HIV versus the Terminator: Drug resistance of HIV reverse transcriptase with mutations at the connection subdomain

Abstract only availableAntiretroviral drug therapy can prolong the life of an HIV-infected individual, but this treatment also promotes drug-resistance mutations. The replicative enzyme of HIV, reverse transcriptase (RT), is a primary target for anti-HIV drug therapy because it is responsible for converting the single stranded RNA genome of HIV into double stranded DNA for integration into the host genome. Many current anti-HIV drugs belong to two classes of inhibitors that target RT: nucleoside reverse transcriptase inhibitors (NRTIs) incorporate into and chain-terminate nascent transcription products of RT, whereas non-nucleoside reverse transcriptase inhibitors (NNRTIs) alter enzyme-nucleic acid interactions, thereby affecting the efficiency of DNA polymerization. Here, we focus on NRTI resistance mutations that are located at the connection subdomain of the enzyme in the presence and absence of thymidine analog associated mutations (TAMs). TAMs cause resistance to the commonly prescribed chain terminator 3'-azido-3'-deoxythymidine (AZT) through excision of the incorporated AZT-monophosphate. Mutations in the connection domain, such as N348I, confer resistance to NRTIs and NNRTIs and augment AZT resistance when present in combination with TAMs. Although the underlying mechanism of N348I resistance remains elusive, it has been suggested that the mutation compromises ribonuclease (RNase) H activity, which is responsible for cleaving the viral genomic RNA of the RNA/DNA heterodimeric intermediate. Changes in RNase H cleavage affect the availability of AZT-terminated primers to be excised, thereby increasing the unblocking of template/primer and NRTI resistance. Our investigation attempts to determine if AZT-resistance mutations affect resistance to other commonly prescribed NRTIs, as well as to competitive substrate inhibitors currently in development, through changes in template/primer processing. In addition, we are examining the effects of NRTI and NNRTI cocktails on the RNase H activity of RT possessing connection domain mutations. Our findings should provide insight for screening novel inhibitors for their efficacy against emergent strains of drug-resistant HIV.Life Sciences Undergraduate Research Opportunity Progra

Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?

The human immunodeficiency virus type 1 (HIV-1) develops resistance to all available drugs, including the nucleoside analog reverse transcriptase inhibitors (NRTIs) such as AZT. ATP-mediated excision underlies the most common form of HIV-1 resistance to AZT. However, clinical data suggest that when HIV-2 is challenged with AZT, it usually accumulates resistance mutations that cause AZT resistance by reduced incorporation of AZTTP rather than selective excision of AZTMP. We compared the properties of HIV-1 and HIV-2 reverse transcriptase (RT) in vitro. Although both RTs have similar levels of polymerase activity, HIV-1 RT more readily incorporates, and is more susceptible to, inhibition by AZTTP than is HIV-2 RT. Differences in the region around the polymerase active site could explain why HIV-2 RT incorporates AZTTP less efficiently than HIV-1 RT. HIV-1 RT is markedly more efficient at carrying out the excision reaction with ATP as the pyrophosphate donor than is HIV-2 RT. This suggests that HIV-1 RT has a better nascent ATP binding site than HIV-2 RT, making it easier for HIV-1 RT to develop a more effective ATP binding site by mutation. A comparison of HIV-1 and HIV-2 RT shows that there are numerous differences in the putative ATP binding sites that could explain why HIV-1 RT binds ATP more effectively. HIV-1 RT incorporates AZTTP more efficiently than does HIV-2 RT. However, HIV-1 RT is more efficient at ATP-mediated excision of AZTMP than is HIV-2 RT. Mutations in HIV-1 RT conferring AZT resistance tend to increase the efficiency of the ATP-mediated excision pathway, while mutations in HIV-2 RT conferring AZT resistance tend to increase the level of AZTTP exclusion from the polymerase active site. Thus, each RT usually chooses the pathway best suited to extend the properties of the respective wild-type enzymes

Characterization of the mechanism of action of the ultra-potent HIV inhibitor 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine [abstract]

