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

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UST McNair Scholars Program Research Journal share a 60% homology in the amino acid sequence of reverse transcriptase. However, HIV-2 has a lower transmissibility than HIV-1, which explains the low prevalence of HIV-2 infections outside of West Africa (Schim van der Loeff and Aaby, 1999). Since the discovery of AZT as an antiretroviral drug against HIV-1, several drugs have been developed for use in HIV-1 treatment (Menendez-Arias, 2002). No treatment has been developed for HIV-2; management of HIV-2 is based on HIV-1 treatment guidelines. Both virus types develop resistance to drug therapies, but available data on HIV-2 drug resistance is lacking. The present research project will study factors that influence HIV-1 drug resistance and investigate if these factors present the same influence in HIV-2. Research on HIV-2 drug resistance is essential to the development of potential treatment options for the HIV-2 infection. Drugs currently used to treat HIV inhibit the activity of enzymes that are essential to viral replication. Monotherapy with antiretroviral drugs has given way to combination therapy because acquisition of resistance occurs in HIV (Menendez-Arias, 2002). Highly active antiretroviral therapy, HAART, combines three or more drug classes, usually two reverse transcriptase inhibitors and a protease inhibitor, to suppress viral replication. The accumulation of mutations that lead to drug resistance exacerbates antiretroviral therapy. HIV has a high mutation rate, 3 x 10 -5 mutations/base pair/cycle for HIV-1, which introduces one mutation every three genomes produced (Mansky, 1996; Mansky, 2002). The reverse transcriptase enzyme is a major generator of mutations because it has no proofreading ability. In normal DNA synthesis, DNA polymerase proofreads and removes incorrect base pairs or nucleosides from the growing DNA strand. RT synthesizes DNA from viral RNA and its polymerase activity is largely error prone (Goff, 1990). This research investigates mutations that lead to resistance to nucleoside reverse transcriptase inhibitors (NRTIs), a drug class that inhibits RT activity. NRTIs are nucleoside analogs; they are similar to normal nucleosides and can be incorporated into growing DNA. However, they act as DNA chain terminators and inhibit the attachment of additional nucleosides because they lack a 3’OH group in the ribose ring (Isel, Ehresmann, Walter, Ehresmann & Marquet, 2001; Menendez, 2002). Resistance to NRTIs occurs when 42 RT selects for mutations that prevent the incorporation of nucleoside analogs. Antiretroviral drugs can stimulate drug resistance in HIV. Studies show that antiretroviral drugs influence the mutation frequency of HIV-1 (mutation frequency is correlated to mutation rate and they are often used interchangeably). Mansky and Bernard (2002) investigated the influence of the antiretroviral drugs AZT and 3TC and AZT- or 3TC-resistant RT on the rate of HIV-1 mutation. Results from this study found replication in the presence of either AZT or 3TC increased the HIV-1 mutation rate. AZT resistant variants also increased the mutation rate. The selection of mutations that confer resistance increases in the presence of antiretroviral drugs, which allows resistance to occur at a rapid rate. HIV-1 develops drug resistance through two mechanism pathways. In the first pathway, mutations that inhibit RT from incorporating nucleoside analogs arise. A study by Sarafianos, Das, Hughes and Arnold (2004) identified residues, such as K65 and Q151 that play a role in positioning the incoming nucleoside; mutations at these residues lead to resistance to NRTIs. In the second pathway, mutations occur that promote adenosine triphosphate, ATP, to remove nucleoside analogs from the blocked DNA (Menendez, 2008). Previous studies have found that resistance by ATP excision is common in antiretroviral therapies that include thymidine analog NRTIs. A study conducted by Isel, Ehresmann, Walter, Ehresmann and Marquet (2001) found resistance to the drug zidovudine (AZT), associated with mutations such as M41L, D67N, K70R, T215F/Y and K219E/Q, is caused by selective excision of the drug. In another study, Lin et al. (1994) found the same set of mutations in viral isolates from patients under stavudine (d4T) therapy. AZT and d4T are both thymidine analogs, justifying their acquired mutations as thymidine analog mutations (TAMs). HIV-1 uses one pathway more frequently than the other in the presence of certain NRTIs (Boyer et al., 2006) HIV-2 appears to acquire resistance by the first pathway only. Previous studies identified genetic changes responsible for HIV-2 drug resistance. These studies, which sequenced isolates from HIV-2 infected patients under NRTI therapy, found patients acquired HIV-1 resistance conferring mutations that correspond to the first resistance pathway. One trend found observed such studies were the frequent emergence of the K65R and Q151M mutations
