These studies were funded by US Public Health Services grants P01AI057005, R01AI047734, R01AI111806 and the Functional Profiling and Computational Biology Core of the University of Washington&#8217;s Center for AIDS Research (P30 AI027757).

<ol>
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The authors declare that they have no competing financial interests. The opinions expressed herein are those of the authors and should not be construed as official or representing the views of the US Department of Defense or the Department of the Army.&#160;

The protocols presented consisted of two main parts: construction of recombinant HIV-1 molecular clones and growth competition assays. In order to distinguish two viruses in a dually infected cell culture, it is important that the competing molecular clones contain sequence tags, which can be detected by a RT-qPCR primer-probe assay or by Sanger sequencing. This protocol makes use of the VifA and VifB tags, which occupy the same region of HIV-1 NL4-3 vif and encode the same amino acid sequence but differ by six synonymous mutations. These mutations were shown not to affect viral replication fitness20. The PCR-based cloning method used in this protocol provides more flexibility in selecting cloning sites, compared to restriction site based cloning. However, the efficiency of PCR cloning decreases as the insert size increases. The current limit of the insert size is ~5 kb34. For PCR-based cloning and site directed mutagenesis, the use of a high-fidelity DNA polymerase is crucial to reduce the likelihood of extra mutations. Use of the minimal number of PCR cycles needed to yield adequate amounts of products is also recommended. In our experience, we did not detect any extra mutations after the PCR-based cloning and site-directed mutagenesis steps described here. Nevertheless the PCR products should be sequenced to check for any undesired mutations. Ideally, the entire HIV coding region should be resequenced.
At least three sampling time points should be examined within the exponential growth phase9. The viral growth kinetics must first be established using daily sampling to determine the appropriate culture period and sampling time points for the growth competition. One factor that affects viral growth kinetics is the multiplicity of infection (MOI). This protocol uses a total MOI of 0.005 for both monoinfection and growth competition, as it was shown to yield more robust results than lower MOIs9. Nonetheless, lower MOIs can be used to obtain a longer exponential growth phase if necessary, but at the expense of result consistency. This protocol suggests an initial infection ratio of 50:50, assuming that the fitness differences of the viruses are generally unknown beforehand. However, the use of unequal input ratios are appropriate when there is preliminary data suggesting significant differences in viral replication kinetics. In these cases, an infection ratio of 70:30 is recommended to allow for the detection of a large fitness difference where the less fit virus is placed in excess9.
The protocol for determining the TCID50, viral growth kinetics, and for performing the growth competition assays were optimized using HIV-1 subtype B, NL4-3 COTB-p24, and PBMCs from a single donor whose PBMC have demonstrated consistent susceptibility to HIV-1 infection in vitro. The culture period and sampling time points presented in this protocol are likely to be suitable for studying HIV-1 group M viruses in human PBMCs. Using PBMCs from a single source is highly recommended to obtain consistent results as the viral replication can vary in different donor cells35. Although less desirable, PBMCs pooled from multiple donors can be used as a substitution provided that the same pool is used across all experiments. Another alternative is the use of cell lines. The protocol presented here was used successfully with the T-cell line CEMx17423,36. However, it is important that the numbers of cells seeded are re-optimized to achieve consistent cell growth. Viral growth kinetics must also be re-established, as it is likely to vary in different cell lines, to determine the appropriate sampling time points in the growth competition steps.
Two different methods to determine the viral ratio for calculating fitness are included in the protocol. The first uses RT-qPCR to measure viral cDNA copy number at each sampling time point. Viral replication fitness was then calculated from the viral ratio, based on cDNA copy number. Alternatively, the viral ratio can be determined based on the ratio of chromatogram peak height at the VifAB tag sites. The two methods yielded comparable results (Figure 4). The chromatogram peak height method can be applied to other HIV-1 strains without an engineered sequence tag. For RT-qPCR, the use of primers or probes to distinguish viral variants must first be carefully evaluated (see20). Nevertheless, RT-qPCR provides better sensitivity for samples with a small amount of viral RNA, such as those from the first time point within the exponential growth phase. Direct measurement of viral cDNA also allows detection of technical problems that may arise from the RNA extraction and cDNA synthesis. Using molecular clones with sequence tags provides a cost effective solution to the RT-qPCR methods, as only two pairs of primers and probes are needed to study multiple viruses. This strategy also avoids the problem of peak-height variation in sequence chromatograms due to neighboring bases37, as sequencing is done at the same site across all viruses in the study. The PCR products produced for RT-qPCR and Sanger sequencing can also be use with other methods to determine viral ratio such as bulk sequencing of individual clones12,13, HTA4,7,17,19, or oligonucleotide ligation assay (OLA)38.