Abstract only availableRetroviruses rely on the enzyme reverse transcriptase (RT) to perform the reverse transcription of its genome from single-stranded RNA into double-stranded DNA, which can then be integrated into the host's genome by the action of the viral integrase enzyme. There are currently sixteen antiretroviral agents used for the treatment of HIV infections. Highly active antiviral therapy (HAART) is based on a combination of at least 3 anti-HIV drugs. It has slowed down the progression of AIDS and decreased mortality. RT is one of the main targets for these antiretroviral drugs. One class of drugs targeting the reverse transcriptase is the nucleoside analogue RT inhibitors (NRTIs). NRTIs compete with natural nucleotides for incorporation in the elongating DNA chain by HIV-1 RT. Once incorporated, they act as chain-terminators because they lackthe 3'OH group which is required for further nucleotide incorporation. Prolonged use of these drugs leads to drug-resistant HIV strains. To overcome drug resistance, novel inhibitors that are active against NRTI-resistant viruses are being developed. NRTIs containing a modification at the 4' position of their sugar moiety have been synthesized by Hiroaki Mitsuya and his colleagues. One of these analogues, 4'ethynyl-2-fluoro-2'-deoxyadenosine (4'-E-2-F dA) was shown to be ultra-potent against wild-type and drug resistant HIV-1. Unlike other nucleoside analogues, 4'-E-2-F dA has a hydroxyl group at the 3' position. The purpose of this project is to understand the mechanism of RT inhibiton by 4'-E-2-F dA. In order to determine its mechanism of action, in vitro primer extension assays as well as gel mobility shift assays were used. Using primer extension assays, we determined that the active form of 4'-E-2-F dA, 4'-E-2-F dA-triphosphate (TP), acts as a chain terminator at physiological concentrations of nucleotides, despite the presence of a 3'OH. We first hypothesized that the presence of 4'-E-2-F dA-monophosphate (MP) at the 3' end of the primer destabilized the RT/DNA complex. The RT/DNA complex is not affected by the presence of 4'-E-2-F dAMP as observed in gel mobility shift assays. We next hypothesised that RT was not able to bind to the next incoming nucleotide to form a ternary complex. Indeed, we found that the presence of 4'-E-2-F dAMP at the 3' end of the primer severly impair the formation of a stable ternary complex. In conclusion, we found that 4'-E-2-F dA inhibits DNA elongation by RT by acting as chain-terminator despite the presence of the 3'OH. In order to do so, 4'-E-2-F dAMP blocks the binding of the next incoming nucleotide. This is a novel mechanism of inhibition that results in the most efficient blocking of HIV activity reported to date for any NRTI. Our findings have generated interest from two pharmaceutical companies that wish to develop it as a next-generation therapeutic for the treatment of HIV infection

Touching the heart of HIV-1 drug resistance: the fingers close down on the dNTP at the polymerase active site

AbstractComparison of the recently solved structure of HIV-1reverse transcriptase (RT)-DNA-dNTP ternary complex with the previously solved structure of RT-DNA binary complex suggests mechanisms by which the HIV-1 RT becomes resistant to nucleoside-analog inhibitors, drugs currently used in the treatment of AIDS

Characterization of polyoxometalate I as an inhibitor of RNA-dependent RNA polymerase of Foot and Mouth Disease virus [abstract]

Abstract only availableFoot and Mouth Disease (FMD) is a highly contagious disease that affects a variety of domesticated cloven-hoofed animals including cattle, swine, sheep and goats, as well as several wild animal species. FMD outbreaks are currently controlled with mass-extermination of livestock. The financial cost of potential outbreaks would be immense. This disease is caused by foot-and-mouth disease virus (FMDV), a non-enveloped, single-stranded, positive-sense RNA virus. The purpose of our investigation is to identify chemicals that interfere with the replication of FMDV. As part of this effort we have identified a polyoxometalate inhibitor (polyoxometalate I). We have cloned, expressed and purified FMDV RdRp. We use steady-state kinetic experiments and polymerization assays to characterize the inhibitory activity of the polyoxometalate I, determining the precise inhibitory potential and the mechanism of inhibition. Preliminary results show that polyoxometalate I inhibits the FMDV RdRp surprisingly efficiently with an IC50 of 0.5uM. Current experiments are focusing on a detailed kinetic characterization of the mechanism of action for this inhibitor. This research may provide insights that lead to new treatment options to prevent the further spread of FMD to unaffected animals.USD

Novel inhibition mechanism and potent antiviral activity of translocation-deficient reverse transcriptase inhibitors [abstract]

