20 research outputs found
Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors
<div><p>Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.</p></div
HCV NS5B polymerase nonnucleoside inhibitors binding sites and NS5B constructs used in studies.
<p>(<b>A</b>) Thumb site I and thumb site II are located on the thumb domain (green); palm site I and palm site II are at the interface of the three domains, thumb, palm (blue) and fingers (red). GS-9669 inhibitor bound in the thumb site II pocket is shown in stick representation (grey, description of crystal structure of NS5B bound to thumb site II inhibitor GS-9669 will be published elsewhere). The active site is indicated by the cyan circle. The other main structural features shown are the C-terminal tail residues (magenta) which contact the β-loop (yellow). (<b>B</b>) 2D representation of domain structure of polymerase and C-terminal truncation sites Δ21, Δ39, Δ47, Δ55, as well as the β-loop deletion mutant Δ21-Δ8 (deleted residues shown in yellow) and LWF triple A mutant F550A/W551A/L553A. Δ55 is a tag free construct and all others contain C6-His. (<b>C</b>) Location of the mutations relative to the tertiary protein structure. (<b>D</b>) Close-up view of interface between LWF motif (magenta, stick representation) and β-loop (yellow) which is dominated by hydrophobic contacts on the surface of the protein.</p
Equilibrium dissociation constants (K<sub>D</sub>) for binding of NNIs to Δ21 and fold-shifts in K<sub>D</sub> for association to Δ55 and Δ21-Δ8 determined by SPR.
<p><sup>a</sup> Sensorgrams for the binding of NNIs to Δ21, Δ55 and Δ21-Δ8 along with a table of equilibrium and kinetic parameters of interaction are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084808#pone.0084808.s002" target="_blank">Figure S2</a> and Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084808#pone.0084808.s006" target="_blank">File S1</a>, respectively.</p><p><sup>b</sup> Fold shift in K<sub>D</sub> for association of NNIs to Δ55 and Δ21-Δ8 was calculated relative to the K<sub>D</sub> determined for binding towards Δ21.</p><p><sup>c</sup> Where K<sub>D</sub> values could not be obtained either due to complex binding kinetics (Palm Site II inhibitor binding to Δ55 and Δ21-Δ8) or super-stoichiometric binding (Palm Site I inhibitor binding to Δ21-Δ8), numerical values have been replaced by n/a (not applicable).</p
IC<sub>50</sub> values for inhibition of RdRp activity of NS5B by Nuc (3′-d′CTP) and NNIs.
<p>Numbers represent mean and standard deviation of IC<sub>50</sub> values determined for each inhibitor against the set of NS5B constructs. Heatmap is colored according to IC<sub>50</sub> fold shift relative to Δ21 to show changes in inhibition profile for given NNI across NS5B mutant constructs. Thumb site II inhibitors begin to show significant loss of inhibitory potency as interactions between β-loop and C-terminal residues are disrupted by mutations in the interface (Δ21-AAA, truncations past Δ39 and Δ21-Δ39 mutations). Palm site I inhibitor is affected as well, which is explained by disruption of the inhibitor's interaction with the β-loop and C-terminal residues. Inhibition by thumb site I remains for the most part unaffected by NS5B mutations whereas inhibition by palm site II NNI is reduced due to decrease in binding to NS5B with truncated β-loop and/or C-terminal.</p
Entry of Tat or R5 Env-VLPs in MDDCs and block by anti-integrin antibodies.
<p>(<b>A</b>) Entry of wt Tat into MDDCs from 3–10 different donors (depending on the anti-integrin mAbs used), in the presence of mAbs against the indicated integrins alone or combined, a control isotype mAb, or nil (buffer). (<b>B</b>) Entry of cys<sub>22</sub> Tat into MDDCs from 3 different donors and block by the combined anti-integrin mAbs versus an Ig control isotype mAb. The percentages of Tat positive cells +/− standard deviations are shown. (<b>C</b>) Entry of VLP-R5Env (BaL) in MDDCs in the presence of Tat and block by anti-integrin mAbs or an Ig control isotype mAb. The percentages of fluorescent cells are shown. A representative experiment out of 4 performed is shown.</p
Tat released by producing T cells increases entry and infection of a Tat-independent replication-incompetent SF162 pseudovirus.
<p>CEMss cells were either infected (CEMss-Tat) or not infected (CEMss) with VSV-G/HIV. After 24 h cells were co-cultured for 4 days with TZM-bl cells in the presence (TZM-bl/VP-SF162) or absence (TZM-bl) of a Tat-independent GFP-expressing single-cycle HIV-1 SF162 virus (VP/SF162), and then GFP expression evaluated by flow cytometry. Results are expressed as MFI fold increase with respect to the TZM-bl + CEMss co-culture (baseline MFI: 1.9).</p
Uptake of trimeric ΔV3 Env by MDDCs and block by anti-integrins mAbs.
<p>Uptake of ΔV2 Env or ΔV3 Env pre-incubated with buffer or increasing concentrations of Tat, in the presence or absence of anti-integrin mAbs (10 µg/mL each) or a control isotype mAb (30 µg/mL). Trimeric ΔV2 Env: circles; trimeric ΔV3 Env: squares. Control isotype Ab: open symbols; anti-integrin blocking mAbs combined: filled symbols.</p
Tat/Env complex and ternary Tat/Env/αvβ3 complex by modeling-docking calculations.
<p>(<b>A</b>) ribbon representation of the Tat/Env complex showing that the Env CD4 binding site and the RGD domain of Tat are both exposed. Color code: ΔV1-2 Env: blue; Tat: red; Tat-RGD: yellow. (<b>B</b>) Surface representation of the ternary Tat/Env/αvβ3 complex. Color code: ΔV1-2 Env: green; Tat: purple; αvβ3 integrin: grey. See experimental procedures for details.</p
Tat/Env binding kinetics and thermodynamics.
<p>(<b>A</b>) Binding kinetics of trimeric wt Env (tw Env), trimeric ΔV2 Env (tΔV2 Env), monomeric wt Env (mwt Env) or monomeric ΔV2 Env (mΔV2 Env) to Tat bound to the Biacore chip. The range of concentrations of Env molecules is indicated. (<b>B</b>) Tat/Env binding thermodynamics by ITC. Tat was titrated with trimeric wt Env. The top panel shows calorimetric data versus time; the lower panel shows integrated areas normalized to the number of moles of Env subunits injected at each injection step.</p
Tat-mediated entry of Env molecules from different clades in MDDCs and block by anti-integrins antibodies.
<p>(<b>A</b>) Clade B trimeric wt Env (twt Env), trimeric ΔV2 Env (tΔV2 Env), monomeric wt Env (mwt Env), or monomeric ΔV2 Env (mΔV2 Env) and (<b>B</b>) clade A and C trimeric wt Env (twt Env) molecules were incubated with control buffer or increasing concentrations of Tat, and then added to MDDCs at 1∶100 final dilution. Cells were then stained for intracellular Env. Open circles: control isotype mAb; filled circles: anti-integrin mAbs directed against the α5β1, αvβ3 and αvβ5 integrins (10 µg/mL each). Data from the same donor out of 3–8 donors tested are shown. Results are expressed as the percentage of Env-positive cells.</p