dr. ronald e. mcnair acknowledgements - University of St. Thomas

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UST McNair Scholars Program Research Journal 24 h after transfection. Virus was harvested 24 h later by filtration of the cell supernatant through a 0.2-mm filter. INFECTION OF TARGET CELLS AND FLOW CYTOMETRY U373-MAGI-CXCR4C E M cells, maintained at 37°C in 5% CO2 in selection medium composed of DMEM with 10% FC3 serum, 1 mg/ml puromycin, 0.1 mg/ml hygromycin, and 0.2 mg/ml neomycin, were plated in a 12-well culture dish 24 h prior to infection. After pretreatment, the viral stock (500 ml) was added to each well. Cells were harvested for analysis 48 h after infection. Cells were then analyzed for fluorescence at 488 nm and 568 nm. Quadrants were drawn using non-infected cells to determine background levels of fluorescence. Cells expressing both HSA and GFP were used to determine the percentage of infected cells. Replication assay protocol was adapted from Mansky, Pearl and Gajary (2006). FIG. 1 (Adapted from Clouser, Patterson and Mansky, 2010) Single round replication assay use to assess mutation frequency. HIV-2 virus is produce in 293T cells by transfection of two plasmid constructs: the HIV-2 envelope-deficient vector contains two marker genes that are used to measure mutation frequency. The second plasmid encodes for HIV envelope. After transfection, the supernatant containing virus is collected and added to target cells. Cells are harvested after infection and flow cytometry is used to examine expression of marker genes. RESULTS Resistance conferring mutations have been shown to increase mutation rate of HIV-1. Since HIV-1 and HIV-2 have similar reverse transcriptase, we asked if HIV-2 resistant RT would influence the mutation rate of HIV-2. Mutation rate determines how many mutations are occurring per replication cycle and contributes to the emergence of drug resistance in HIV. To examine mutation 44 frequency, we used an assay that detects HIV-infection through the expression of two marker genes, HAS and GFP. The assay, Fig 1, uses a HIV-2 vector construct with a mutated Env gene, which limits the virus on replication cycle. 93T cells are used to produce the virus, which is used to infect target cells, U373-MAGI-CXCR4C E M cells. The expression of target genes is assessed by flow cytometry to determine mutation frequency. The two target genes, HSA and GFP, were used to simultaneously detect HIV infectivity and mutation frequency. To determine mutation frequency, the flow data were divided into four quadrants based on the expression of GFP and/or HAS (Fig 2). Cells infected with the wildtype HIV-2 construct were expected to express both GFP and HSA, whereas cells infected with a mutant HIV-2 construct express either one or no marker gene. Cells that express only one marker gene have been infected with mutant HIV-2 that inhibits the expression of the other marker gene. Therefore, cells infected with mutant HIV were detected as cells expressing either HSA or GFP, but not both. The relative mutation frequency was then calculated as a fraction by dividing the percentage of cells infected with mutant virus by the total percentage of cells infected. Results shown in Table 1 demonstrate that HIV- 2 had an average mutant frequency of 0.239 ± 0.041 in the first replication assay. Table 2 shows that HIV-2 produced an average mutant frequency of 0.273 ± 0.033 in a second replication assay. Once HIV-2Q151M mutants are generated, we can produce a HIV-2 resistant virus and repeat replication assay protocol to observe if resistanceconferring mutations influence HIV-2 mutation frequency. a)
Mondraya Howard Pharmacy HIV-1 Resistant Conferring Mutations b) FIG. 2. Flow data of single round replication assay use to assess the mutation frequency. (a) Flow data from 1 st replicate. (b) Flow data from 2 nd replicate Flow data were divided into 4 quadrants based on expression of HSA and GFP genes. Mutation frequency was calculated by dividing the number of cells infected with mutant virus (Q4) by the total number of infected cells (Q1- Q13). Values from this calculation expressed the mutation frequency in each replicate (Table 1, 2). HIV- 2Replicate 1 st replicate 2 nd replicate Table 1. Summary of infection percentage and mutant frequency from flow cytometry data DISCUSSION Average % infection (n=3) ± sd Average mutant frequency (n=3)± sd χ 2 (df = 1) p-value 19.6 ±4.1 0.239 ±0.041 12.52 0.0004 12.0 ± 2.9 0.273 ±0.033 65.02
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