Cross-Clade Inhibition of Recombinant Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus SIVcpz Reverse Transcriptases by RNA Pseudoknot Aptamers

Abstract

Reverse transcriptase (RT) remains a primary target in therapies directed at human immunodeficiency virus type 1 (HIV-1). RNA aptamers that bind RT from HIV-1 subtype B have been shown to protect human cells from infection and to reduce viral infectivity, but little is known about the sensitivity of the inhibition to amino sequence variations of the RT target. Therefore, we assembled a panel of 10 recombinant RTs from phylogenetically diverse lentiviral isolates (including strains of HIV-1, simian immunodeficiency virus SIVcpz, and HIV-2). After validating the panel by measuring enzymatic activities and inhibition by small-molecule drugs, dose-response curves for each enzyme were established for four pseudoknot RNA aptamers representing two structural subfamilies. All four aptamers potently inhibited RTs from multiple HIV-1 subtypes. For aptamers carrying family 1 pseudoknots, natural resistance was essentially all-or-none and correlated with the identity of the amino acid at position 277. In contrast, natural resistance to aptamers carrying the family 2 pseudoknots was much more heterogeneous, both in degree (gradation of 50% inhibitory concentrations) and in distribution across clades. Site-directed and subunit-specific mutagenesis identified a common R/K polymorphism within the p66 subunit as a primary determinant of resistance to family 1, but not family 2, pseudoknot aptamers. RNA structural diversity therefore translates into a nonoverlapping spectrum of mutations that confer resistance, likely due to differences in atomic-level contacts with RT.The reverse transcriptases (RTs) of the human and simian immunodeficiency viruses (HIVs and SIVs, respectively) are encoded by the viral pol gene and are expressed from viral mRNA as part of the multifunctional Gag-Pol polyprotein. Mature RT is the product of proteolytic processing of the polyprotein, first into an asymmetric homodimer and then into the mature heterodimer (21). Due to its central role in HIV type 1 (HIV-1) replication and the early clinical availability of anti-RT compounds, RT has long been an established therapeutic target. Of the 22 anti-HIV compounds currently approved by the U.S. Food and Drug Administration, 15 target the viral RT (60). The nucleoside analogue RT inhibitors (NRTIs) are deoxynucleoside triphosphate analogues that result in chain termination when incorporated by RT into a growing DNA strand. The nonnucleoside RT inhibitors (NNRTIs) bind RT near the active site and disrupt enzymatic activity by allosteric inhibition (38, 59). The search for new anti-RT compounds is fueled by cytotoxicity and clinically selected resistance-resulting from drug exclusion or from postincorporation excision (45)-which are persistent problems for both classes of RT inhibitors.Nucleic acid aptamers, ribozymes, antisense RNA, and small interfering RNA exhibit potent antiviral effects and are under development as potential gene therapy adjuvants to smallmolecule therapeutics (25,34), and several of these have recently entered into clinical trials (1,39,4...

Active site binding and sequence requirements for inhibition of HIV-1 reverse transcriptase by the RT1 family of single-stranded DNA aptamers

“…Mutation R277K has been shown to be resistant to Type I RNA pseudoknot aptamers but not to RT1t49 [(37), data not shown]. Based on our modeling, this mutation faces away from the helix and may have only minimal effect RT1t49 binding (data not shown).…”

Aptamers against pathogenic microorganisms

“…Reverse transcriptase of HIV-1 virus plays one of the key roles in viral replication, the synthesis of the DNA copy of the viral RNA genome before its integration into the DNA of the host cell. At present, a wide spectrum of aptamers has been obtained for effective reverse transcriptase binding and/or the inhibition of its activity in vitro (Burke et al, 1996; DeStefano & Cristofaro, 2006; Ditzler et al, 2011; Green et al, 1995; Held et al, 2006, 2007; Kensch et al, 2000; Kissel et al, 2007a, b; Lai & DeStefano, 2012; Li et al, 2008; Michalowski et al, 2008; Mosing et al, 2005; Tuerk et al, 1992; Whatley et al, 2013). Furthermore, several aptamers were able to inhibit the viral replication in cells (Chaloin et al, 2002; Joshi & Prasad, 2002; Joshi et al, 2005; Lange, 2012).…”

Aptamers that recognize drug-resistant HIV-1 reverse transcriptase

“…In contrast and as a positive control, TPK1.1 readily inhibited primer extension, as has been previously described (9). Similarly, the selected aptamers did not inhibit the RNase H activity of WT HIV-1 RT, although TPK1.1 again showed strong inhibition (Figure 7c) (14).

Figure 7.Inhibitory activities of anti-RT aptamers.

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