine (C/G) dimer for the PBS mutation (Figure 6a). Sequence analysis from samples obtained within the first 24 h revealed that ~50% of all sequences contained the suboptimal dimer (T/A), and ~10% contained the optimal pairing (C/G). The remaining ~40% of all sequences contained a mismatched pairing of C/A at position 860 of the PBS. Since these sequences were obtained from DNA by SGA, where only a single, double stranded template is amplified for any given reaction, we concluded that following reverse transcription and integration, the viral genome contains a mismatched pairing at the suboptimal PBS position. Careful examination of the complex reverse transcription process with a tRNA3Lys primer actually predicts a mixed base within the PBS (Figure 7). Interestingly, while the fraction of cells with mixed bases was constant over the 8-day culture, we found that the proportion of the suboptimal pairing (T/A) decreased from ~50% of the sequences down to less than 10% and the proportion of the optimal nucleotide bases (C/G) increased in the exact opposite amount from nearly 10% to over 50% (Figure 6a). The rate of loss of the suboptimal nucleotide and the reciprocal gain of the optimal nucleotide was ~5% per day.Figure 6

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus