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Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes
已表达前病毒的HIV-1包膜糖蛋白120 (gp120) 和nef基因的单基因组测序   

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Abstract

The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al., 2016a and 2016b; Rose et al., 2016). This method optimizes protocols from previous publications (Palmer et al., 2005; Norström et al., 2012; Lamers et al., 2015; 2016a and 2016b; Rife et al., 2016) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.

Keywords: HIV-1(HIV-1), Single genome sequencing(单基因组测序), SGS(SGS), Gene amplification(基因扩增), Nested PCR(巢式PCR)

Background

Over thirty years ago, HIV infection and its clinical manifestation, Acquired Immunodeficiency Syndrome (AIDS), emerged as a worldwide epidemic. Since then, significant understanding of HIV pathogenesis has occurred and the development of drug treatments now significantly extend patients’ lives. Current cART regimens encompass a variety of drugs that inhibit viral replication in several ways, which allows for the almost complete suppression of viral particles found in the blood and recovery of a healthy CD4+ T-cell population (CD4+) (Autran et al., 1997). However, the persistence of very low levels of HIV in plasma of cART treated patients, even those treated for decades, suggests the presence of a cell based ‘viral reservoir’. Viral reservoirs contain infected cells that do not release infectious virus (i.e., are latently infected), but can do so following activation, which may occur under a variety of conditions (Chun et al., 1995 and 1997). HIV latency is primarily attributed to proviral HIV DNA in resting memory CD4+ T cells (Anderson et al., 2011; Ho et al., 2013), although recent reviews highlight a breadth of research into other potential reservoirs (Abbas et al., 2015; Kandathil et al., 2016; Rothenberger et al., 2016; Sacha and Ndhlovu, 2016). The resting memory CD4+ T cells can live for long periods of time, contribute to low-level persistent viremia during cART and viral rebound after treatment interruption, and produce viral variants with escape mutations (Chun et al., 1997; Finzi et al., 1997). Methods to determine the effectiveness of antiretroviral therapy and latency-reversing agents by measuring the circulating resting memory CD4+ T cells have been developed and evaluated (Ericksson et al., 2013; Crooks et al., 2015). However, it is pertinent to consider that less than 2% of the total body lymphocyte population resides in peripheral blood (Svincher et al., 2014), making the evaluation of HIV persistence of tissue-resident lymphocyte populations in anatomical reservoirs critically important.
   The use of single genome sequencing or SGS (also known as single genome amplification or SGA) has become the routine way to generate sequences for examination of HIV intrahost evolution (Kearney et al., 2014; Lamers et al., 2016; Rose et al., 2016), compartmentalization (Sturdevant et al., 2012; Evering et al., 2014), phyloanatomy (Salemi and Rife, 2016), persistence (Josephsson et al., 2013; Buzon et al., 2014; Boritz et al., 2016), and rebound dynamics (Kearney et al., 2015; Bednar et al., 2016). In contrast to bulk PCR methods wherein many targets are amplified together in the same tube, SGS uses end-point dilution to amplify from only one template. While some studies have demonstrated that bulk PCR and SGS produce sequences that are similar by certain metrics and the techniques can be used interchangeably (Jordan et al., 2010; Etemad et al., 2015), some analyses can only yield accurate results with sequences generated from SGS. These include identifying identical HIV sequences that may arise from clonally-expanding cells rather than PCR resampling (Wagner et al., 2013; Simonetti et al., 2016), determining proportions of viral variants in a sample through sequencing (Iyer et al., 2015), estimating evolutionary rate from point-mutations that occur only from viral reverse-transcriptase rather than PCR Taq errors (Novitsky et al., 2013), and evaluating recombination rates in vivo without including PCR-mediated recombination (Brown et al., 2011; Sanborn et al., 2015).
   We used SGS to generate linked gp120 envelope and nef gene sequences from single starting templates to assess viral expression, compartmentalization and evolution in RNA and DNA extracted from a collection of fresh frozen tissues obtained from HIV-infected patients on cART who died with no detectable viral load in their plasma or cerebral spinal fluid at the time of death (Lamers et al., 2016a and 2016b; Rose et al., 2016). Our data demonstrated that a privileged environment exists in some tissues of these patients wherein expression of HIV continues; however, in other tissues, only unexpressed proviral DNA copies were identified. The inferred evolutionary rate of the tissue-based HIV sequences was not significantly different than previously reported rates of replicating virus in cART-negative subjects, suggesting on-going evolution.

Materials and Reagents

  1. RNA and DNA extraction
    1. Pipette tips
    2. TissueRuptor disposable probes (QIAGEN, catalog number: 990890 )
    3. Fresh frozen tissue sections (30-50 ng)
    4. ELIMINaseTM Decontaminant (Fisher Scientific, catalog number: 04-355-32 )
    5. AllPrep DNA/RNA Mini Kit (QIAGEN, catalog number: 80204 )
    6. RNeasy MinElute Cleanup Kit (QIAGEN, catalog number: 74204 )
    7. Qubit 2.0 fluorometer (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32857 )
    8. Ethyl alcohol pure (200 Proof molecular biology grade) (Sigma-Aldrich, catalog number: E7023 )
    9. Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
    10. Qubit® RNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32852 )

  2. cDNA synthesis
    1. 0.2 ml PCR 8-tube FLEX-FREE strip, attached clear flat caps, natural (USA Scientific, catalog number: 1402-4700 )
    2. SuperScript® III First-Strand Synthesis System (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080051 ). The SuperScript® III First-Strand Synthesis System is supplied with the following:
      1. Oligo(dT)20 (50 µM), 50 µl
      2. Random hexamers (50 ng/µl), 250 µl
      3. 10x RT buffer, 1 ml
      4. 0.1 M DTT, 250 µl
      5. 25 mM magnesium chloride (MgCl2), 500 µl
      6. 10 mM dNTP mix, 250 µl
      7. SuperScript® III RT (200 U/µl), 50 µl
      8. RNase-OUTTM (40 U/µl), 100 µl
      9. E. coli RNase H (2 U/µl), 50 µl
      10. DEPC-treated water, 1.2 ml
      11. Total HeLa RNA (10 ng/µl), 20 µl
      12. Sense Control Primer (10 µM), 25 µl
      13. Antisense Control Primer (10 µM), 25 µl

  3. Single genome sequencing of gp120 and nef
    1. 24 PCR wells
    2. Pipette tips
    3. TempPlate semi-skirted polypropylene 0.2 ml 96-well PCR plate (USA Scientific, catalog number: 1402-9220 )
    4. Posi-Click 1.7 ml microcentrifuge tube, 1.7 ml natural color (Denville Scientific, catalog number: C2170 )
    5. Molecular biology grade sterile purified water (RNase, DNase, proteinase free)
    6. EB buffer (QIAGEN, catalog number: 19086 )
    7. Platinum® Blue PCR SuperMix (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12580023 )
    8. Agarose (Fisher Scientific, catalog number: BP160-500 )
    9. Ethidium bromide (Fisher Scientific, catalog number: BP102-1 )
    10. Tris-base (Sigma-Aldrich, catalog number: T1378 )
    11. Acetic acid, glacial (Fisher Scientific, catalog number: A38-212 )
    12. Ethylenediaminetetraacetic acid, EDTA, 0.5 M solution/pH 8.0 (Fisher Scientific, catalog number: BP2482-500 )
    13. Milli-Q quality water (RNase, DNase free water [dH2O])
    14. Primers listed in Table 1

      Table 1. Primers


    15. 50x TAE stock solution (see Recipes)
    16. 1x TAE buffer(see Recipes)

Equipment

  1. TissueRupter rotor-stator homogenizer (QIAGEN, model: TissueRupter, catalog number: 9001271 )
  2. Matrix multichannel electronic pipette (Range: 2-125 µl; 12-channel) (Fisher Scientific, catalog number: 14-387-117 )*
  3. Matrix multichannel electronic pipette (Range: 1-30 µl; 12-channel) (Thermo Fisher Scientific, catalog number: 14-387-137 )*
  4. Matrix multichannel electronic pipette (Range: 2-125 µl; 12-channel) (Thermo Fisher Scientific, catalog number: 14-387-138 )*
  5. Eppendorf RepeaterTM stream electronic pipette (Eppendorf, catalog number: 4987000118 )
  6. Eppendorf ResearchTM Plus adjustable-volume pipettes: 0.1-2.5 µl, 2-20 µl, 20-200 µl, 100-1,000 µl (Eppendorf, catalog number: 022575442 )
  7. Tape pads (QIAGEN, catalog number: 19570 )
  8. Sub-CellTM Model 192 electrophoresis system (Bio-Rad Laboratories, model: Model 192, catalog number: 1704507 )
  9. 51-Well comb (Bio-Rad Laboratories, catalog number: 1704529 )
  10. Comb holder (Bio-Rad Laboratories, catalog number: 1704525 )
  11. UV-Transparent gel tray (Bio-Rad Laboratories, catalog number: 1704524 )
  12. Model 192 gel caster (Bio-Rad Laboratories, model: Model 192, catalog number: 1704517 )
  13. Centrifuge 5424, non-refrigerated, with Rotor FA-45-24-11, keypad, 230 V/50 -60 Hz (Eppendorf, model: 5424 , catalog number: 5424000010)
  14. IsotempTM Digital Dry Bath incubator (Fisher Scientific, catalog number: 11-718-2Q )*
  15. T100TM Thermal cycler (Bio-Rad Laboratories, model: T100TM, catalog number: 1861096 )
  16. DNA oligonucleotides were obtained from Invitrogen
  17. Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific, Applied BiosystemsTM, model: 3730xl DNA Analyser , catalog number: 3730XL)

*Note: These products have been discontinued.

