Active Viremia in Rotavirus-Infected Mice

Rotavirus circulates extraintestinally in animals used as models for rotavirus infection and in children. Rotavirus infection in mice was used to define host or viral factors that affect rotavirus viremia. Antigenemia was observed with homologous and heterologous rotaviruses, and neither age nor mouse strain genetics altered the occurrence of rotavirus antigenemia or viremia. Rotavirus RNA and infectious virus were present in sera and associated with the plasma fraction of blood in all infected mice. These findings indicate that antigenemia/viremia occurs routinely in rotavirus infections and imply that infectious rotavirus has access to any extraintestinal cell within contact of blood

Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase▿

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain

Antiviral nucleotide incorporation by recombinant human mitochondrial RNA polymerase is predictive of increased in vivo mitochondrial toxicity risk

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2′-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 μM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 μM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, β, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated

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_D) for binding of NNIs to Δ21 and fold-shifts in K_D for association to Δ55 and Δ21-Δ8 determined by SPR.

<p>^a 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>^b Fold shift in K_D for association of NNIs to Δ55 and Δ21-Δ8 was calculated relative to the K_D determined for binding towards Δ21.</p><p>^c Where K_D 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

IC₅₀ values for inhibition of RdRp activity of NS5B by Nuc (3′-d′CTP) and NNIs.

<p>Numbers represent mean and standard deviation of IC₅₀ values determined for each inhibitor against the set of NS5B constructs. Heatmap is colored according to IC₅₀ 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