Viral replication fitness is defined as the capacity of a virus to produce infectious progeny in a given environment1 and is an important contributing factor in determining the prevalence of a virus variant at the population level over time2. As in vivo fitness studies are not feasible with pathogenic human viruses, such as HIV-1, various in vitro and ex vivo replication fitness assays have been developed to study the effects on fitness arising from drug resistance and immune escape mutations, epistasis and the evolution of viral populations3-6. Among different fitness assays, growth competition assays are recognized to yield more sensitive and valid measures of fitness differences, as two or more viral variants compete for the same cell population under precisely the same environmental conditions, as occurs in vivo1,7,8. Before starting growth competition experiments, several variables need to be determined, including the use of different multiplicities of infection (MOI), viral input ratio, and timing of sampling for analysis. We have studied the effects of these parameters on viral growth kinetics and on the outcome of competition experiments, and have identified key factors necessary for robust measurements of HIV-1 fitness in cell culture9.
In addition to assay variables, there are a variety of methods for quantitating viral variants in growth competition experiments. Bulk10,11 or clonal sequencing12,13 has been used to determine the ratio of the competing viruses based on the nucleotide frequencies at the site(s) of interest. Relative fitness is derived from changes in this ratio over time. This method is convenient as DNA sequencing services are widely available. The parallel allele-specific sequencing (PASS) method enables sequencing at multiple sites and the detection of recombinants14, but it also requires specifically developed reagents and detection systems. Essentially, these methods were developed to study viral strains with a small number of nucleotide differences in a region of interest. Other methods use a small reporter gene15 or synonymous mutations4,16-18 as tags to distinguish the competing viruses by sequencing, heteroduplex tracking assays (HTA)4,7,17,19 or reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)15,16,18,20, all of which can be made applicable to study competing strains regardless of sequence similarity. An additional step is required to introduce tags into viral genomes and the RT-qPCR assay also requires specific reagents and instrumentation. We have found that bulk Sanger sequencing yields comparable results9.
Following growth competition, viral replication fitness is presented as relative fitness, or a fitness ratio between two viral variants. The relative fitness of a virus can be defined as the final proportion of a viral variant normalized by its initial proportion in the inoculum or as the net growth rate difference between the two competing viruses. We found that the latter method, using longitudinal data points only within the exponential growth phase, produced the most robust results9,20.
In vitro fitness assays are used primarily to study biological clones6-8 and infectious molecular clones of HIV-1. The latter, being amenable to genetic manipulation, are often employed to study the effect on fitness from particular mutations or specific sequences of interest3-5,21,22. The following protocols describe a workflow from the point of constructing new full-length infectious HIV-1 molecular clones using HIV-1 vectors containing a sequence tag, introducing mutations of interest, making viral stocks and establishing viral growth kinetics, to performing the growth competition assay and calculating relative fitness (Figure 1).
Using our optimized procedures, we created three recombinant HIV-1 mutants and determined their replication fitness. The recombinant molecular clone was first constructed by replacing the HIV-1 gag-p24 gene region of pNL4-3, a plasmid containing a full length infectious genome of HIV-1 lab strain NL4-3, with a synthetic COTB (Center-Of-Tree, subtype B) gag-p24 sequence23 to create the prototype strain. Single amino changes (T186M, T242N, and I256V) were then introduced to create three mutant clones. Each mutant was competed against the prototype virus to observe the fitness impact of each mutation in the given genetic background. The three mutants demonstrated varying levels of replication fsitness from slight to significantly lower than the prototype virus. The T242N mutation was previously reported to have a moderate fitness cost24-26, similar to the result shown in this study. The fitness cost of the other two mutations had not been reported previously.