Software

  1. Geneious R7 software package (Biomatters http://www.geneious.com)
  2. MEGA5

Procedure

  1. RNA and DNA extraction
    1. Thoroughly clean work surfaces and equipment before and after use with ELIMINase Decontaminant.
      Note: RNA and DNA extractions, cDNA synthesis and first round PCR set-up should be performed using filtered pipette tips and must be conducted in a restricted-access amplicon-free room with separate air-handling and laboratory equipment where no amplified PCR products or recombinant cloned plasmids are allowed. If no such room is available, conduct steps before amplification in a cell biology-grade clean hood equipped with separate air-handling mechanisms.
    2. Total RNA and genomic DNA are isolated separately and simultaneously from each tissue section (30-50 ng) using the AllPrep DNA/RNA Mini Kit following manufacturer’s guidelines. Two final 50 μl elutions using RNase-free water are performed during the last step of RNA isolation, totaling a final volume of 100 μl.
    3. Tissues are homogenized just prior to extraction using a TissueRupter rotor-stator homogenizer with a fresh sterile disposable probe for each sample.
    4. The 100 μl final volume of RNA is concentrated using RNeasy MinElute Cleanup Kit according to manufacturer’s instructions. A single final elution of 20 μl RNase-free water is used.
    5. Quantification of the resulting RNA and DNA is performed to determine the success of the extraction protocol and the concentration, utilizing the Qubit 2.0 fluorometer and either the Qubit RNA HS Assay Kit or Qubit dsDNA HS Assay Kit where appropriate. Failure to detect DNA or RNA, or a yield of less than 1 ng/μl for either RNA or DNA, indicates a failed extraction and the extraction should be repeated until more than 1 ng/μl of RNA and DNA is detected.

  2. cDNA synthesis
    1. cDNA is created immediately from the RNA of each sample using the SuperScript® III First-Strand Synthesis System using the provided oligo(dT)20 primer according to manufacturer’s recommendations with slight modifications, detailed below, to increase product length.
    2. In two identical reactions for each sample, 8 μl RNA is incubated at 65 °C for 5 min with deoxynucleoside triphosphates (0.5 mM [each]) and 5 µM oligo (dT)20, then cooled quickly to 4 °C.
      Note: Use thermocycler for accurate temperatures and hold times. cDNA synthesis reactions are conducted in 0.2 ml PCR 8-tube FLEX-FREE strips.
    3. First-strand cDNA synthesis will continue in a 20 µl reaction volume containing 1x reverse transcription buffer (10 mM Tris-HCl [pH 8.4], 25 mM KCl), 5 mM MgCl2, 10 mM ditiothreitol, 2 U/µl of RNase-OUTTM (RNase inhibitor), and 10 U/µl SuperScript® III RT. The reaction is heated to 45 °C for 90 min, and then 85 °C for 5 min.
    4. The reaction is then cooled to 37 °C and 0.1 U/µl of E. coli RNase H is added, followed by a 20-min incubation.
    5. The two reactions for each sample are combined with gentle pipette mixing to avoid shearing the cDNA. cDNA is stored at -20 °C until needed.


  3. Single genome sequencing of gp120 and nef
    1. cDNA and genomic DNA (gDNA) dilutions using EB buffer are usually performed to achieve 30% or less of positive nested PCR reactions, which indicates the positive reactions will have a greater than 80% chance of one starting template.
      Notes:
      1. For patients on cART, it is practical to start with 1:3 and 1:9 dilutions of cDNA and gDNA, with 24 PCR wells for each dilution. For patients not on cART, higher dilutions can be used.
      2. Stock and dilutions must be kept on ice after thawing and mixing, and frozen at -20 °C when not in use. Pipette mix or flick mix samples and dilutions, do not vortex to mix.
      3. Serial dilutions and first round PCR setup must be done in the amplicon-free room and always use filtered pipette tips.
      4. Two rounds of PCR are required to generate enough product for visualization, quantification and sequencing when starting with only a single template.
    2. During the first round PCR, 1 µl of diluted cDNA or genomic DNA is amplified in 20 µl reactions containing 1x Platinum® Blue PCR SuperMix and 0.05 µM of each primer: BEF1, 5’-TAATAGCAATAGTTGTGTGG-3’ and BNR1, 5’-AGCTCCCAGGCTCAGATCT-3’ (6,111-6,130 and 9,558-9,576 bp of HIV-1 HXB2 respectively).
      1. The first round primers are at 0.05 µM concentration in the reaction volume to eliminate unused excess first round primer carryover into the second round PCR. Excess first round primers in the second round PCR produces non-specific PCR products and reduces the amount of the desired product. See Figure 1 for an example of the non-specific PCR products generated by first round PCR primer carryover.


        Figure 1. Example of non-specific primer binding to genomic DNA. This gel provides an example of experiments where the concentration of the first round PCR primers was 0.2 µM in the first round PCR. First round primers at this concentration resulted in non-specific product formation in the second round PCR, as seen by the fainter bands found in most wells, whether or not those wells have a bright band that corresponds to the size of the positive control. When the first round PCR primers were used at 0.05 µM, these secondary products are no longer visible while the positive PCR products of the correct size are still visible, resulting in easier to interpret results and direct sequencing of the second round products without further gel purification to isolate a single band. 

    3. First round PCR cycling parameters–an initial denaturation 94 °C for 3 min, then 40 cycles of 94 °C for 30 sec, 56 °C for 30 sec,72 °C for 4 min, followed by a final extension of 72 °C for 10 min.


      Notes:
      1. PCRs are conducted in a 96-well format using TempPlate semi-skirted polypropylene 0.2 ml 96-well PCR plates and Tape pads. Large batches of PCR plates containing premixed SuperMix and primers are created and frozen for future use to reduce inter-experiment variability.
      2. Positive PCR controls should be selected carefully and diluted enough to produce only a single band after nested PCR is complete–therefore a band will not be visible after the first round PCR. Very concentrated positive controls can easily contaminate the PCR plates and diluted to a workable level.
      3. Using automated pipettes reduces the possibility of error and cross-contamination. We use Matrix multichannel electronic pipette (Range: 1-30 µl; 12-channel) and Eppendorf Repeater stream electronic pipette.
      4. The amount of primer in first round PCR is reduced significantly to reduce non-specific binding and primer carry-over during second round PCR. Extension times and cycle number are increased to generate an increased number of complete products.
    4. Second round gp120 PCR consists of 2 µl of the first round PCR product added to a 20 µl second round reaction consisting of 1x Platinum® Blue PCR SuperMix and 0.2 µM of each primer: BEF2, 5’-CAATAGTTGTGTGGTCCATAG-3’ and BER2, 5’-CAACAGATGCTGTTGCGC-3’ (6,117-6,137 bp and 7,905-7,922 bp of HIV-1 HXB2 respectively)
    5. Second round gp120 PCR cycling parameters–an initial denaturation 94 °C for 3 min, then 40 cycles of 94 °C for 30 sec, 56 °C for 30 sec, 72 °C for 3 min, followed by a final extension of 72 °C for 10 min.


    6. Second round gp120 PCR products are visualized on 1% agarose gels stained with ethidium bromide run at 150 V for 30 min in 1x TAE buffer.
      Notes:
      1. This second round PCR generates a 1.8 Kb product when positive, containing a complete gp120 sequence. Products from positive wells are sent sequencing with BEF2 and BER2 primers. This protocol produces single specific PCR products that can be directly sequenced, and do not require PCR purification. See Figure 2 for an example of successful gp120 second round PCR with two different patient samples and a dilution series for DNA from another tissue.
      2. We use Platinum® Blue PCR SuperMix to direct load second round products on agarose gels rather than mixing loading dye in each reaction. We use Matrix multichannel electronic pipette (Range: 2-125 µl; 12-channel) to automated loading on a Sub-CellTM Model 192 electrophoresis system.


      Figure 2. Example of Second Round gp120 PCR plate agarose gel image. The two samples used on the top row of the gel are undiluted genomic DNA from spleen tissue of two patients. Both samples have a total number of positive wells that equals less than 30%, indicating that the positive wells are most likely the result of nested PCR amplification of gp120 from a single integrated proviral genome in the DNA present in that well. The bottom row (HC09SPd1 DIL_1) provides an example of serial dilution testing to assess the correct SGS dilution. Four dilutions are tested here, and while all four dilutions are high enough to generate the amplification of a single integrated provirus in a positive well, all four are too high of a dilution to get many positive reactions resulting in wasted reagents. The ideal situation would be to find a dilution where 20-30% of the wells are positive, so lower dilutions must be tested to find an optimal dilution. The negative control, while not labeled on the gel, is in well A1, and the positive control (labeled POS) is in well H12. The negative control has 1 µl of the water used for dilution of the DNA, and the positive control is diluted genomic DNA from a patient who was not on cART that was PCR positive in previous experiments. 

    7. Subsequently, the first round reactions that corresponded to positive second round gp120 PCRs were used to amplify the nef gene sequence; second round nef PCR consisted of 2 μl of the first round PCR added to a 20 μl second round reaction consisting of 1x Platinum® Blue PCR SuperMix and 0.2 µM of each primer: BNF1, 5’-CTGGCTGTGGAAAGATACCT-3’ and BNR2, 5’-ATCTGAGGGCTCGCCACT-3’ (7,965-7,984 bp and 9,488-9,505 of HIV-1 HXB2 respectively).
    8. Nef cycling parameters–an initial denaturation 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 sec, 58 °C for 30 sec, 72 °C for 2 min, followed by a final extension at 72 °C for 10 min.


    9. Second round nef PCR products were visualized on 1% agarose gels stained with ethidium bromide run at 150 V for 30 min in 1x TAE buffer.
      Note: Reactions containing single 1.5 Kb products were considered positive and selected for sequencing with BNF1 and BNR2. This protocol produces single specific PCR products that can be directly sequenced, and do not require PCR purification.

