A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein

Abstract

Although a highly effective vaccine is available, the number of yellow fever cases has increased over the past 2 decades, which highlights the pressing need for antiviral therapeutics. In a high-throughput screening campaign, we identified an acetic acid benzodiazepine (BDAA) compound which potently inhibits yellow fever virus (YFV). Interestingly, while treatment of YFV-infected cultures with 2 μM BDAA reduced the virion production by greater than 2 logs, the compound was not active against 21 other viruses from 14 different viral families. Selection and genetic analysis of drug-resistant viruses revealed that replacement of the proline at amino acid 219 (P219) of the nonstructural protein 4B (NS4B) with serine, threonine, or alanine conferred YFV with resistance to BDAA without apparent loss of replication fitness in cultured mammalian cells. However, replacement of P219 with glycine conferred BDAA resistance with significant loss of replication ability. Bioinformatics analysis predicts that the P219 amino acid is localized at the endoplasmic reticulum lumen side of the fifth putative transmembrane domain of NS4B, and the mutation may render the viral protein incapable of interacting with BDAA. Our studies thus revealed an important role and the structural basis for the NS4B protein in supporting YFV replication. Moreover, in YFV-infected hamsters, oral administration of BDAA protected 90% of the animals from death, significantly reduced viral load by greater than 2 logs, and attenuated virus infection-induced liver injury and body weight loss. The encouraging preclinical results thus warrant further development of BDAA or its derivatives as antiviral agents to treat yellow fever. IMPORTANCE Yellow fever is an acute viral hemorrhagic disease which threatens approximately 1 billion people living in tropical areas of Africa and Latin America. Although a highly effective yellow fever vaccine has been available for more than 7 decades, the low vaccination rate fails to prevent outbreaks in at-risk regions. It has been estimated that up to 1.7 million YFV infections occur in Africa each year, resulting in 29,000 to 60,000 deaths. Thus far, there is no specific antiviral treatment for yellow fever. To cope with this medical challenge, we identified a benzodiazepine compound that selectively inhibits YFV by targeting the viral NS4B protein. To our knowledge, this is the first report demonstrating in vivo safety and antiviral efficacy of a YFV NS4B inhibitor in an animal model. We have thus reached a critical milestone toward the development of specific antiviral therapeutics for clinical management of yellow fever.

FIG 1

In vitro antiviral activity of BDAA against YFV. (A) Structure of BDAA. (B) Huh7.5 cells seeded in 96-well plates were infected with YFV strain 17D at an MOI of 0.1 for 1 h, followed by treatment with the indicated concentrations of BDAA for 48 h. YFV E protein was visualized by In-Cell immunostaining (green). Cell viability was determined using DRAQ 5 and Sapphire 700 stains (red). (C and D) Huh7.5 cells seeded in 96-well plates were infected with YFV at an MOI of 0.01 for 1 h, followed by treatment with the indicated concentrations of BDAA for 48 h. Total intracellular RNAs were extracted to determine the amount of YFV RNA by qRT-PCR. YFV RNAs were normalized to β-actin mRNA and presented as the percentage of mock treated control (C). Data represented the mean results from four independent replicates (± standard deviations). Culture media were harvested from infected cells treated with various concentrations of BDAA to determine the virus titer in a plaque assay in Vero cells (D). Values represent average results from a triplicate experiment (± standard deviations). EC₅₀, EC₉₀, and CC₅₀ values were calculated using Prism 5 (GraphPad Software, Inc.).

FIG 2

Antiviral activity of BDAA enantiomers. (A) Structures of BDAA and its enantiomers. (B) Determination of the antiviral activities of BDAA and its enantiomers from a qRT-PCR assay. Huh7.5 cells were cultured and treated as described for Fig. 1C. YFV RNAs were normalized to β-actin mRNA levels and are presented as percentages of mock-treated control results. Values represent the mean results from four independent replicates (± standard deviations). EC₅₀ and EC₉₀ values were calculated using Prism 5 (GraphPad Software, Inc.).

FIG 3

The anti-YFV and psychiatric activities of benzodiazepines require distinct structural features. (A) Structures of six representative clinically psychoactive benzodiazepines. (B) Determination of the antiviral activities of clinically used benzodiazepines based on a qRT-PCR assay. Huh7.5 cells were cultured and treated as described for Fig. 1C. YFV RNA levels were normalized to β-actin mRNA levels and are presented as the percentage of the mock-treated control values. Values represent the mean results from four independent replicates (± standard deviations). (C) The affinities of BDAA to central and peripheral nervous system benzodiazepine receptors were measured in radioligand competitive binding assays. The binding of [³H]flunitrazepam and [³H]PK11195 to the corresponding benzodiazepine receptors was measured in the presence or absence of 10 μM BDAA, and the effects of BDAA on radioligand banding are expressed as the percentage of the mock-treatment control. The reference compounds diazepam and nonlabeled PK11195 with known benzodiazepine receptor binding activities were tested at 0.3 μM. Values represent the mean results from duplicate experiments (± standard deviations).

FIG 4

Time-of-addition analysis of BDAA antiviral activity against YFV. Huh7.5 cells were infected with YFV at an MOI of 10 for 1 h. BDAA (5 μM) was present either transiently for 2 h before infection (−2 to 0), during infection (0, 1), or added at the indicated time points until 48 h postinfection. For each time point, 1% DMSO was added to mock-treated controls. Total intracellular RNAs were extracted, and culture media samples were collected at 48 h postinfection to determine the amounts of YFV RNA in a qRT-PCR assay (A) and virus titers were determinedin a plaque assay (B). YFV RNAs were normalized to β-actin mRNA levels. The graph denotes the percentages of YFV RNA relative to that of the mock-treated control added at the time of infection (time point 0). Virus titers in the culture media were measured by plaque assay in Vero cells. Each data point is the mean results from four independent replicates (± standard deviation).