Abstract only availableNucleoside RT inhibitors (NRTIs) are among the most potent anti-HIV agents and act as chain terminators because they lack a 3'OH. However, this feature can reduce affinity for RT compared to the analogous dNTP substrate, as well as reduced intracellular conversion to the active dNTP. To overcome this, it was shown that certain nucleosides that retain the 3'OH and have substitutions at the 4' ribose and 2 position of the base have exceptional antiviral properties. One of these compounds, 4'-ethynyl, 2-fluoro deoxy-adenosine (4'E-2FdA) is the most potent NRTI inhibitor against wild-type and multi-drug resistant HIV viruses described to date. We have recently reported that 4'E-2FdA acts as a chain terminator despite the presence of an accessible 3'OH. We show that after 4'E-2FdA-MP incorporation, RT does not bind the next incoming dNTP. We analyzed RT translocation on different sequences terminated with 4'E-2FdA-MP, and found that even at sequences when RT is naturally found post-translocated, the inhibitor prevents translocation. This decrease in translocation efficiency explains the reduced binding of the next incoming dNTP and the termination of elongation. While the inhibitor stabilizes the pre-translocated 4'E-2FdA-MP-terminated primer, the pyrophosphate-dependent excision rate of 4'E-2FdA-MP was not very high compared to ddAMP. In conclusion, this highly potent chain termination activity arises from difficulty of the primer 3'-terminus to translocate following incorporation of the compound, and not from simple steric hindrance due to the 4' substitution. Therefore, we propose that 4'E-2FdA is a Translocation-Deficient Reverse Transcriptase Inhibitor (TDRTI) that acts by a novel mechanism.NIH grant to S. Sarafiano

Effectiveness of current anti-HIV regimen in low-and middle-income countries

Nevirapine (NVP) is a first-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1). However, with the emergence of resistance mutations due to a low genetic barrier under NVP pressure, new (second generation) NNRTIs have been approved. Rilpivirine (RPV), a second generation NNRTI, is not frequently used in low- and middle- income countries (LMICs) that bear the major HIV burden. RPV has been co-formulated with tenofovir (TDF) and emtricitabine (FTC) and has been recommended for patients with viral loads <100,000 copies/mL, inhibiting viruses that are resistant to NVP. It is now being considered in many LMICs. To understand RPV efficacy in HIV-1 subtypes prevalent in LMICs, we cloned RT genes from patients infected with four different HIV-1 subtypes: subtype B (HIV-1B), subtype C (HIV-1C), and recombinant forms CRF01_AE and CRF02_AG. HIV-1B is most prevalent in western countries and accounts for only ~12% of all infections. However, HIV-1C, which accounts for ~52% of all HIV infections, is most prevalent in LMICs. In vitro inhibition assays were performed with the four patient-derived RTs. Our results show that overall, NVP binds RTs with lower affinity than RPV, suggesting that NVP has lower effectiveness than RPV. However, NV binds 02_AG RT with better affinity than RPV. Hence, NVP may still be effective for patients infected with 02_AG. Furthermore, RPV binding affinity with HIV-1C is lower than other subtypes. This result is consistent with clinical results, showing less efficacy of RPV among HIV-1C infected patients

Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action

Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase

The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5′→3′ polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ∼280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis

High Resolution Crystal Structure of KD-247, a Humanized Antibody that Inhibits HIV Entry [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionHighly active antiretroviral therapy (HAART) has been very efficient in reducing the rate of mortality of human immunodeficiency virus type 1 (HIV-1) infected patients. However, resistance to clinically used drugs inevitably develops and impairs the potency of these drugs. There is also no vaccine available to prevent the spread of the virus. Our collaborators, Dr. Shuzo Matsushita and his colleagues have developed a monoclonal antibody, KD-247, that is currently in Phase Ib clinical trials for the treatment of HIV-1 infections. KD-247 blocks virus entry into host cells by binding to the V3 loop of the surface glycoprotein of HIV. It is the first humanized antibody shown to neutralize a wide range of subtype B HIV viruses (Matsushita et al. Hum. Antibodies, 14, 81) and to prevent HIV infection in cell culture and in a chimpanzee model (Eda et al. J. Virol., 80, 5563). KD-247 reacts exclusively with subtype B viruses (Eda et al. J. Virol., 80, 5552). In order to understand the molecular basis of this specificity we have solved the crystal structure of KD-247 at 1.5 Å resolution, the highest resolution structure for any humanized antibody reported to date. The present structure reveals in atomic detail the molecular boundaries of a pocket formed by the antigen-binding region of the antibody. Molecular docking experiments of a pre-existing structure of the V3-loop target at the presumed binding pocket on KD-247 suggest possible molecular interactions involved in HIV resistance to KD-247 and clade B specificity. A G314E V3 loop mutation that has been reported to confer resistance to KD-247 (Yoshimura et al., AIDS 20, 2065) appears to result in steric interactions between the tip of the V3 loop and residues of the heavy chain of KD-247. Further, Arg315, a residue critical for clade B specificity, appears to form extensive interactions with multiple residues of KD-247. Analysis of these interactions has provided insights into the design of second-generation antibodies with broader subtype specificity and improved ability to evade resistance mutations. This work is a product of collaborations between the University of Missouri and researchers at Kumamoto University, an academic institution in Japan, and the Chemo-Sero-Therapeutic Research Institute, an industrial partner in Japan, working to commercialize the antibody and further its progress in clinical trials