Data analysis

Sequencing was performed on an Applied Biosystems 3730xl DNA Analyzer at the University of Florida Interdisciplinary Center for Biotechnology Research (UF ICBR) using with BEF2/BER2 primers for gp120 and BNF1/BNR2 primers for nef sequencing. Forward and reverse chromatograms for each PCR product sequenced were assembled with the Geneious R7 software package (Biomatters http://www.geneious.com) and optimized by hand where possible to resolve ambiguous sequencing calls assigned by the sequencer or spurious gaps from the assembly algorithm. Forward and reverse chromatograms that did not assemble using the Geneious automated assembly algorithm with default settings were discarded, usually these were sequences of very poor quality (too many overlapping peaks for accurate base calling by the sequencer) or of very short length (less than 600 base pairs). For assemblies where multiple chromatogram peaks were found in two or more base pairs, indicating either multiple starting templates or multiple PCR errors in the initial amplification of the starting template, these sequences were removed from further analysis. A consensus sequence was extracted from each optimized assembly using the Geneious software package. Consensus sequences were aligned using ClustalW (Thompson et al., 1997) in MEGA5 (Tamura et al., 2011) with further optimization performed by hand to remove spurious gaps created by the alignment algorithm. The final env and nef alignments spanned from positions 6,213-7,823 and positions 8,797-9,411 relative to the HXB2 genome, respectively. Hypervariable regions in env (V1, V2 and V4 domains) were excluded due to a large number of naturally occurring insertions and deletions that are typically problematic to align and may bias phylogenetic analysis. A preliminary maximum-likelihood phylogeny for each gene was estimated using PhyML (http://www.atgc-montpellier.fr/phyml/) and sequences from all participants to ensure no cross-contamination of patients occurred. Sequences were tested for the presence of hyper-mutations using the HYPERMUTE tool (http://www.lanl.gov); sequences with a P-value of < 0.01 were removed from the alignments. Example sequences generated with this protocol have been submitted to GenBank (Accession numbers KU708874-KU709831).

Notes

When considering the results of SGS experiments, it is important to keep in mind several ideas:

  1. Primer binding efficiency might vary by patient, subtype, or viral gene targeted based on variations of the viral genome. Screening each patient with multiple sets of primers specific for the subtype of the patient and finding concordant results will increase confidence in the sequencing results. Tissues found to be negative for the SGS protocol for gp120-nef presented here should also be assessed with primers in more conserved regions of the HIV genome like gag (Norström et al., 2012) or pol (Palmer et al., 1999; Shafer et al., 2000) to confirm the absence of virus. Using a program like QUALITY (Rodrigo et al., 1997) to estimate copy number based on SGS dilutions (Rife et al., 2016) with SGS results from different primer sets can also provide data on the binding efficiency of each set. In addition, alternative gp120/nef primers (Lamers et al., 2016b) can be used to confirm that some variants are not missed due to primer binding efficiency of the primers presented here. Sequences generated from these alternative primers can be included in phylogenic analysis to evaluate the efficiency of the original primers at capturing the landscape of viral diversity in the tissue. Real-time or quantitative PCR can also be used to evaluate positive or negative SGS results (Lamers et al., 2016a).
  2. Tissue type can affect the DNA and RNA isolation. The Qiagen Allprep kit has some detailed instructions on altering methods to boost isolation efficiency for different tissue types. Alternative kits or protocols should be considered for tissues that consistently result in low yields. Tissue preservation will also affect isolation results and great care in handling tissues should be exercised to prevent premature thawing. Separate isolations conducted on multiple tissue sections will increase confidence in SGS results. Tissues from the same patient should be processed separately where possible, as cross-contamination of samples from the same patient will not be as easily recognized as mixing between patients during initial phylogenetic analysis of all sequences generated.

Recipes

  1. 50x TAE stock solution
    To prepare 1 L of 50x TAE dissolve following components:
    in 600 ml of deionized water:
    242 g Tris base (FW = 121)
    57.1 ml glacial acetic acid
    100 ml 0.5 M EDTA (pH 8.0)
  2. 1x TAE buffer
    40 mM Tris (pH 7.6)
    20 mM acetic acid
    1 mM EDTA
    Dilute 1:50 for 1x TAE buffer for gel electrophoresis

Acknowledgments

Funding for development of this HIV-1 sequencing protocol was provided by the National Institutes of Health (NIH) through the R01 MH100984 grant, and is based on methods developed on a previous NIH grant, R01 NS063897. We would like to thank the National Neurological AIDS Bank (NNAB, UM1 CA181255) and the AIDS and Cancer Specimen Resource (ACSR, UM1 CA181255) to providing the tissues used to develop the protocol and generate data for publication, specifically Elyse Singer (NNAB), Debra Garcia (ACSR) and Salman Mahboob (ACSR). We would also like to acknowledge and thank Melissa Norström (formerly of Karolinska Institutet) for initially teaching us the HIV-1 single genome sequencing technique for the gag gene. This method adapted from protocols from previous publications (Palmer et al., 2005; Norström et al., 2012; Lamers et al., 2015; Rife et al., 2016).

References

  1. Abbas, W., Tariq, M., Iqbal, M., Kumar, A. and Herbein, G. (2015). Eradication of HIV-1 from the macrophage reservoir: an uncertain goal? Viruses 7(4): 1578-1598.
  2. Anderson, J. A., Archin, N. M., Ince, W., Parker, D., Wiegand, A., Coffin, J. M., Kuruc, J., Eron, J., Swanstrom, R. and Margolis, D. M. (2011). Clonal sequences recovered from plasma from patients with residual HIV-1 viremia and on intensified antiretroviral therapy are identical to replicating viral RNAs recovered from circulating resting CD4+ T cells. J Virol 85(10): 5220-5223.
  3. Autran, B., Carcelain, G., Li, T. S., Blanc, C., Mathez, D., Tubiana, R., Katlama, C., Debre, P. and Leibowitch, J. (1997). Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science 277(5322): 112-116.
  4. Bednar, M. M., Hauser, B. M., Zhou, S., Jacobson, J. M., Eron, J. J., Jr., Frank, I. and Swanstrom, R. (2016). Diversity and tropism of HIV-1 rebound virus populations in plasma level after treatment discontinuation. J Infect Dis 214(3): 403-407.
  5. Boritz, E. A., Darko, S., Swaszek, L., Wolf, G., Wells, D., Wu, X., Henry, A. R., Laboune, F., Hu, J., Ambrozak, D., Hughes, M. S., Hoh, R., Casazza, J. P., Vostal, A., Bunis, D., Nganou-Makamdop, K., Lee, J. S., Migueles, S. A., Koup, R. A., Connors, M., Moir, S., Schacker, T., Maldarelli, F., Hughes, S. H., Deeks, S. G. and Douek, D. C. (2016). Multiple origins of virus persistence during natural control of HIV infection. Cell 166(4): 1004-1015.
  6. Brown, R. J., Peters, P. J., Caron, C., Gonzalez-Perez, M. P., Stones, L., Ankghuambom, C., Pondei, K., McClure, C. P., Alemnji, G., Taylor, S., Sharp, P. M., Clapham, P. R. and Ball, J. K. (2011). Intercompartmental recombination of HIV-1 contributes to env intrahost diversity and modulates viral tropism and sensitivity to entry inhibitors. J Virol 85(12): 6024-6037.
  7. Buzon, M. J., Sun, H., Li, C., Shaw, A., Seiss, K., Ouyang, Z., Martin-Gayo, E., Leng, J., Henrich, T. J., Li, J. Z., Pereyra, F., Zurakowski, R., Walker, B. D., Rosenberg, E. S., Yu, X. G. and Lichterfeld, M. (2014). HIV-1 persistence in CD4+ T cells with stem cell-like properties. Nat Med 20(2): 139-142.
  8. Chun, T. W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J. A., Taylor, H., Hermankova, M., Chadwick, K., Margolick, J., Quinn, T. C., Kuo, Y. H., Brookmeyer, R., Zeiger, M. A., Barditch-Crovo, P. and Siliciano, R. F. (1997). Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387(6629): 183-188.
  9. Chun, T. W., Finzi, D., Margolick, J., Chadwick, K., Schwartz, D. and Siliciano, R. F. (1995). In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat Med 1(12): 1284-1290.
  10. Crooks, A. M., Bateson, R., Cope, A. B., Dahl, N. P., Griggs, M. K., Kuruc, J. D., Gay, C. L., Eron, J. J., Margolis, D. M., Bosch, R. J. and Archin, N. M. (2015). Precise quantitation of the latent HIV-1 reservoir: Implications for eradication strategies. J Infect Dis 212(9): 1361-1365.
  11. Eriksson, S., Graf, E. H., Dahl, V., Strain, M. C., Yukl, S. A., Lysenko, E. S., Bosch, R. J., Lai, J., Chioma, S., Emad, F., Abdel-Mohsen, M., Hoh, R., Hecht, F., Hunt, P., Somsouk, M., Wong, J., Johnston, R., Siliciano, R. F., Richman, D. D., O'Doherty, U., Palmer, S., Deeks, S. G. and Siliciano, J. D. (2013). Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog 9(2): e1003174.
  12. Etemad, B., Ghulam-Smith, M., Gonzalez, O., White, L. F. and Sagar, M. (2015). Single genome amplification and standard bulk PCR yield HIV-1 envelope products with similar genotypic and phenotypic characteristics. J Virol Methods 214: 46-53.
  13. Evering, T. H., Kamau, E., St Bernard, L., Farmer, C. B., Kong, X. P. and Markowitz, M. (2014). Single genome analysis reveals genetic characteristics of Neuroadaptation across HIV-1 envelope. Retrovirology 11: 65.
  14. Finzi, D., Hermankova, M., Pierson, T., Carruth, L. M., Buck, C., Chaisson, R. E., Quinn, T. C., Chadwick, K., Margolick, J., Brookmeyer, R., Gallant, J., Markowitz, M., Ho, D. D., Richman, D. D. and Siliciano, R. F. (1997). Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278(5341): 1295-1300.
  15. Ho, Y. C., Shan, L., Hosmane, N. N., Wang, J., Laskey, S. B., Rosenbloom, D. I., Lai, J., Blankson, J. N., Siliciano, J. D. and Siliciano, R. F. (2013). Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 155(3): 540-551.
  16. Jordan, M. R., Kearney, M., Palmer, S., Shao, W., Maldarelli, F., Coakley, E. P., Chappey, C., Wanke, C. and Coffin, J. M. (2010). Comparison of standard PCR/cloning to single genome sequencing for analysis of HIV-1 populations. J Virol Methods 168(1-2): 114-120.
  17. Josefsson, L., von Stockenstrom, S., Faria, N. R., Sinclair, E., Bacchetti, P., Killian, M., Epling, L., Tan, A., Ho, T., Lemey, P., Shao, W., Hunt, P. W., Somsouk, M., Wylie, W., Douek, D. C., Loeb, L., Custer, J., Hoh, R., Poole, L., Deeks, S. G., Hecht, F. and Palmer, S. (2013). The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time. Proc Natl Acad Sci U S A 110(51): E4987-4996.
  18. Kandathil, A. J., Sugawara, S. and Balagopal, A. (2016). Are T cells the only HIV-1 reservoir? Retrovirology 13(1): 86.
  19. Kearney, M. F., Spindler, J., Shao, W., Yu, S., Anderson, E. M., O'Shea, A., Rehm, C., Poethke, C., Kovacs, N., Mellors, J. W., Coffin, J. M. and Maldarelli, F. (2014). Lack of detectable HIV-1 molecular evolution during suppressive antiretroviral therapy. PLoS Pathog 10(3): e1004010.
  20. Kearney, M. F., Wiegand, A., Shao, W., Coffin, J. M., Mellors, J. W., Lederman, M., Gandhi, R. T., Keele, B. F. and Li, J. Z. (2015). Origin of rebound plasma HIV includes cells with identical proviruses that are transcriptionally active before stopping of antiretroviral therapy. J Virol 90(3): 1369-1376.
  21. Lamers, S. L., Nolan, D. J., Rife, B. D., Fogel, G. B., McGrath, M. S., Burdo, T. H., Autissier, P., Williams, K. C., Goodenow, M. M. and Salemi, M. (2015). Tracking the emergence of host-specific simian immunodeficiency virus env and nef populations reveals nef early adaptation and convergent evolution in brain of naturally progressing rhesus macaques. J Virol 89(16): 8484-8496.
  22. Lamers, S. L, Rose, R., Maidji, E., Agsalda-Garcia, M., Nolan, D. J., Fogel, G. B., Salemi, M., Garcia, D. L., Bracci, P., Yong, W., Commins, D., Said, J., Khanlou, N., Hinkin, C. H., Sueiras, M. V., Mathisen, G., Donovan, S., Shiramizu, B., Stoddart, C. A., McGrath, M. S. and Singer, E. J. (2016a). HIV DNA is frequently present within pathologic tissues evaluated at autopsy from combined antiretroviral therapy-treated patients with undetectable viral loads. J Virol 90(20): 8968-83.
  23. Lamers, S. L., Rose, R., Nolan, D. J., Fogel, G. B., Barbier, A. E., Salemi, M. and McGrath, M. S. (2016b). HIV-1 evolutionary patterns associated with metastatic Kaposi's sarcoma during AIDS. Sarcoma 2016: 4510483.
  24. Iyer, S., Casey, E., Bouzek, H., Kim, M., Deng, W., Larsen, B. B., Zhao, H., Bumgarner, R. E., Rolland, M. and Mullins, J. I. (2015). Comparison of major and minor viral SNPs identified through single template sequencing and pyrosequencing in acute HIV-1 infection. PLoS One 10(8): e0135903.
  25. Norström M. M., Buggert, M., Tauriainen, J., Hartogensis, W., Prosperi, M. C., Wallet, M. A., Hecht, F. M., Salemi, M. and Karlsson, A. C. (2012). Combination of immune and viral factors distinguishes low-risk versus high-risk HIV-1 disease progression in HLA-B*5701 subjects. J Virol 86(18): 9802-9816.
  26. Novitsky, V., Wang, R., Rossenkhan, R., Moyo, S. and Essex, M. (2013). Intra-host evolutionary rates in HIV-1C env and gag during primary infection. Infect Genet Evol 19: 361-368.
  27. Palmer, S., Kearney, M., Maldarelli, F., Halvas, E. K., Bixby, C. J., Bazmi, H., Rock, D., Falloon, J., Davey, R. T., Jr., Dewar, R. L., Metcalf, J. A., Hammer, S., Mellors, J. W. and Coffin, J. M. (2005). Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis. J Clin Microbiol 43(1): 406-413.
  28. Palmer, S., Shafer, R. W. and Merigan, T. C. (1999). Highly drug-resistant HIV-1 clinical isolates are cross-resistant to many antiretroviral compounds in current clinical development. AIDS 13(6): 661-667.
  29. Rife, B. D., Nolan, D. J., Lamers, S. L., Autissier, P., Burdo, T., Williams, K. C. and Salemi, M. (2016). Evolution of neuroadaptation in the periphery and purifying selection in the brain contribute to compartmentalization of simian immunodeficiency virus (SIV) in the brains of rhesus macaques with SIV-associated encephalitis. J Virol 90(13): 6112-6126.
  30. Rodrigo, A. G., Goracke, P. C., Rowhanian, K. and Mullins, J. I. (1997). Quantitation of target molecules from polymerase chain reaction-based limiting dilution assays. AIDS Res Hum Retroviruses 13(9): 737-742.
  31. Rose, R., Lamers, S. L., Nolan, D. J., Maidji, E., Faria, N. R., Pybus, O. G., Dollar, J. J., Maruniak, S. A., McAvoy, A. C., Salemi, M., Stoddart, C. A., Singer, E. J. and McGrath, M. S. (2016). HIV maintains an evolving and dispersed population in multiple tissues during suppressive combined antiretroviral therapy in individuals with cancer. J Virol 90(20): 8984-8993.
  32. Rothenberger, M. K., Keele, B. F., Wietgrefe, S. W., Fletcher, C. V., Beilman, G. J., Chipman, J. G., Khoruts, A., Estes, J. D., Anderson, J., Callisto, S. P., Schmidt, T. E., Thorkelson, A., Reilly, C., Perkey, K., Reimann, T. G., Utay, N. S., Nganou Makamdop, K., Stevenson, M., Douek, D. C., Haase, A. T. and Schacker, T. W. (2015). Large number of rebounding/founder HIV variants emerge from multifocal infection in lymphatic tissues after treatment interruption. Proc Natl Acad Sci U S A 112(10): E1126-1134.
  33. Sacha, J. B. and Ndhlovu, L. C. (2016). Strategies to target non-T-cell HIV reservoirs. Curr Opin HIV AIDS 11(4): 376-382.
  34. Salemi, M. and Rife, B. (2016). Phylogenetics and phyloanatomy of HIV/SIV intra-host compartments and reservoirs: the key role of the central nervous system. Curr HIV Res 14(2):110-20.
  35. Sanborn, K. B., Somasundaran, M., Luzuriaga, K. and Leitner, T. (2015). Recombination elevates the effective evolutionary rate and facilitates the establishment of HIV-1 infection in infants after mother-to-child transmission. Retrovirology 12: 96.
  36. Shafer, R. W., Warford, A., Winters, M. A. and Gonzales, M. J. (2000). Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1-infected individuals. J Virol Methods 86(2): 143-153.
  37. Simonetti, F. R., Sobolewski, M. D., Fyne, E., Shao, W., Spindler, J., Hattori, J., Anderson, E. M., Watters, S. A., Hill, S., Wu, X., Wells, D., Su, L., Luke, B. T., Halvas, E. K., Besson, G., Penrose, K. J., Yang, Z., Kwan, R. W., Van Waes, C., Uldrick, T., Citrin, D. E., Kovacs, J., Polis, M. A., Rehm, C. A., Gorelick, R., Piatak, M., Keele, B. F., Kearney, M. F., Coffin, J. M., Hughes, S. H., Mellors, J. W. and Maldarelli, F. (2016). Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo. Proc Natl Acad Sci U S A 113(7): 1883-1888.
  38. Sturdevant, C. B., Dow, A., Jabara, C. B., Joseph, S. B., Schnell, G., Takamune, N., Mallewa, M., Heyderman, R. S., Van Rie, A. and Swanstrom, R. (2012). Central nervous system compartmentalization of HIV-1 subtype C variants early and late in infection in young children. PLoS Pathog 8(12): e1003094.
  39. Svicher, V., Ceccherini-Silberstein, F., Antinori, A., Aquaro, S. and Perno, C. F. (2014). Understanding HIV compartments and reservoirs. Curr HIV/AIDS Rep 11(2): 186-194.
  40. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10): 2731-2739.
  41. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24): 4876-4882.
  42. Wagner, T. A., McKernan, J. L., Tobin, N. H., Tapia, K. A., Mullins, J. I. and Frenkel, L. M. (2013). An increasing proportion of monotypic HIV-1 DNA sequences during antiretroviral treatment suggests proliferation of HIV-infected cells. J Virol 87(3): 1770-1778.

