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 border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name of Material/ Equipment</td>
			<td style="width:484px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:196px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Construction of recombinant clones</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Chimeric primer/Mutagenic/Sequencing primers</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom DNA oligos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">pNL4-3VifA and pNL4-3VifB plasmid</td>
			<td style="width:484px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-Fidelity DNA polymerase</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-fidelity buffer</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal cycler</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems&#174; GeneAmp&#174; PCR System 9700</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA loading dye</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td style="width:137px;">R0631</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1kb Plus DNA ladder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAquick PCR Purification Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick gel extraction kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">DpnI enzyme</td>
			<td style="width:484px;">New England Biolabs</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">TOP10 chemically competent Escherichia coli</td>
			<td style="width:484px;">Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Luria Broth base powder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">12795-084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Carbenicillin</td>
			<td style="width:484px;">Research Products International</td>
			<td style="width:137px;">C46000-25.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep kit&#160;</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">27104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">12362</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">X-tremeGENE 9 DNA transfection reagent</td>
			<td style="width:484px;">Roche</td>
			<td>6365787001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HEK 293T-17</td>
			<td style="width:484px;">ATCC</td>
			<td style="width:137px;">CRL-11268</td>
			<td>http://www.atcc.org/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">0.22um filter top tube</td>
			<td style="width:484px;">VWR International</td>
			<td>89220-716&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#8242;s Modified Dulbecco&#8242;s Medium (IMDM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>31980-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RPMI-1640 media</td>
			<td style="width:484px;">Life Technologies</td>
			<td>61870-036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phytohemagglutinin (PHA)</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>R30852801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Human Interleukin-2</td>
			<td style="width:484px;">Roche</td>
			<td>11147528001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum</td>
			<td style="width:484px;">JR Scientific</td>
			<td style="width:137px;">43640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Penicillin and Streptomycin</td>
			<td style="width:484px;">Corning Cellgro</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">73520-906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">62407-338</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well flat-bottomed plate</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td style="width:137px;">T-3015-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well round-bottomed plate</td>
			<td style="width:484px;">BD Falcon</td>
			<td style="width:137px;">353077</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 ml Microcentrifuge Tube</td>
			<td style="width:484px;">Mt. Baker Bio</td>
			<td style="width:137px;">MBD-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 mL tubes w/ O-ring</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">89004-290</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">50 mL conical tube</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td>C-3317-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Triton-X 100</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phosphate buffer saline (PBS)</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum (FBS)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>F0392</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">glycerol</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td style="width:137px;">G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Tween 20</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-HIV-1 SF2 p24 antiserum</td>
			<td style="width:484px;">NIH AIDS Reagent Program</td>
			<td style="width:137px;">4250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit HRP</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">474-1516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMB Microwell Peroxidase Substrate</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">52-00-01</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">H2SO4</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 minutes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HIV p24 standard</td>
			<td style="width:484px;">AIDS and Cancer Virus program (NCI-Frederick)</td>
			<td style="width:137px;">N/A</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 height="20">
			<td height="20" style="height:20px;width:355px;">Microplate reader</td>
			<td style="width:484px;">Molecular Devices</td>
			<td style="width:137px;">N/A</td>
			<td>VMax Kinetic ELISA Microplate Reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA isolation cDNA synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxtractor</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">N/A</td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAamp Viral RNA Mini Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">52906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">SuperscriptIII</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">First-strand buffer&#160;</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Rnase inhibitor</td>
			<td style="width:484px;">Roche</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RnaseH</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18021-071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Real-time qPCR machine</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems 7300 Real-Time PCR&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan&#174; Gene Expression Master Mix</td>
			<td style="width:484px;">Life Technologies</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Forward, reverse primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Probe</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">optical 96 well reaction plate</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">I19N3Q216</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR+sequencing</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Taq DNA polymerase (Biolase)</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NH4 buffer&#160;</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Sequencing primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxcel</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;"></td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA sequencing service provider</td>
			<td>http://www.htseq.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GRC web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/grc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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

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JoVE Journal

Immunology and Infection

11.0K 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.