Enzymatic Characterization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Resistant to Multiple 2′,3′-Dideoxynucleoside 5′-Triphosphates

Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC 50 s and IC 90 s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC 50 s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culturebased, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC 50 s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine.

supporting

confidence: 82%

mentioning

confidence: 99%

Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors

Qari

Magre

Garcı́a-Lerma

et al. 2001

“…To make matters even more confusing, there are mutations (for example, the multidrug resistance mutation Q151M) that do confer considerable resistance to AZTTP in simple in vitro polymerization reactions (19,23). A number of possible solutions to this dilemma have been suggested, the most promising of which is this: although many of the mutations that confer resistance to nucleoside analogs do so by interfering with nucleoside incorporation into DNA, AZT resistance mutations lead to enhanced excision of AZT from the nascent DNA strand after it has been incorporated (1,15).…”

mentioning

confidence: 99%

Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

Boyer

Sarafianos

Arnold

et al. 2001

244

358

Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

“…HIV-1 also develops high levels of resistance against multiple antiviral drugs by accumulating a variety of amino acid substitutions near (and beyond) the active sites of target viral enzymes (5,20,(35)(36)(37), whereas such multiple mutations can often compromise the enzymatic functions of the viral protease and reverse transcriptase (RT) (7,10,17,24,34,39). In the case of HIV-1 resistance to an RTI, amino acid changes in the polymerase are virtually fully responsible for the viral acquisition of resistance to RTIs.…”

mentioning

confidence: 99%

Amino Acid Insertions near Gag Cleavage Sites Restore the Otherwise Compromised Replication of Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

Tamiya

Mardy

Kavlick

et al. 2004

A variety of amino acid substitutions in the protease and Gag proteins have been reported to contribute to the development of human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors. In the present study, full-length molecular infectious HIV-1 clones were generated by using HIV-1 variants isolated from heavily drug-experienced and therapy-failed AIDS patients. Of six full-length infectious clones generated, four were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites, in addition to the known resistance-related multiple amino acid substitutions within the protease. The addition of such Gag inserts mostly compromised the replication of wild-type HIV-1, whereas the primary multidrug-resistant HIV infectious clones containing inserts replicated significantly better than those modified to lack the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type protease was impaired by the presence of the inserts, whereas that by mutant protease was substantially improved. The present study represents the first report clearly demonstrating that the inserts seen in the proximity of the Gag cleavage sites in highly multi-PI resistant HIV-1 variants restore the otherwise compromised enzymatic activity of mutant protease, enabling the multi-PI-resistant HIV-1 variants to remain replication competent.