简介

目前的研究提供了详细的方案,用于扩增完整的HIV-1 gp120和nef基因,从单个拷贝的表达或综合的HIV存在于新鲜冷冻尸检组织中,在联合抗逆转录病毒治疗(cART)而死亡的患者中,没有可检测的血浆病毒 死亡时负荷(pVL)(Lamers等,2016a和2016b; Rose等,2016)。 该方法优化了以前的出版物(Palmer等,2005;Norström等,2012; Lamers等,2015; 2016a和2016b; Rife等,2016)的方案,以产生可以直接的单独不同的PCR产物 测序并包括若干成本节约和时间有效的修改。
【背景】三十多年前,艾滋病毒感染及其临床表现,即获得性免疫缺陷综合征(AIDS),已成为全球流行病。此后,对艾滋病病毒发病机制的认识已经出现,药物治疗的发展显着延长了患者的生命。目前的cART方案包括以几种方式抑制病毒复制的各种药物,其允许几乎完全抑制血液中发现的病毒颗粒和恢复健康的CD4 + T细胞群体(CD4 +)(Autran等人,1997 )。然而,cART治疗患者血浆中持续存在非常低水平的艾滋病毒,即使是经过数十年治疗的患者,也表明存在一种以病毒为基础的细胞库。病毒储库包含不释放感染性病毒(即被潜在感染)的感染细胞,但可以在活化后进行,这可能在各种条件下发生(Chun等,1995和1997)。 HIV延迟主要归因于静息记忆CD4 + T细胞中的病毒性HIV DNA(Anderson et al。,2011; Ho et al。,2013),尽管最近的评论强调了对其他潜在水库的广泛研究(Abbas et al。,2015 ; Kandathil等人,2016; Rothenberger等人,2016; Sacha和Ndhlovu,2016)。休息记忆CD4 + T细胞可以长时间存活,在治疗中断之前导致cART期间的低水平持续病毒血症和病毒反弹,并产生具有逃避突变的病毒变体(Chun等,1997; Finzi等, 1997年)。已经开发和评估了通过测量循环静息记忆CD4 + T细胞来确定抗逆转录病毒疗法和潜伏逆转剂的有效性的方法(Ericksson等人,2013; Crooks等,2015)。然而,考虑到全身淋巴细胞总数的不到2%存在于外周血中(Svincher等,2014)是有必要的,因此对解剖学水库中组织常住淋巴细胞群体HIV持续性的评估至关重要。
单基因组测序或SGS(也称为单基因组扩增或SGA)的使用已经成为产生用于检测HIV内源性进化的序列的常规方法(Kearney等,2014; Lamers等,2016; Rose等(Sturdevant et al。,2012; Evering et al。,2014),Phyloanatomy(Salemi and Rife,2016),持久性(Josephsson等,2013; Buzon等,2014; Boritz等,2016)和反弹动态(Kearney等,2015; Bednar等,2016)。与其中许多靶在同一管中一起放大的体PCR方法相反,SGS使用终点稀释仅从一个模板扩增。虽然一些研究已经证明,批量PCR和SGS产生与某些指标相似的序列,并且该技术可以互换使用(Jordan等人,2010; Etemad等人,2015),但一些分析只能产生具有序列的准确结果由SGS生成。这些包括识别可能由克隆扩增的细胞而不是PCR重采样产生的相同的HIV序列(Wagner等,2013; Simonetti等,2016),通过测序确定样品中病毒变体的比例(Iyer等,估计来自仅从病毒逆转录酶而不是PCR Taq错误发生的点突变的进化速率(Novitsky等人,2013),并评估体内重组率而不包括PCR介导的重组(Brown等人, 2011; Sanborn等,2015)。
我们使用SGS从单个起始模板生成链接的gp120包膜和nef基因序列,以评估从获自HIV感染患者的cART中从无可检测病毒死亡的一组新鲜冷冻组织提取的RNA和DNA的病毒表达,区室化和进化死亡时血浆或脑脊髓液中的负荷(Lamers等,2016a和2016b; Rose等,2016)。我们的数据表明,在这些患者的一些组织中存在特殊的环境,其中HIV的表达继续存在;然而,在其他组织中,仅鉴定未表达的前病毒DNA拷贝。推测的基于组织的HIV序列的进化速率与以前报道的cART阴性受试者中复制病毒的比率没有显着差异,这表明持续进化。

关键字:HIV-1, 单基因组测序, SGS, 基因扩增, 巢式PCR

材料和试剂

  1. RNA和DNA提取
    1. 移液器提示
    2. TissueRuptor一次性探针(QIAGEN,目录号:990890)
    3. 新鲜冷冻组织切片(30-50ng)
    4. 消毒剂消毒剂(Fisher Scientific,目录号:04-355-32)
    5. AllPrep DNA/RNA Mini Kit(QIAGEN,目录号:80204)
    6. RNeasy MinElute清理套件(QIAGEN,目录号:74204)
    7. Qubit 2.0荧光计(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32857)
    8. 纯乙醇(200分子生物学级)(Sigma-Aldrich,目录号:E7023)
    9. Qubit ® dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32854)
    10. Qubit ® RNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32852)

  2. cDNA合成
    1. 0.2 ml PCR 8管FLEX-FREE条,附有透明平盖,天然(USA Scientific,目录号:1402-4700)
    2. SuperScript III第一链合成系统(Thermo Fisher Scientific,Invitrogen TM,目录号:18080051)。 SuperScript ® III第一级合成系统提供以下功能:
      1. Oligo(dT)<20>(50μM),50μl
      2. 随机六聚体(50ng /μl),250μl
      3. 10x RT缓冲液,1 ml
      4. 0.1 M DTT,250μl
      5. 25mM氯化镁(MgCl 2/2),500μl
      6. 10mM dNTP混合物,250μl
      7. SuperScript ® III RT(200 U /μl),50μl
      8. RNase-OUT TM(40U /μl),100μl
      9. 电子。大肠杆菌核糖核酸酶H(2U /μl),50μl
      10. DEPC处理水,1.2 ml
      11. 总HeLa RNA(10 ng /μl),20μl
      12. 感应控制引物(10μM),25μl
      13. 反义对照引物(10μM),25μl

  3. gp120 和
    的单基因组测序
    1. 24个PCR孔
    2. 移液器提示
    3. TempPlate半裙式聚丙烯0.2 ml 96孔PCR板(USA Scientific,目录号:1402-9220)
    4. Posi-Click 1.7ml微量离心管,1.7 ml自然色(Denville Scientific,目录号:C2170)
    5. 分子生物学级无菌纯化水(RNase,DNase,不含蛋白酶)
    6. EB缓冲液(QIAGEN,目录号:19086)
    7. 蓝色PCR SuperMix(Thermo Fisher Scientific,Invitrogen TM,目录号:12580023)
    8. 琼脂糖(Fisher Scientific,目录号:BP160-500)
    9. 溴化乙锭(Fisher Scientific,目录号:BP102-1)
    10. Tris-base(Sigma-Aldrich,目录号:T1378)
    11. 乙酸,冰川(Fisher Scientific,目录号:A38-212)
    12. 乙二胺四乙酸,EDTA,0.5M溶液/pH 8.0(Fisher Scientific,目录号:BP2482-500)
    13. Milli-Q质量水(RNase,DNase free water [dH <2> O])
    14. 底漆列于表1

      表1.引物


    15. 50x TAE储备溶液(见配方)
    16. 1x TAE缓冲液(参见食谱)

设备

  1. TissueRupter转子 - 定子匀浆器(QIAGEN,型号:TissueRupper,目录号:9001271)
  2. Matrix多通道电子移液器(范围:2-125μl; 12通道)(Fisher Scientific,目录号:14-387-117)*
  3. Matrix多通道电子移液器(范围:1-30μl; 12通道)(Thermo Fisher Scientific,目录号:14-387-137)*
  4. Matrix多通道电子移液器(范围:2-125μl; 12通道)(Thermo Fisher Scientific,目录号:14-387-138)*
  5. Eppendorf Repeater TM 流式电子移液器(Eppendorf,目录号:4987000118)
  6. Eppendorf Research TM Plus可调量移液管:0.1-2.5μl,2-20μl,20-200μl,100-1,000μl(Eppendorf,目录号:022575442)
  7. 胶带(QIAGEN,目录号:19570)
  8. Sub-Cell TM 192型电泳系统(Bio-Rad Laboratories,型号:192型,目录号:1704507)
  9. 51-Well comb(Bio-Rad Laboratories,目录号:1704529)
  10. 梳子架(Bio-Rad Laboratories,目录号:1704525)
  11. UV透明凝胶托盘(Bio-Rad Laboratories,目录号:1704524)
  12. 192型凝胶脚轮(Bio-Rad Laboratories,型号:型号192,目录号:1704517)
  13. 离心机5424,非冷藏,带转子FA-45-24-11,键盘,230 V/50-60 Hz(Eppendorf,型号:5424,目录号:5424000010)
  14. Isotemp TM数字干浴培养箱(Fisher Scientific,目录号:11-718-2Q)*
  15. T100 TM热循环仪(Bio-Rad Laboratories,型号:T100 TM,目录号:1861096)
  16. DNA寡核苷酸得自Invitrogen
  17. Applied Biosystems 3730xl DNA分析仪(Thermo Fisher Scientific,Applied Biosystems ,型号:3730 DNA分析仪,目录号:3730XL)

