Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors.

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 µM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 µM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

Dolutegravir inhibits HIV-1 Env evolution in primary human cells.

OBJECTIVE: In treatment-naive HIV-positive individuals, the integrase strand-transfer inhibitor dolutegravir (DTG) has not been associated with emergent drug-resistance mutations, neither against this drug nor against other antiretroviral drugs that were used in combination with it. This is in contrast to all other antiretroviral drugs tested so far, including the integrase strand-transfer inhibitors raltegravir (RAL) and elvitegravir that can lead to treatment failure with the emergence of drug-resistance mutations. DESIGN: These observations suggest that DTG may provide an additional protection against resistance compared to other drugs by decreasing HIV-1 genetic evolution. METHODS: Here, we tested this hypothesis by measuring the genetic and amino-acid diversity of Env/gp160 from two HIV-1 primary isolates that were grown in the presence of increasing concentrations of DTG or RAL over the course of 38-55 weeks. RESULTS: The results show that treatment with DTG led to less HIV-1 genetic and amino-acid diversification over time, as compared to treatment with RAL or the absence of drug. CONCLUSION: These results may help to explain the absence of emergent resistance mutations in treatment-naive individuals treated with DTG.

Distinct resistance patterns to etravirine and rilpivirine in viruses containing nonnucleoside reverse transcriptase inhibitor mutations at baseline.

OBJECTIVE: The current in-vitro study examined HIV-1 drug resistance patterns following etravirine (ETR) and rilpivirine (RPV) drug pressure in viruses containing baseline nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations. DESIGN AND METHOD: Several baseline mutations were introduced into NL-4.3 (subtype B clone) by site-directed mutagenesis. This virus, together with two subtype C clinical isolates containing baseline mutations, was passaged in increasing drug pressure of NNRTIs in cord blood mononuclear cells. Genotypic analysis was performed at different weeks. Phenotypic resistance for ETR, RPV, and efavirenz (EFV) and the replication capacity of several mutations and combinations were tested. RESULTS: In wild-type viruses and viruses containing K103N alone at baseline, E138K or E138G mutations were observed following pressure with either ETR or RPV prior to the appearance of other NNRTI resistance mutations. These changes were observed regardless of viral subtype. However, subtype B viruses containing Y181C generated V179I/F or A62V/A but not E138K following exposure to ETR or RPV, respectively, whereas subtype C viruses containing Y181C developed E138V together with Y188H and V179I under ETR pressure. The addition of mutations at position 138 to Y181C did not significantly enhance levels of resistance to ETR or RPV. The replicative capacity of viruses containing Y181C and either E138K or E138A was similar to that of viruses containing either E138K or E138A alone. CONCLUSION: These results demonstrate that ETR and RPV are likely to select for E138K as a major resistance mutation if no or very few other resistance mutations are present and that Y181C may be antagonistic to E138K.

[L'usage de papier buvard de type « FTATM Classic ¼ pour la détection et le génotypage du VIH chez des hommes ayant des relations sexuelles avec d'autres hommes à Montréal (Québec, Canada)]. / L'usage de papier buvard de type « FTATM Classic ¼ pour la détection et le génotypage du VIH chez des hommes ayant des relations sexuelles avec d'autres hommes à Montréal (Québec, Canada).

As part of a program for HIV-1 detection in the gay community of Montreal, blood sampling on "FTA Classic" papers (DBS "dried blood spot") has been tested and validated. It turns out to be sensitive (up to 1 000 copies of proviral DNA/mL) and reliable. Thus, this approach should be considered when a blood sampling is subject to various constraints.