Mondraya Howard Pharmacy HIV-1 Resistant Conferring Mutations in HIV-2. A study by Boyer, Sarafianos, Clark, Arnold and Hughes (2006) found the Q151M to be the primary mutation associated with AZT therapy. The Q151M mutation has also been shown to induce resistance to almost all NRTIs in HIV-1(Rhodes et al., 2000; van der Ende et al., 2000). Another study conducted by Descamps et al. (2004) found frequent emergence of the K65R mutation in patients under 3TC therapy. In their study, Smith et al. (2009) determined that the K65R and Q151M mutations, together, promote class wide NRTI resistance. The residues that these mutations take place play a role in positioning the incoming nucleoside during DNA synthesis. Resistance with these mutations follows the first mechanism pathway of HIV-1. There was a low prevalence of TAMs in HIV-2 resistance indicating that HIV-2 does not employ the excision pathway to confer drug resistance (Smith et al., 2009; Boyer et al., 2006). The primary goal of this project is to create HIV-2 resistant RT by incorporating HIV-1 resistance conferring mutations into HIV-2 RT. Drug susceptibility decreases and mutation frequency increases in the presence of HIV- 1 resistant RT. Since HIV-1 and HIV-2 have genetically and structurally similar RT, I hypothesize that HIV-2 resistant RT will confer resistance to NRTIs and increase the mutation frequency of HIV-2. Mutation frequency of HIV-2 was determined by producing the virus in a HIV- 2env- vector containing two marker genes, HSA and GFP, and using flow cytometer to calculate the percentage of cells expressing the marker genes (mutant frequency). Using site-directed mutagenesis, the Q151M and K65R mutations will be introduced into HIV-2 RT. Mutant HIV- 2 can be produced and replicated the HIV-2env- vector and flow cytometry will examine the mutation frequency. Drug susceptibility will be observed by replicating HIV-2 resistant RT in presence of NRTIs. Research on the HIV- 2 infection is limited to a handful of studies because of the restricted spread of the virus. This research provides a better understanding of HIV-2 drug resistance and can contribute to development of potential treatment options for HIV-2. MATERIALS AND METHODS CELL LINES AND PLASMIDS. HIV-2ROD viral DNA was obtained from the Mansky Lab, Institute of Molecular Virology (University of Minnesota). The 293T cell line was obtained from the American Type Culture Collection. Antibody to mouse heat-stable antigen protein (HSA) was purchased from BD Pharmingen (San Diego, CA). The Purelink Quick Plasmid miniprep kit was obtained from Invitrogen (Grand Island, NY). HIV-2env- vector was obtained from Hu Wei-Shau. pIRES2-EGFP was obtained from Clontech (Mountain View, CA). Restriction enzymes were purchased from New England Biolabs (Ipswich, MA). The pCR-18S plasmid was a gift from Mauro Magnani (Universita’ Degli Studi Di Urbino). CONSTRUCTION OF THE HIV-2 VECTOR FOR MUTATION DETECTION. HIV-2 vector (obtained from Wei Shau) was modified by restoring the gfp and HIV-2 vpr genes via site-directed mutagenesis. The vector contains the gene for HSA as well as a frame-shift mutation at the 5’end of env, which limits the virus to one round of replication. The internal ribosome entry site (IRES)-green fluorescent protein (GFP) fragment was PCR amplified from pIRES2-EGFP and subcloned into pCR2.1. This plasmid, as well as HIV-2env- was restriction digested with XhoI. Following purification, HIV-2env- and the IRES-enhanced GFP (EGFP) fragments were ligated and then transformed using DH5α cells. Restriction digestion and DNA sequencing analysis was used to verify the clones. TRANSFECTION OF 293T CELLS 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal clone 3 (FC3) serum (HyClone, Logan, UT) and penicillin/ streptomycin at 37°C in 5% CO2. 293T cells were plated on poly-L-lysine- coated 10-cm culture dishes 24 h before transfection. The cells were then transfected by calcium phosphate coprecipitation with 10m g of the HIV-2 vector (HIG) and 1m g of a plasmid encoding the HIV envelope, VSVG. The medium was replaced with 6 ml of DMEM containing 10% FC3 serum and penicillin/streptomycin 43
Page 1 and 2: DR. RONALD E. MCNAIR Ronald E. McNa
Page 3 and 4: ABSTRACT The purpose of the present
Page 5 and 6: Oluwademilade Adediran Psychology M
Page 21 and 22: ABSTRACT Early childhood education
Page 23 and 24: Kesha Berg Public Policy Enhancing
Page 43: ABSTRACT Human immunodeficiency vir
Page 47 and 48: Mondraya Howard Pharmacy HIV-1 Resi
Page 49 and 50: ABSTRACT Food waste in the United S
Page 51 and 52: Bridgette Kelly Environmental Scien
Page 63 and 64: ABSTRACT This research discusses he
Page 65 and 66: Chia Lee Public Health Health care
Page 75 and 76: Mai-Eng Lee Social Work Teachers’
Page 83 and 84: APPENDIX B INTERVIEW QUESTIONS Back

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

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