注意:这些产品已经停产。

软件

  1. Geneious R7软件包(Biomatters http://www.geneious.com
  2. MEGA5

程序

  1. RNA和DNA提取
    1. 使用ELIMINase去污剂前后彻底清洁工作台面和设备。
      注意:应使用过滤的吸管头进行RNA和DNA提取,cDNA合成和第一轮PCR设置,并且必须在具有单独的空气处理和实验室设备的无限制扩增子的空间中进行,其中没有扩增允许PCR产物或重组克隆质粒。如果没有这样的房间可用,在装有单独的空气处理机构的细胞生物级洁净罩中进行放大前的步骤。
    2. 使用AllPrep DNA/RNA Mini Kit按照制造商的指南,从每个组织切片(30-50ng)分别并同时分离总RNA和基因组DNA。在RNA分离的最后一步中进行使用无RNA酶的水的两个最终的50μl洗脱液,总计最终体积为100μl。
    3. 在使用TissueRupter转子 - 定子匀浆器提取之前,将组织均匀化,每个样品都装有新鲜的无菌一次性探头。
    4. 根据制造商的说明书,使用RNeasy MinElute Cleanup Kit将100μl终体积的RNA浓缩。使用20μl无RNase的水的单次最终洗脱。
    5. 使用Qubit 2.0荧光计和Qubit RNA HS测定试剂盒或Qubit dsDNA HS测定试剂盒在适当的情况下,进行所得RNA和DNA的定量以确定提取方案和浓度的成功。未检测到DNA或RNA,或对于RNA或DNA,产量低于1 ng /μl,表明提取失败,应重复提取,直到检测到超过1 ng /μl的RNA和DNA。 >
  2. cDNA合成
    1. 根据制造商的建议,使用所提供的寡核苷酸(dT)二级引物,使用SuperScript III型第一链合成系统立即从每个样品的RNA产生cDNA。 ,详细如下,以增加产品长度。
    2. 在每个样品的两个相同的反应中,将8μlRNA与脱氧核苷三磷酸(0.5mM [各])和5μM寡聚(dT)<20>在65℃孵育5分钟,然后迅速冷却至4°C。
      注意:使用热循环仪获得准确的温度和保持时间。 cDNA合成反应在0.2ml PCR 8管FLEX-FREE条中进行。
    3. 第一链cDNA合成将在含有1x逆转录缓冲液(10mM Tris-HCl [pH 8.4],25mM KCl),5mM MgCl 2,10mM二氨基苏糖醇的20μl反应体积中继续, 2U /μlRNase-OUT TM(RNase抑制剂)和10U /μlSuperScript III RT。将反应物加热至45℃90分钟,然后加热85℃5分钟。
    4. 然后将反应冷却至37℃和0.1U /μl的E。大肠杆菌加入核糖核酸酶H,然后孵育20分钟。
    5. 将每个样品的两个反应与温和的移液管混合结合,以避免剪切cDNA。将cDNA存储在-20℃直至需要。


  3. gp120 和
    的单基因组测序
    1. 通常进行使用EB缓冲液的cDNA和基因组DNA(gDNA)稀释液以达到30%以下的阳性嵌套PCR反应,这表明阳性反应将具有一个起始模板的大于80%的几率。
      注意:
      1. 对于cART患者,首先从1:3和1:9稀释的cDNA和gDNA开始,每个稀释用24个PCR孔。对于不在cART上的患者,可以使用更高的稀释度。
      2. 解冻和混合后,储存和稀释液必须保存在冰上,并在不使用时在-20°C下冷冻。移液器混合或轻弹混合样品和稀释液,不要涡旋混合。
      3. 连续稀释和第一轮PCR设置必须在无扩增子的房间内完成,并始终使用过滤的吸管头。

      4. 需要两轮PCR来产生足够的产品,用于可视化,量化和排序。
    2. 在第一轮PCR期间,将1μl稀释的cDNA或基因组DNA在含有1×Platinum Blue PCR SuperMix的20μl反应物和0.05μM的每种引物:BEF1,5'-TAATAGCAATAGTTGTGTGG-3'和BNR1,5'-AGCTCCCAGGCTCAGATCT-3'(分别为6,111-6,130和9,558-9,576bp的HIV-1 HXB2)。
      1. 第一轮引物在反应体积中为0.05μM浓度,以消除未用过量的第一轮引物携带到第二轮PCR中。第二轮PCR中过量的第一轮引物产生非特异性PCR产物并减少所需产物的量。参见图1,了解通过第一轮PCR引物携带产生的非特异性PCR产物的实例

        图1.非特异性引物与基因组DNA结合的实施例。该凝胶提供了第一轮PCR引物的浓度在第一轮PCR中为0.2μM的实验实例。该浓度的第一轮引物在第二轮PCR中产生非特异性产物形成,如大多数孔中发现的较弱条带所见,无论这些孔是否具有对应于阳性对照的大小的亮带。当第一轮PCR引物以0.05μM使用时,这些次级产物不再可见,而正确大小的阳性PCR产物仍然可见,导致更容易解释结果并直接测序第二轮产品而无需进一步的凝胶纯化隔离单一乐队。 

    3. 第一轮PCR循环参数 - 初始变性94℃3分钟,然后40个循环94℃30秒,56℃30秒,72℃4分钟,最后延伸72℃ 10分钟。


      注意:
      1. 使用TempPlate半裙状聚丙烯0.2ml 96孔PCR板和带垫以96孔格式进行PCR。制备含有预混合SuperMix和引物的大批PCR板,并冷冻以备将来使用以减少实验间变异性。
      2. 应积极选择正确的PCR对照,并将其稀释至足以在嵌套PCR完成后仅产生一个条带,因此在第一轮PCR后,条带将不可见。非常浓缩的阳性对照可以很容易地污染PCR板并稀释到可行的水平。
      3. 使用自动移液器可以减少错误和交叉污染的可能性。我们使用Matrix多通道电子移液器(范围:1-30μl; 12通道)和Eppendorf Repeater流式电子移液器。
      4. 第一轮PCR中引物的量显着减少,以减少第二轮PCR期间的非特异性结合和引物携带。扩展时间和周期数增加,以产生更多的完整产品。
    4. 第二轮gp120 PCR由2μl第一轮PCR产物加入到由1x Platinum Blue PCR SuperMix组成的20μl第二回合反应和0.2μM每种引物中:BEF2,5'-CAATAGTTGTGTGGTCCATAG-3'和BER2,5'-CAACAGATGCTGTTGCGC-3'(分别为6,117-6,137bp和7,905-7,922bp的HIV-1 HXB2)
    5. 第二轮gp120 PCR循环参数 - 初始变性94℃3分钟,然后40个循环94℃30秒,56℃30秒,72℃3分钟,随后最终延长72°C 10分钟。


    6. 第二轮gp120 PCR产物在1%琼脂糖凝胶上显色,所述琼脂糖凝胶在1×TAE缓冲液中以150V运行30分钟。
      注意:
      1. 这种第二轮PCR在阳性时产生1.8Kb的产物,其含有完整的gp120序列。来自阳性井的产物用BEF2和BER2引物进行测序。该方案产生可直接测序的单特异性PCR产物,不需要PCR纯化。参见图2,其中有两个不同的患者样本的成功的gp120第二轮PCR和来自另一个组织的DNA的稀释系列。
      2. 我们使用白蛋白蓝色PCR SuperMix将负载第二轮产品引导到琼脂糖凝胶上,而不是在每个反应中混合加载染料。我们使用Matrix多通道电子移液器(范围:2-125μl; 12通道)在Sub-CellTM 192型电泳系统上自动加载。


      图2.第二轮gp120的实例PCR板琼脂糖凝胶图像。凝胶顶行使用的两个样品是两个患者的脾组织的未稀释的基因组DNA。两个样品具有等于小于30%的正极孔的总数,表明阳性孔最有可能来自存在于DNA中的单个整合的原始病毒基因组的巢式PCR扩增的gp120 那好底排(HC09SPd1 DIL_1)提供了连续稀释测试的例子,以评估正确的SGS稀释度。在这里测试四种稀释液,并且当所有四种稀释度都足够高以在正性阱中产生单个整合的原病毒的扩增时,所有四种稀释度都太高,以获得许多阳性反应,导致浪费的试剂。理想情况是找到稀释液,其中20-30%的孔是阳性的,因此必须测试较低的稀释度以找到最佳稀释度。凝胶上没有标记阴性对照,A1为良好,阳性对照(标记为POS)为H12。阴性对照具有1μl用于稀释DNA的水,阳性对照是来自不在以前实验中为阳性的cART的患者的稀释的基因组DNA。 

    7. 随后,使用对应于阳性第二轮gp120 PCR的第一轮反应来扩增基因序列; PCR第二轮PCR由2μl第一轮PCR加入到由1×Platinum Blue PCR SuperMix组成的20μl第二轮反应中,每种引物0.2μM: BNF1,5'-CTGGCTGTGGAAAGATACCT-3'和BNR2,5'-ATCTGAGGGCTCGCCACT-3'(分别为7,965-7,984bp和9,488-9,505 HIV-1 HXB2)。
    8. 循环参数 - 初始变性94℃3分钟,接着是40个循环,94℃30秒,58℃30秒,72℃2分钟,然后是最终在72℃延伸10分钟

    9. 第二轮在1×TAE缓冲液中,PCR产物在1%琼脂糖凝胶上显色,用溴化乙锭在150V下运行30分钟。
      注意:含有单个1.5 Kb产物的反应被认为是阳性的,并选择用于BNF1和BNR2的测序。该方案产生可直接测序的单特异性PCR产物,不需要PCR纯化。

数据分析

在佛罗里达大学生物技术研究中心(UF ICBR)的Applied Biosystems 3730xl DNA分析仪上进行测序,使用BEF2/BER2引物用于gp120和BNF1/BNR2引物用于nef 的测序。使用Geneious R7软件包(Biomatters http:/...)组装每个测序PCR产物的正向和反向色谱图, /www.geneious.com ),并在可能的情况下用手进行优化,以解决由序列器分配的模糊排序调用或装配算法的虚假间隔。没有使用具有默认设置的Geneious自动装配算法组装的正向和反向色谱图被丢弃,通常这些是质量非常差的序列(由定序器精确调色的太多重叠峰)或非常短的长度(小于600碱基对)。对于在两个或更多个碱基对中发现多个色谱峰的组合,在起始模板的初始扩增中指示多个起始模板或多个PCR错误,从进一步分析中除去这些序列。使用Geneious软件包从每个优化组合中提取共有序列。使用MEGA5(Tamura等人,2011)中的ClustalW(Thompson等人,1997),利用手工进一步优化来消除共识序列,以消除由对齐算法。分别位于相对于HXB2基因组的位置6,213-7,823和位置8,797-9,411的最终 env 和 nef 对齐。由于大量的天然存在的插入和缺失,通常有问题地排列并可能偏向系统发育分析,排除了env (V1,V2和V4域)中的高变区。使用PhyML估计每个基因的初步最大似然系统发育( http://www.atgc-montpellier.fr/phyml/)和来自所有参与者的序列,以确保不发生患者的交叉污染。使用HYPERMUTE工具测试序列是否存在超突变( http://www.lanl .GOV );具有< P<> - 值的序列<从对准中除去0.01。使用该协议生成的实例序列已提交给GenBank(登录号KU708874-KU709831)。