FIG 5

Selection and characterization of BDAA-resistant YFV. (A) Schematic representation of the procedure to select BDAA-resistant YFV. At the 18th passage, CPE was observed in the culture treated with BDAA. The culture medium was harvested as a BDAA-resistant YFV stock. (B) Effects of BDAA on parental YFV 17D and BDAA-resistant virus were determined in a virus yield reduction assay. Values represent average results from two independent wells with duplication for virus titer titration. EC₅₀ and EC₉₀ values were calculated using Prism 5 (GraphPad Software, Inc.). (C) Alignment of the amino acid sequence flanking the putative NS4B protein transmembrane domain 5 (pTMD5) of YFV and other flaviviruses. The predicted membrane topology of YFV NS4B is shown at the top of amino acid sequence. Amino acid P219 of YFV is highlighted. GenBank accession numbers for the listed viruses are as follows: YFV strain 17D (X03700.1), YFV strain BeH655417 (JF912190.1), DENV-1 Western Pacific strain (U88536.1), DENV-2 New Guinea C strain (AF038403.1), DENV-3 H87 strain (KU050695.1), DENV-4 rDEN4 clone (AF326825.1), WNV NY99 (NC_009942.1), JEV JaOArS982 strain (M18370.1), and ZIKV strain BeH819966 (KU365779.1). The amino acid position of NS4B is numbered according to that for YFV 17D.

FIG 6

Antiviral activity of BDAA against replication-competent recombinant YFV with P219 mutations in Huh7.5 cells. (A and B) Replication kinetics of recombinant viruses with different P219 mutations. Huh7.5 cells were infected at an MOI of 0.1 for 1 h. Total cellular RNAs were extracted, and the culture media were collected from cells at the indicated time points postinfection. YFV RNA was detected by a qRT-PCR assay and normalized to β-actin mRNA levels. The YFV RNA arbitrary units are the amount relative to that of the RNA level in uninfected cells (A). Supernatants from the cultures were used to determine the virus yields in a plaque assay (right panel). Data presented are mean results from two independent replicates with duplication in the plaque assay (± standard deviations) (B). (C and D) Sensitivity of recombinant viruses to BDAA. Huh7.5 cells were infected with YFV 17D or mutant virus at an MOI of 0.1 and mock treated or treated with indicated concentrations of BDAA for 48 h. Total cellular RNAs were extracted to detect YFV RNA in a qRT-PCR assay, using β-actin as an internal control. Values are the percentage relative to mock-treated controls (C). Supernatants from the cultures were harvested to determine the virus yields in a plaque assay in Vero cells (D). Data are mean results from four independent replicate experiments (± standard deviation).

FIG 7

Antiviral activity of BDAA against recombinant YFV with a P219G mutation in Huh7.5 cells. (A to C) Replication kinetics of P219G recombinant YFV. Huh7.5 cells were infected with cell-associated viruses harvested from YFV 17D or P219G RNA-transfected Huh7.5 cells at an MOI of 0.1 for 1 h. Intracellular viral RNA (A) and yields of cell-associated virions (B) and secreted infectious virions (C) at the indicated times postinfection were quantified in a qRT-PCR assay and plaque assay, respectively. YFV RNA was normalized as described for Fig. 6A. (D and E) Sensitivity of YFV P219G to BDAA. Huh7.5 cells were infected with cell-associated YFV 17D or P219G YFV at an MOI of 0.1 and mock treated or treated with the indicated concentrations of BDAA for 48 h. Total cellular RNA was extracted to detect YFV RNA by qRT-PCR using β-actin as an internal control. Values are percentages relative to mock-treated controls (D). Supernatants from the cultures were harvested to determine the virus yields in a plaque assay in Vero cells (E). Data presented are means from four independent replicate experiments (± standard deviation).

FIG 8

Sensitivity of YFV to BDAA in C6/C36 cells. C6/C36 cells were infected with YFV 17D or mutant virus at an MOI of 0.1 and mock treated or treated with indicated concentrations of BDAA for 48 h. Total cellular RNAs were extracted to detect YFV RNA in a qRT-PCR assay, using Aedes albopictus S7 ribosomal mRNA as the internal control. Values are percentages relative to mock-treated controls. Data presented are means from four independent replicates (± standard deviation).

FIG 9

In vivo efficacy of BDAA in a hamster model of lethal YFV infection. Female Syrian golden hamsters were challenged with Jimenez, a hamster-adapted YFV strain, via i.p. injection (10 CC₅₀/animal). Three groups of infected animals were treated with escalated doses of BDAA twice daily (BID) via oral administration for 7 days, beginning 4 h prior to infection. Animals treated with ribavirin served as positive controls, and a placebo-treated group served as a negative control. Each infected group had 10 animals. Sham-infected controls were either treated with the highest dose of BDAA to monitor toxicity or placebo treated (n = 5). A group of 5 animals without any treatment served as normal controls. (A) The effect of treatment on mortality of hamsters infected with YFV. *, P < 0.05 compared with placebo. (B) Weight change between 3 and 6 dpi from hamsters infected with YFV and treated with various doses of BDAA or with ribavirin. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (compared with placebo). (C) The effect of BDAA at various doses or ribavirin on serum ALT at 6 dpi from hamsters infected with YFV. ***, P < 0.001; **, P < 0.01. (D) Virus titers in serum on day 4 of YFV-infected hamsters treated with various doses of BDAA or with ribavirin. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 10

Flavivirus NS4B membrane topology and locations of drug-resistant amino acid substitutions.