<table><tbody><tr><td>Construction of recombinant clones</td><td></td><td></td><td></td></tr><tr><td>Chimeric primer/Mutagenic/Sequencing primers</td><td>IDT</td><td></td><td>Custom DNA oligos</td></tr><tr><td>pNL4-3VifA and pNL4-3VifB plasmid</td><td></td><td></td><td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td></tr><tr><td>High-Fidelity DNA polymerase</td><td>Thermo Scientific</td><td>F-549S</td><td></td></tr><tr><td>High-fidelity buffer</td><td>Thermo Scientific</td><td>F-549S</td><td></td></tr><tr><td>dNTP</td><td>Bioline</td><td>Bio-39026</td><td></td></tr><tr><td>Thermal cycler</td><td>Life Technologies</td><td></td><td>Applied Biosystems&amp;reg; GeneAmp&amp;reg; PCR System 9700</td></tr><tr><td>DNA loading dye</td><td>Thermo Scientific</td><td>R0631</td><td></td></tr><tr><td>1 kb Plus DNA ladder</td><td>Invitrogen</td><td>10787-018</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>QIAquick gel extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>DpnI enzyme</td><td>New England Biolabs</td><td>R0176S</td><td></td></tr><tr><td>TOP10 chemically competent &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Invitrogen</td><td>C4040-10</td><td></td></tr><tr><td>Luria Broth base powder</td><td>Invitrogen</td><td>12795-084</td><td></td></tr><tr><td>Carbenicillin</td><td>Research Products International</td><td>C46000-25.0</td><td></td></tr><tr><td>QIAprep Spin Miniprep kit&amp;nbsp;</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Transfection</td><td></td><td></td><td></td></tr><tr><td>X-tremeGENE 9 DNA transfection reagent</td><td>Roche</td><td>6365787001</td><td></td></tr><tr><td>HEK 293T-17</td><td>ATCC</td><td>CRL-11268</td><td>http://www.atcc.org/</td></tr><tr><td>0.22 &amp;#956;m filter top tube</td><td>VWR International</td><td>89220-716&amp;nbsp;</td><td></td></tr><tr><td>Cell culture</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>10566016</td><td></td></tr><tr><td>Iscove&amp;prime;s Modified Dulbecco&amp;prime;s Medium (IMDM)</td><td>Life Technologies</td><td>31980-030</td><td></td></tr><tr><td>RPMI-1640 media</td><td>Life Technologies</td><td>61870-036</td><td></td></tr><tr><td>Phytohemagglutinin (PHA)</td><td>Thermo Scientific</td><td>R30852801</td><td></td></tr><tr><td>Human Interleukin-2</td><td>Roche</td><td>11147528001</td><td></td></tr><tr><td>Fetal bovine serum</td><td>JR Scientific</td><td>43640</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Corning Cellgro</td><td>30-001-CI</td><td></td></tr><tr><td>6 well plate</td><td>VWR Scientific</td><td>73520-906</td><td></td></tr><tr><td>48 well plate</td><td>VWR Scientific</td><td>62407-338</td><td></td></tr><tr><td>96 well flat-bottomed plate</td><td>ISC Bioexpress</td><td>T-3015-4</td><td></td></tr><tr><td>96 well round-bottomed plate</td><td>BD Falcon</td><td>353077</td><td></td></tr><tr><td>1.5 ml Microcentrifuge Tube</td><td>Mt. Baker Bio</td><td>MBD-1500</td><td></td></tr><tr><td>1.5 ml tubes w/ O-ring</td><td>VWR Scientific</td><td>89004-290</td><td></td></tr><tr><td>50 ml conical tube</td><td>ISC