笔记

在考虑SGS实验的结果时,请记住几个想法:

  1. 基于病毒基因组的变化,引物结合效率可能因患者,亚型或病毒基因而异。筛选每组具有针对患者亚型特异性的多组引物的每个患者,并发现一致的结果将增加测序结果的置信度。发现在本文中提出的用于gp120-nef 的SGS方案的阴性组织也应该使用在HIV基因组的更保守区域中的引物进行评估,如gag (Norstr? et al。,2012)或 pol (Palmer等人,1999; Shafer等人,2000)以确认没有病毒。使用诸如QUALITY(Rodrigo等人,1997)的程序根据SGS稀释(Rife等人,2016)估算不同引物组的SGS结果的拷贝数也可以提供每组的绑定效率的数据。此外,替代的gp120/nef引物(Lamers等人,2016b)可以用于确认由于引物的引物结合效率而导致的一些变体不被遗漏这里。从这些替代引物产生的序列可以包括在系统发育分析中,以评估原始引物捕获组织中病毒多样性景观的效率。实时或定量PCR也可用于评估SGS结果的正或负值(Lamers等人,2016a)。
  2. 组织型可影响DNA和RNA的分离。 Qiagen Allprep试剂盒有一些关于改变不同组织类型提高隔离效率的方法的详细说明。对于始终导致低产量的组织,应考虑替代试剂盒或方案。组织保存也会影响分离结果,并且应该谨慎处理组织以防止过早解冻。在多个组织切片上进行的分离分离将增加对SGS结果的信心。来自同一患者的组织应尽可能分开处理,因为来自同一患者的样品的交叉污染物将不会像所有生成的所有序列的初始系统发育分析期间容易地识别为患者之间的混合。

食谱

  1. 50x TAE库存解决方案
    准备1L 50x TAE溶解以下组分:
    在600毫升去离子水中:
    242g Tris碱(FW = 121)
    57.1毫升冰醋酸
    100ml 0.5M EDTA(pH8.0)
  2. 1x TAE缓冲区
    40mM Tris(pH 7.6)
    20mM乙酸
    1 mM EDTA
    稀释1:50用于1×TAE缓冲液进行凝胶电泳

致谢

这项HIV-1测序方案的开发资金由美国国家卫生研究院(NIH)通过R01 MH100984资助提供,并基于以前的NIH授权R01 NS063897开发的方法。我们要感谢国家神经系统艾滋病银行(NNAB,UM1 CA181255)和艾滋病和癌症标本资源(ACSR,UM1 CA181255)提供用于开发协议的组织,并生成出版数据,特别是Elyse Singer(NNAB) ,Debra Garcia(ACSR)和Salman Mahboob(ACSR)。我们还要感谢并感谢MelissaNorström(以前的Karolinska Institutet),最初向我们介绍了用于"gag"基因的HIV-1单基因组测序技术。该方法根据先前出版物的协议(Palmer等人,2005;Norström等人,2012; Lamers等人,2015年) ; Rife等人,2016)。

参考

  1. Abbas,W.,Tariq,M.,Iqbal,M.,Kumar,A.and Herbein,G。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/25835530"target ="_ blank">从巨噬细胞储库中去除HIV-1:一个不确定的目标?病毒 7(4):1578-1598 。
  2. Anderson,JA,Archin,NM,Ince,W.,Parker,D.,Wiegand,A.,Coffin,JM,Kuruc,J.,Eron,J.,Swanstrom,R。和Margolis,DM(2011) ; 从残留HIV-1病毒血症患者血浆中回收的克隆序列强化的抗逆转录病毒治疗与从循环静息的CD4 + T细胞中回收的复制性病毒RNA相同。 85(10):5220-5223。 br />
  3. Autran,B.,Carcelain,G.,Li,TS,Blanc,C.,Mathez,D.,Tubiana,R.,Katlama,C.,Debre,P。和Leibowitch,J。(1997) a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/9204894"target ="_ blank">联合抗逆转录病毒治疗对CD4 + T细胞体内平衡和功能在晚期艾滋病病毒。 科学 277(5322):112-116。
  4. Bednar,MM,Hauser,BM,Zhou,S.,Jacobson,JM,Eron,JJ,Jr.,Frank,I.and Swanstrom,R。(2016)。  治疗停止后血浆中HIV-1反弹病毒群体的多样性和趋向性。感染Dis 214(3):403-407。
  5. Boritz,EA,Darko,S.,Swaszek,L.,Wolf,G.,Wells,D.,Wu,X.,Henry,AR,Laboune,F.,Hu,J.,Ambrozak,D.,Hughes, MS,Hoh,R.,Casazza,JP,Vostal,A.,Bunis,D.,Nganou-Makamdop,K.,Lee,JS,Migueles,SA,Koup,RA,Connors,M.,Moir, Schacker,T.,Maldarelli,F.,Hughes,SH,Deeks,SG和Douek,DC(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih .gov/pubmed/27453467"target ="_ blank">在艾滋病毒感染自然控制期间病毒持续存在的多重起因。 166(4):1004-1015。
  6. Brown,RJ,Peters,PJ,Caron,C.,Gonzalez-Perez,MP,Stones,L.,Ankghuambom,C.,Pondei,K.,McClure,CP,Alemnji,G.,Taylor,S.,Sharp, PM,Clapham,PR和Ball,JK(2011)。  HIV-1的间室间重组有助于内源性多样性,并调节病毒向性和对入侵抑制剂的敏感性。 85(12):6024-6037 。
  7. Buzon,MJ,Sun,H.,Li,C.,Shaw,A.,Seiss,K.,Ouyang,Z.,Martin-Gayo,E.,Leng,J.,Henrich,TJ,Li,JZ,Pereyra ,F.,Zurakowski,R.,Walker,BD,Rosenberg,ES,Yu,XG and Lichterfeld,M。(2014)。< a class ="ke-insertfile"href ="http://www.ncbi .nlm.nih.gov/pubmed/24412925"target ="_ blank">具有干细胞样特性的CD4 + T细胞中的HIV-1持久性。/20(2):139-142。
  8. Chun,TW,Carruth,L.,Finzi,D.,Shen,X.,DiGiuseppe,JA,Taylor,H.,Hermankova,M.,Chadwick,K.,Margolick,J.,Quinn,TC,Kuo,YH ,Brookmeyer,R.,Zeiger,MA,Barditch-Crovo,P。和Siliciano,RF(1997)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 HIV-1感染中潜伏性组织储库的定量和总体病毒载量的定量。自然 387(6629):183-188。 br />
  9. Chun,TW,Finzi,D.,Margolick,J.,Chadwick,K.,Schwartz,D。和Siliciano,RF(1995)。< a class ="ke-insertfile"href ="https: .ncbi.nlm.nih.gov/pubmed/7489410"target ="_ blank"> HIV-1感染的T细胞的体内命运:向稳定潜伏期的转变的定量分析。 > Nat Med 1(12):1284-1290。
  10. Crooks,AM,Bateson,R.,Cope,AB,Dahl,NP,Griggs,MK,Kuruc,JD,Gay,CL,Eron,JJ,Margolis,DM,Bosch,RJ和Archin,NM(2015) 潜伏HIV-1水库的精确定量:对根除策略的影响。 J Infect Dis 212(9):1361-1365。
  11. Eriksson,S.,Graf,EH,Dahl,V.,Strain,MC,Yukl,SA,Lysenko,ES,Bosch,RJ,Lai,J.,Chioma,S.,Emad,F.,Abdel-Mohsen,M 。,Hoh,R.,Hecht,F.,Hunt,P.,Somsouk,M.,Wong,J.,Johnston,R.,Siliciano,RF,Richman,DD,O'Doherty,U.,Palmer,S 。,Deeks,SG和Siliciano,JD(2013)。  HIV-1根除研究中病毒储库测量的比较分析。 PLoS Pathog 9(2):e1003174。
  12. Etemad,B.,Ghulam-Smith,M.,Gonzalez,O.,White,LF和Sagar,M。(2015)。  单基因组扩增和标准体积PCR产生具有相似基因型和表型特征的HIV-1信封产品。 ViV方法 214:46-53。
  13. Ever,TH,Kamau,E.,St Bernard,L.,Farmer,CB,Kong,XP和Markowitz,M。(2014)。潜在的水库中复制的非诱导性原始病毒增加了HIV-1治愈的障碍。 Cell 155(3):540-551。
  14. 约旦,MR,Kearney,M.,Palmer,S.,Shao,W.,Maldarelli,F.,Coakley,EP,Chappey,C.,Wanke,C.and Coffin,JM(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20451557"target ="_ blank">用于分析HIV-1群体的标准PCR /克隆与单基因组测序的比较 J Virol Methods 168(1-2):114-120。
  15. Josefsson,L.,von Stockenstrom,S.,Faria,NR,Sinclair,E.,Bacchetti,P.,Killian,M.,Epling,L.,Tan,A.,Ho,T.,Lemey, Shao,W.,Hunt,PW,Somsouk,M.,Wylie,W.,Douek,DC,Loeb,L.,Custer,J.,Hoh,R.,Poole,L.,Deeks,SG,Hecht,F 。和Palmer,S。(2013)。艾滋病毒在8名长期抑制性抗逆转录病毒治疗患者中,-1水平稳定,随着时间的推移几乎没有遗传变化。"美国国家科学院院刊"110(51):E4987-4996。 >
  16. Kandathil,AJ,Sugawara,S.and Balagopal,A。(2016)。 T细胞是否是唯一的HIV-1水库?逆行病学13 /(13):86.
  17. Kearney,MF,Spindler,J.,Shao,W.,Yu,S.,Anderson,EM,O'Shea,A.,Rehm,C.,Poethke,C.,Kovacs,N.,Mellors,JW,Coffin ,JM和Maldarelli,F.(2014)。缺乏在抑制性抗逆转录病毒治疗期间可检测的HIV-1分子进化。 PLoS Pathog 10(3):e1004010。
  18. Kearney,MF,Wiegand,A.,Shao,W.,Coffin,JM,Mellors,JW,Lederman,M.,Gandhi,RT,Keele,BF和Li,JZ(2015)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26581989"target ="_ blank">反弹血浆HIV的起源包括在停止抗逆转录病毒治疗之前具有转录活性的相同前驱病毒的细胞。 J Virol 90(3):1369-1376。
  19. Lamers,SL,Nolan,DJ,Rife,BD,Fogel,GB,McGrath,MS,Burdo,TH,Autissier,P.,Williams,KC,Goodenow,MM和Salemi,M。(2015) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26041280"target ="_ blank">跟踪主机特定猿猴免疫缺陷病毒的出现env 和 nef 群体显示自然进行的恒河猴颅脑早期适应和融合进化。 J Virol 89(16) :8484-8496。
  20. Lamers,S.L,Rose,R.,Maidji,E.,Agsalda-Garcia,M.,Nolan,DJ,Fogel,GB,Salemi,M.,Garcia,DL,Bracci,P.,Yong, Commins,D.,Said,J.,Khanlou,N.,Hinkin,CH,Sueiras,MV,Mathisen,G.,Donovan,S.,Shiramizu,B.,Stoddart,CA,McGrath,MS and Singer,EJ(   HIV DNA经常存在于联合抗逆转录病毒治疗 - 治疗不能检测到病毒载量的患者。 90(20):8968-83。
  21. Lamers,SL,Rose,R.,Nolan,DJ,Fogel,GB,Barbier,AE,Salemi,M.and McGrath,MS(2016b)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/27651732"target ="_ blank"> HIV-1与艾滋病期间转移性Kaposi氏肉瘤相关的进化模式。肉瘤 2016:4510483。
  22. Iyer,S.,Casey,E.,Bouzek,H.,Kim,M.,Deng,W.,Larsen,BB,Zhao,H.,Bumgarner,RE,Rolland,M。和Mullins,JI(2015)。   通过单一模板测序鉴定的主要和次要病毒SNP的比较和焦虑测序在急性HIV-1感染中。 10(8):e0135903。
  23. NorströmMM,Buggert,M.,Tauriainen,J.,Hartogensis,W.,Prosperi,MC,Wallet,MA,Hecht,FM,Salemi,M.and Karlsson,AC(2012)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22761389"target ="_ blank">免疫和病毒因子的组合区分了低风险与高风险的HIV-1疾病进展HLA-B * 5701名受试者。 J Virol 86(18):9802-9816。
  24. Novitsky,V.,Wang,R.,Rossenkhan,R.,Moyo,S.and Essex,M。(2013)。初次感染期间HIV-1C 和 gag 内的宿主进化率。感染Genet Evol 19:361-368。
  25. Palmer,S.,Kearney,M.,Maldarelli,F.,Halvas,EK,Bixby,CJ,Bazmi,H.,Rock,D.,Falloon,J.,Davey,RT,Jr.,Dewar,RL,Metcalf ,JA,Hammer,S.,Mellors,JW and Coffin,JM(2005)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15635002 "target ="_ blank">通过标准基因型分析错过了治疗有经验的患者的多重连接的人类免疫缺陷病毒1型耐药性突变。J Clin Microbiol。43(1):406 -413。
  26. Palmer,S.,Shafer,RW和Merigan,TC(1999)。  目前临床发展中,高耐药性HIV-1临床分离物与许多抗逆转录病毒化合物具有交叉耐药性。 13(6):661-667。 >
  27. Rife,BD,Nolan,DJ,Lamers,SL,Autissier,P.,Burdo,T.,Williams,KC和Salemi,M。(2016)。  外周神经适应的进化和脑中的纯化选择有助于猕猴脑中的猿猴免疫缺陷病毒(SIV)的分化与SIV相关的脑炎。 J Virol 90(13):6112-6126。
  28. Rodrigo,AG,Goracke,PC,Rowhanian,K.and Mullins,JI(1997)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/9171217"target ="_ blank">从基于聚合酶链反应的有限稀释测定法定量靶分子。艾滋病抗逆转录病毒13(9):737-742。
  29. Rose,R.,Lamers,SL,Nolan,DJ,Maidji,E.,Faria,NR,Pybus,OG,Dollar,JJ,Maruniak,SA,McAvoy,AC,Salemi,M.,Stoddart,CA,Singer,EJ和McGrath,MS(2016)。艾滋病毒维持不断发展并且在患有癌症的个体的抑制性联合抗逆转录病毒治疗期间将人群分散在多个组织中。 90(20):8984-8993。
  30. Rothenberger,MK,Keele,BF,Wietgrefe,SW,Fletcher,CV,Beilman,GJ,Chipman,JG,Khoruts,A.,Estes,JD,Anderson,J.,Callisto,SP,Schmidt,TE,Thorkelson, ,Reilly,C.,Perkey,K.,Reimann,TG,Utay,NS,Nganou Makamdop,K.,Stevenson,M.,Douek,DC,Haase,AT和Schacker,TW(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25713386"target ="_ blank">大量的反弹/创始人艾滋病病毒变体在治疗后淋巴组织多灶感染中出现中断。 Proc Natl Acad Sci USA 112(10):E1126-1134。
  31. Sacha,JB和Ndhlovu,LC(2016)。策略靶向非T细胞HIV水库。 Curr Opin HIV AIDS 11(4):376-382。
  32. Salemi,M。和Rife,B.(2016)。 HIV/SIV宿主间隔室和水库的系统发育和系统解剖学:中枢神经系统的关键作用。 14(2):110-20。
  33. Sanborn,KB,Somasundaran,M.,Luzuriaga,K.和Leitner,T。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26573574"target ="_ blank">重组提高了有效的进化速率,并促进了母婴传播后婴儿艾滋病毒1感染的建立。逆行病学 12:96 。
  34. Shafer,RW,Warford,A.,Winters,MA and Gonzales,MJ(2000)。  来自重度治疗的HIV-1感染个体的血浆样品的人类免疫缺陷病毒1型(HIV-1)蛋白酶和逆转录酶测序的重现性。 86(2):143-153。
  35. Simonetti,FR,Sobolewski,MD,Fyne,E.,Shao,W.,Spindler,J.,Hattori,J.,Anderson,EM,Watters,SA,Hill,S.,Wu,X.,Wells, ,Su,L.,Luke,BT,Halvas,EK,Besson,G.,Penrose,KJ,Yang,Z.,Kwan,RW,Van Waes,C.,Uldrick,T.,Citrin,DE,Kovacs,J ,Polis,MA,Rehm,CA,Gorelick,R.,Piatak,M.,Keele,BF,Kearney,MF,Coffin,JM,Hughes,SH,Mellors,JW和Maldarelli,F。(2016) 克隆扩增的CD4 + T细胞可以在体内产生感染性HIV-1 。 Proc Natl Acad Sci USA 113(7):1883-1888。
  36. Sturdevant,CB,Dow,A.,Jabara,CB,Joseph,SB,Schnell,G.,Takamune,N.,Mallewa,M.,Heyderman,RS,Van Rie,A.and Swanstrom,R。(2012)。   早期HIV-1亚型C变体的中枢神经系统划分和幼年感染迟发。 PLoS Pathog 8(12):e1003094。
  37. Svicher,V.,Ceccherini-Silberstein,F.,Antinori,A.,Aquaro,S。和Perno,CF(2014)。< a class ="ke-insertfile"href ="http://www.ncbi .nlm.nih.gov/pubmed/24729094"target ="_ blank">了解艾滋病毒病毒和水库艾滋病毒/艾滋病感染者报告11(2):186-194。 />
  38. Tamura,K.,Peterson,D.,Peterson,N.,Stecher,G.,Nei,M.and Kumar,S。(2011)。 MEGA5:使用最大似然,进化距离和最大简约方法进行分子进化遗传学分析。 Mol Biol Evol 28(10):2731-2739。
  39. Thompson,JD,Gibson,TJ,Plewniak,F.,Jeanmougin,F.和Higgins,DG(1997)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih .gov/pubmed/9396791"target ="_ blank"> CLUSTAL_X Windows界面:通过质量分析工具辅助的多序列比对的灵活策略。 Nucleic Acids Res 25(24):4876 -4882。
  40. Wagner,TA,McKernan,JL,Tobin,NH,Tapia,KA,Mullins,JI and Frenkel,LM(2013)。  在抗逆转录病毒治疗期间越来越多的单型HIV-1 DNA序列表明艾滋病毒感染细胞的增殖。 (3):1770-1778。
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引用:Nolan, D. J., Lamers, S. L., Rose, R., Dollar, J. J., Salemi, M. and McGrath, M. S. (2017). Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes. Bio-protocol 7(12): e2334. DOI: 10.21769/BioProtoc.2334.
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