Formation of a quaternary complex of HIV-1 reverse transcriptase with a nucleotide-competing inhibitor and its ATP enhancer.

Nucleotide-competing reverse transcriptase inhibitors were shown to bind reversibly to the nucleotide-binding site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). Here, we show that the presence of ATP can enhance the inhibitory effects of the prototype compound INDOPY-1. We employed a combination of cell-free and cell-based assays to shed light on the underlying molecular mechanism. Binding studies and site-specific footprinting experiments demonstrate the existence of a stable quaternary complex with HIV-1 RT, its nucleic acid substrate, INDOPY-1, and ATP. The complex is frozen in the post-translocational state that usually accommodates the incoming nucleotide substrate. Structure-activity relationship studies show that both the base and the phosphate moieties of ATP are elements that play important roles in enhancing the inhibitory effects of INDOPY-1. In vitro susceptibility measurements with mutant viruses containing amino acid substitutions K70G, V75T, L228R, and K219R in the putative ATP binding pocket revealed unexpectedly a hypersusceptible phenotype with respect to INDOPY-1. The same mutational cluster was previously shown to reduce susceptibility to the pyrophosphate analog phosphonoformic acid. However, in the absence of INDOPY-1, ATP can bind and act as a pyrophosphate donor under conditions that favor formation of the pre-translocated RT complex. We therefore conclude that the mutant enzyme facilitates simultaneous binding of INDOPY-1 and ATP to the post-translocated complex. Based on these data, we propose a model in which the bound ATP traps the inhibitor, which, in turn, compromises its dissociation.

Effects of mutations F61A and A62V in the fingers subdomain of HIV-1 reverse transcriptase on the translocational equilibrium.

Changes of the translocational status of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) can affect susceptibility to antiretroviral drugs. The pyrophosphate analogue phosphonoformic acid (PFA) binds specifically to and traps the pretranslocated complex of HIV-1 RT, while nucleotide-competing RT inhibitors trap the posttranslocated conformation. Here, we attempted to assess the potential role of residues in the fingers subdomain as determinants of polymerase translocation. The fingers can exist in open and closed conformations; however, the relationship between such conformational changes and the translocation status of HIV-1 RT remains elusive. We focused on substitution F61A and the neighboring A62V that is frequently associated with drug-resistance-conferring mutations. The proximity of these residues to the nucleic acid substrate suggested a possible role in translocation for these amino acid changes. We employed site-specific footprinting, binding assays, and DNA-synthesis inhibition experiments to study F61A and A62V, alone and against a background of known drug-resistance mutations. We demonstrate that F61A causes a strong bias to the posttranslocational state, while A62V shows a subtle bias toward pretranslocation regardless of the mutational background. Increases in the population of pretranslocated complexes were accompanied by increases in PFA activity, while F61A is literally resistant to PFA. Our data shed light on equilibria between pre- and posttranslocated complexes with the fingers subdomain in its open or closed conformations. We propose that a binary, pretranslocated complex in a closed conformation is stabilized with A62V and destabilized with F61A.

N348I in HIV-1 reverse transcriptase can counteract the nevirapine-mediated bias toward RNase H cleavage during plus-strand initiation.

Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (+)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.

Connection domain mutations N348I and A360V in HIV-1 reverse transcriptase enhance resistance to 3'-azido-3'-deoxythymidine through both RNase H-dependent and -independent mechanisms.

Thymidine analogue-associated mutations (TAMs) in reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) cause resistance to 3'-azido-3'-deoxythymidine (AZT) through excision of the incorporated monophosphate. Mutations in the connection domain of HIV-1 RT can augment AZT resistance. It has been suggested that these mutations compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains elusive. Here, we focused on connection mutations N348I and A360V that are frequently observed in clinical samples of treatment-experienced patients. We show that both N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase excision of AZT in the presence of the pyrophosphate donor ATP. The TAMs/N348I/A360V mutant accumulates transiently formed, shorter hybrids that can rebind to RT before the template is irreversibly degraded. These hybrids dissociate selectively from the RNase H-competent complex, whereas binding in the polymerase-competent mode is either not affected with N348I or modestly improved with A360V. Both connection domain mutations can compensate for TAM-mediated deficits in processive DNA synthesis, and experiments with RNase H negative mutant enzymes confirm an RNase H-independent contribution to increased levels of resistance to AZT. Moreover, the combination of diminished RNase H cleavage and increased processivity renders the use of both PP(i) and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction. Taken together, our findings demonstrate that distinct, complementary mechanisms can contribute to higher levels of excision of AZT, which in turn can amplify resistance to this drug.

High resolution footprinting of the hepatitis C virus polymerase NS5B in complex with RNA.

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.

The "FAD" (Floating Alveolar Device): a bidirectional distraction system for distraction osteogenesis of the alveolar process.

PURPOSE: The following study illustrates preoperative and perioperative vector management in alveolar distraction using a new distraction system--the "Floating Alveolar Device" (FAD). The FAD is a bidirectional alveolar distractor that allows augmentation of an atrophic alveolar process in several planes, assures easy intraoperative positioning of the planned vector of distraction, and provides correction of the horizontal position of the transported segment during and after vertical distraction. PATIENTS AND METHODS: The FAD is composed entirely of stainless steel and has the following basic components: an upper member, a distraction rod, a lower base plate supporting the vertical force of the distraction rod, a jointed hinge that connects the upper and the lower members, and a tightening rod that provides blocking of the hinge. The clinician can manipulate and adjust the tightening rods, allowing a change in the angle of the hinge, thereby altering the transverse dimension of the vector of distraction. A total of 4 patients aged to 19 to 40 years underwent bidirectional alveolar distraction. All procedures were performed in the mandible. RESULTS: In all treated patients, planned distraction height and direction were achieved. In all cases it was possible to place implants at the planned time. CONCLUSIONS: The most common complication, axial displacement, is easily eliminated by moving the bone with the "floating" rod of the FAD during or immediately after the distraction period, according to the principles of the floating bone concept.

Mutations E44D and V118I in the reverse transcriptase of HIV-1 play distinct mechanistic roles in dual resistance to AZT and 3TC.

Retroviral resistance to AZT and 3TC has been associated with two different mechanisms. The M184V mutation in the reverse transcriptase (RT) of the human immunodeficiency virus, type 1 (HIV-1) diminishes the incorporation of 3TC-monophosphate (3TC-MP), whereas AZT resistance-conferring mutations were shown to facilitate the phosphorolytic excision of incorporated AZT-MP in the presence of ATP. Both mechanisms show a certain degree of incompatibility; however, previous clinical data revealed that mutations E44D and V118I, when present in a background of classical AZT mutations (M41L, D67N, L210W, and T215Y), confer dual resistance to AZT and 3TC. We have purified RT enzymes that contain E44D and V118I either alone or in a background of different combinations of AZT mutations to study the underlying biochemical mechanisms. We found that enzymes containing E44D in a background of these latter mutations increase the efficiency of excision of 3TC-MP. Unexpectedly, V118I-containing enzymes show dramatic reductions in rates of incorporation of AZT-MP and 3TC-MP. The V118I mutant is also associated with diminished rates of ATP-dependent primer unblocking. The additional presence of mutations M41L, D67N, L210W, and T215Y can partially neutralize this deficit, which helps to explain the concurrent presence of these changes in resistant isolates. These biochemical data make clear that mutations E44D and V118I play distinct mechanistic roles in dual resistance to AZT and 3TC. Our findings are consistent with an increasing number of clinical studies suggesting that the V118I cluster constitutes a novel pathway for HIV resistance to multiple nucleotide analogue RT inhibitors.