Bioexpress</td><td>C-3317-6</td><td></td></tr><tr><td>ELISA</td><td></td><td></td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Invitrogen</td><td>14190-250</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F0392</td><td></td></tr><tr><td>glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Rabbit anti-HIV-1 SF2 p24 antiserum</td><td>NIH AIDS Reagent Program</td><td>4250</td><td></td></tr><tr><td>Goat anti-rabbit HRP</td><td>KPL</td><td>474-1516</td><td></td></tr><tr><td>TMB Microwell Peroxidase Substrate</td><td>KPL</td><td>52-00-01</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>A300-500</td><td>Causes severe burns by all exposure routes. Use personal protective equipment. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 min.</td></tr><tr><td>HIV p24 standard</td><td>AIDS and Cancer Virus program (NCI-Frederick)</td><td></td><td>For order details please contact Julian Bess, Jr. (bessjw@mail.nih.gov); http://ncifrederick.cancer.gov/Programs/Science/Acvp/bio/Bess.aspx</td></tr><tr><td>Microplate reader</td><td>Molecular Devices</td><td></td><td>VMax Kinetic ELISA Microplate Reader</td></tr><tr><td>RNA isolation cDNA synthesis</td><td></td><td></td><td></td></tr><tr><td>QIAxtractor</td><td>Qiagen</td><td></td><td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td></tr><tr><td>QIAamp Viral RNA Mini Kit</td><td>Qiagen</td><td>52906</td><td></td></tr><tr><td>dNTP</td><td>Bioline</td><td>Bio-39026</td><td></td></tr><tr><td>SuperscriptIII</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>First-strand buffer&amp;nbsp;</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>Rnase inhibitor</td><td>Roche</td><td>3335402001</td><td></td></tr><tr><td>RnaseH</td><td>Invitrogen</td><td>18021-071</td><td></td></tr><tr><td>qPCR</td><td></td><td></td><td></td></tr><tr><td>Real-time qPCR machine</td><td>Life Technologies</td><td></td><td>Applied Biosystems 7300 Real-Time PCR&amp;nbsp;</td></tr><tr><td>TaqMan&amp;reg; Gene Expression Master Mix</td><td>Life Technologies</td><td>4369016</td><td></td></tr><tr><td>Forward, reverse primer</td><td>IDT</td><td></td><td>Custom order</td></tr><tr><td>Probe</td><td>Life Technologies</td><td></td><td>Custom order</td></tr><tr><td>optical 96 well reaction plate</td><td>Life Technologies</td><td>I19N3Q216</td><td></td></tr><tr><td>PCR+sequencing</td><td></td><td></td><td></td></tr><tr><td>Taq DNA polymerase (Biolase)</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>NH4 buffer&amp;nbsp;</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>Sequencing primer</td><td>IDT</td><td></td><td>Custom order</td></tr><tr><td>QIAxcel</td><td>Qiagen</td><td></td><td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td></tr><tr><td>DNA sequencing service provider</td><td>http://www.htseq.org/</td><td></td><td></td></tr><tr><td>GRC web tool</td><td>http://indra.mullins.microbiol.washington.edu/grc/</td><td></td><td></td></tr><tr><td>ChromatQuan web tool</td><td>http://indra.mullins.microbiol.washington.edu/cgi-bin/chromatquant.cgi</td><td></td><td></td></tr></tbody></table>

pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

To study the fitness impact of single amino acid changes in HIV-1 Gag-p24, we used oligonucleotide directed mutagenesis to introduce mutations into a pNL4-3 plasmid containing the HIV-1 COTB-p24 gene23,32,33. Viral stocks were generated by transfection of 293T cells and harvested after 48 hr. We estimated the 50% tissue culture infectious dose (TCID50) of each viral stock by the Reed-Muench method31. The TCID50 of the prototype and mutant viruses ranged from 104 to 105 IU/ml (Figure 3A).
The growth kinetics of the recombinant viruses were established in PBMCs from a single donor at an MOI of 0.005. RT-qPCR was used to measure viral cDNA copy number daily for six days. All mutant and prototype viruses grew exponentially between day 2 and day 4, following which viral growth slowed, as indicated by a decreased slope of the viral RNA copy number increase (Figure 3B). The decrease in cDNA copy number between day 0 (corresponding to the inoculum) and day 2 is due to the absorption of virus to cells and the removal of unbound virions by the day 1 wash (step 5.1.6). In the exponential growth phase, all three mutants had a slower growth rate (g) than the prototype virus (Figure 3C).
All three mutants were competed against the prototype virus in growth competition assays at a total MOI of 0.005. Viral growth kinetics in dually infected cultures were similar to that of monoinfection; the viral exponential growth phase was between days 2 and 4, and viral growth reached a plateau around day 5 (Figure 4A).
Viral growth rate differences were derived from the change in the viral ratio over time. The viral ratio was calculated based on cDNA copy number of the reference and the mutant viruses using RT-qPCR, and by comparing peak heights in sequence chromatograms at nucleotide sites distinguishing the two viruses (Figure 4B). The growth rate differences determined using the peak-height and viral RNA copy number methods yielded similar results (Figure 4C). All three mutants had lower replication fitness than the prototype viruses with the mutant I256V having the lowest fitness (Figure 4C).
<img alt="Figure 1" src="/files/ftp_upload/52610/52610fig1.jpg" /> 
Figure 1: Flow diagram of the protocols presented in this paper. The Virus Preparation protocols concern construction of HIV-1 recombinant clones and generation of viral stocks. The Fitness Assays protocols are for establishing viral growth kinetics and determining relative viral fitness. Dashed lines represent alternative flows for the protocols. For example, a mutation can be introduced directly into HIV-1 NL4-3 molecular clone without generating a recombinant clone.
<img alt="Figure 2" src="/files/ftp_upload/52610/52610fig2.jpg" /> 
Figure 2: Construction of HIV-1 NL4-3 COTB Gag-p24 recombinant molecular clones using overlap extension PCR.&#160;(A) Design the chimeric primers. The 5&#8217; halves of the primers contain the NL4-3 vector sequence and the 3&#8217; halves contain the ends of the insert sequence. (B) Chimeric primers are used to amplify the insert fragment, COTB Gag-p24. (C) The insert fragments are used as primers for a second PCR to generate a new recombinant plasmid.
<img alt="Figure 3" src="/files/ftp_upload/52610/52610fig3.jpg" /> 
Figure 3: Viral growth characteristics in PBMCs.&#160;(A) Log10 TCID50 of viral stocks. (B) Viral growth kinetics in monoinfections begun at an MOI = 0.005. (C) Viral growth rates over six days, including the exponential growth phase (days 2-4) derived from panel (B). The values shown represent the average of three replicates from one experiment. The error bars represent 95% confidence intervals.
<img alt="Figure 4" src="/files/ftp_upload/52610/52610fig4.jpg" /> 
Figure 4: Viral growth and relative fitness determinations. (A) Viral growth in dually infected PBMC cultures. (B) Ratios of T242N and prototype viruses determined using RT-qPCR or the sequencing peak-height method. (C) Net viral growth rate differences (d) between the mutant and the prototype viruses in dually infected cultures, as shown in (A). d values were calculated from the viral ratio data shown in (B). The values shown represent the average of three replicates from one experiment. The error bars represent 95% confidence intervals.

Watch this Scientific Journal Video about Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses at JoVE.com

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

pairwise-growth-competition-assay-for-determining-replication-fitness

Research

JoVE Journal

Immunology and Infection

11.1K Views.  University of Washington. Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Video: Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Genetics

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Cancer Research

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

Studying ER Stress and UPR Activation During HIV-1 Infection in T-Cells

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. 
Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2&#945;, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

Author Spotlight: Exploring the Role of Unfolded Protein Response in HIV-1 Replication and Infectivity

Here, we describe some established methods to determine endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, with particular emphasis on HIV-1 infection. This article also describes a set of protocols to investigate the effect of ER stress/UPR on HIV-1 replication and virion infectivity.

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Summary

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Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication