Lifecycle modelling systems support inosine monophosphate dehydrogenase (IMPDH) as a pro-viral factor and antiviral target for New World arenaviruses

Abstract

Infection with Junín virus (JUNV) is currently being effectively managed in the endemic region using a combination of targeted vaccination and plasma therapy. However, the long-term sustainability of plasma therapy is unclear and similar resources are not available for other New World arenaviruses. As a result, there has been renewed interest regarding the potential of drug-based therapies. To facilitate work on this issue, we present the establishment and subsequent optimization of a JUNV minigenome system to a degree suitable for high-throughput miniaturization, thereby providing a screening platform focused solely on factors affecting RNA synthesis. Using this tool, we conducted a limited drug library screen and identified AVN-944, a non-competitive inosine monophosphate dehydrogenase (IMPDH) inhibitor, as an inhibitor of arenavirus RNA synthesis. We further developed a transcription and replication competent virus-like particle (trVLP) system based on these minigenomes and used it to screen siRNAs against IMPDH, verifying its role in supporting arenavirus RNA synthesis. The antiviral effect of AVN-944, as well as siRNA inhibition, on JUNV RNA synthesis supports that, despite playing only a minor role in the activity of ribavirin, exclusive IMPDH inhibitors may indeed have significant therapeutic potential for use against New World arenaviruses. Finally, we confirmed that AVN-944 is also active against arenavirus infection in cell culture, supporting the suitability of arenavirus lifecycle modelling systems as tools for the screening and identification, as well as the mechanistic characterization, of novel antiviral compounds.

Keywords: AVN-944; Arenavirus; Inosine monophosphate dehydrogenase (IMPDH); Life cycle modelling systems.

Fig. 1.. Establishment of Junín virus (JUNV) minigenome systems.

(A) Schematic diagram of JUNV minigenome plasmid construction. Minigenomes were based on the S-segment of the JUNV (strain Romero) cloned in cRNA orientation into the pAmp expression vector, which contains a T7 promoter, as well as a Hepatitis Delta Virus (HDV) ribozyme and T7 terminator elements, as indicated. The open reading frames encoding the NP and GP were then excised leaving a unique cloning cassette in each location. Subsequently, either NanoLuciferase (nLuc) or green-fluorescent protein (GFP) was inserted in place of the GPC gene to generate the final minigenome constructs. (B) Schematic diagram of the JUNV minigenome system. Transfection of the minigenomes described in (A), along with pCAGGS constructs encoding the T7 polymerase, JUNV nucleoprotein (NP) and polymerase (L), leads to transcription of these three proteins by RNA polymerase II and subsequent translation. T7 then directs transcription of the minigenome RNA, which is autocatalytically processed by HDV ribozyme cleavage to generate a construct containing authentic JUNV leader and trailer sequences. This genome analogue can then be encapsidated by NP and transcribed and replicated by L. The transcribed minigenome mRNA from the GPC gene encodes an assayable reporter protein, in this case either nLuc or GFP, and the production of this reporter reflects the cumulative viral RNA synthesis (i.e. transcription and replication) taking place in these cells. (C) JUNV minigenome assay for detection of NanoLuciferase expression. The indicated cell lines were transfected as described in (B) with a monocistronic JUNV minigenome encoding nLuc as well as support plasmids, either with or without JUNV L. A control plasmid pCAGGS-Firefly (FF) was also transfected. Cells were harvested 48 h later and measured for both nLuc (viral RNA synthesis) and FF (host cell RNA synthesis) activity. The means and standard deviations of normalized reporter levels (nLuc/FF) are shown and represent data from three independent experiments. The results of a one-way ANOVA analysis to compare sample pairs with and without L are indicated. (D) JUNV minigenome assay for detection of GFP expression. Cells were transfected as described in (C) but using a GFP-expressing minigenome and omitting the pCAGGS-FF control plasmid. Samples were prepared either without (top panel) or with (bottom panel) JUNV L and fluorescence was examined after 48 h.

Fig. 2.. Minigenome optimization, scale-up, and comparison of intracellular and secreted reporter luciferase measurement.

(A) Schematic overview of decoy open reading frame (ORF) location and orientation. Constructs based on the NanoLuciferase (nLuc)-expressing minigenome were generated that encoded an additional promoter-less green fluorescent protein (GFP) in either a forward (5′-3’; right, R) or reverse (3′-5’; left, L) orientation at one or both of two insertion sites located either after the T7 terminator (site 1) or between the HDV ribozyme and the T7 terminator elements (site 2). (B) Reporter activity of decoy ORF-containing minigenome constructs in 6-well format. The decoy ORF-encoding minigenome plasmids described in (A), or the original unmodified construct (−) were transfected into BSR-T7/5 cells along with support plasmids and either with or without JUNV L. A control plasmid, either pCAGGS-Firefly (FF) or pCAGGSCypridina (Cyp), was also transfected. Cells lysates (left panel) were measured for nLuc (viral RNA synthesis) and FF (host cell RNA synthesis), while supernatants (right panel) were measured 48 h later for nLuc (viral RNA synthesis) and Cyp (host cell RNA synthesis) expression. The means and standard deviations of normalized reporter levels [nLuc/FF (lysates) or nLuc/Cyp (supernatants)] are shown and represent data from three independent experiments. Dynamic range [DR: < 2 (red)/2–3 (orange)/> 3 (green)], separation band [SB: < 1(red)/1–2(orange)/> 2(green)] and Z factor [Z’: < 0.2(red)/.2–0.5(orange)/> 0.5(green)] are shown as measures of assay performance. (C) Reporter activity of decoy ORF-containing minigenome constructs in 96-well format. Transfected plasmids amounts were reduced uniformly to account for the smaller surface areas in a 96-well plate, but experiments were otherwise performed and analyzed as described in (B). The means and standard deviations shown represent data for 12 biological replicates from 2 independent experiments.

Fig. 3.. Antiviral compound screening using the JUNV minigenome system.

(A) Schematic of the antiviral compound screening workflow. BSR-T7/5 cells were treated with a small set of 34 drugs, in addition to Ribavirin (RIB) as a control, for 2 h. Subsequently, these cells were transfected with the optimized 1L2L decoy-containing JUNV nLuc-expressing minigenome and helper plasmids in the continued presence of drug. The control plasmid pCAGGS-Firefly (FF) was also transfected. Cell lysates were measured 48 h later for nLuc (viral RNA synthesis) and FF (host cell RNA synthesis). (B) Drug library screening results. Each compound to be tested was applied in three different concentrations (0.1 μM, 1 μM, and 10 μM) according to the workflow described in (A) with 0.02%, 0.2% and 2% DMSO used as controls. The effects of treatment on viral RNA synthesis (nLuc) and cellular RNA synthesis (FF) are shown separately as means and standard deviations representing data from six biological replicates from two independent experiments. (C) Analysis of minigenome inhibition at additional AVN-944 and Ribavirin concentrations. BSR-T7/5 cells were treated with the indicated concentrations of AVN-944 or Ribavirin, DMSO alone, or left untreated for 2 h. Subsequently, these cells were transfected with the optimized 1L2L decoy-containing JUNV nLuc-expressing minigenome and helper plasmids. Untreated controls were run either with or without JUNV L, as indicated. Cell lysates were measured 48 h later for nLuc activity (viral RNA synthesis, left panel), FF activity (cellular RNA synthesis; right panel). Cell viability was also measured based on cellular ATP levels (both panels). Means and standard deviations shown represent at least 4 biological replicates from at least 2 independent experiments. Results of a one-way ANOVA to compare each sample to the DMSO control are indicated when statistically significant. Comparison of AVN-944 (1.6 μM) and Ribavirin (400 μM) treatments are also indicated.

Fig. 4.. Development of a JUNV trVLP assay and testing of siRNA-mediated IMPDH knock-down.

(A) Schematic diagram of the JUNV trVLP system. Transfection of producer cells (p0 cells) with the optimized 1L2L decoy-containing JUNV nLuc-expressing minigenome and helper plasmids encoding the T7 polymerase, JUNV nucleoprotein (NP) and polymerase (L) leads to transcription of these three proteins by RNA polymerase II and subsequent translation. T7 then directs initial transcription of the minigenome RNA, which is autocatalytically processed by HDV ribozyme cleavage to generate a construct containing authentic JUNV leader and trailer sequences. This genome analogue can then be encapsidated by NP and transcribed and replicated by L. The transcribed mRNA from the GPC gene encodes the assayable nLuc reporter protein. The production of reporter in these p0 cells reflects the cumulative RNA synthesis (i.e. transcription and replication) taking place in these cells. An additional second transfection of plasmids encoding the JUNV glycoprotein precursor (GPC) and matrix protein (Z) results in inhibition of further RNA synthesis and budding of transcription and replication competent virus-like particles (trVLPs) that have a structure analogous to actual virus particles, but instead contain only a minigenome as their genetic material. These particles can be used to infect new target cells (p1 cells), which have been pretransfected with pCAGGS-NP and pCAGGS-L, and thus are able to further transcribe and replicate the viral minigenome. As such, if used in drug/siRNA treatment experiments, reporter activity in these p1 cells reflects the cumulative effects of treatment on RNA synthesis (in p0 and p1 cells), trVLP production and trVLP entry. Alternatively, if only the p1 cells are treated, the effects reflect changes in trVLP entry and subsequent p1 cell RNA synthesis only. (B) JUNV trVLP assay for detection of nLuc expression. BSR-T7/5 cells (p0 cells) were transfected as described in (A) either with (+L) or without JUNV L (−L). A control plasmid pCAGGSFirefly (FF) was also transfected in all cells. After 24 h cells were further transfected with pCAGGS-Z and pCAGGS-GPC (+Z, +GP), or with pCAGGS as a control (−Z, −GP). These p0 cells were harvested 48 h later and measured for both nLuc (viral RNA synthesis) and FF (host cell RNA synthesis) activity. In addition, the supernatants from these p0 cells were transferred onto the indicated cell lines, which had been pre-transfected with pCAGGS-NP and pCAGGS-L (p1 cells) as described in (A). After 48 h the p1 cells were harvested and measured for nLuc (viral RNA synthesis). The means and standard deviations of normalized reporter levels (nLuc/FF, p0 cells) or nLuc activity alone (p1 cells) are shown and represent data from two independent experiments. Results of a one-way ANOVA to compare p0 samples or p1 samples either with or without Z and GP among the different cell types tested are shown. (C) Schematic of the siRNA analysis workflow. Huh7 cells were first transfected with siRNAs for 24 h, after which they were further transfected with pCAGGS-NP and pCAGGS-L helper plasmids. After 24 h the cells were infected with trVLPs containing supernatants produced as described in (A) and (B). After an additional 48 h cell lysates were measured for nLuc (viral RNA synthesis) activity. (D) Cells were transfected with 2 pmol of commercially available validated siRNAs against IMPDH2 (Supplemental Fig. 1), or positive and negative control siRNAs, according to the workflow described in (A). The effects of siRNA treatments on viral RNA synthesis (nLuc) in p1 cells are shown as means and standard deviations of three independent experiments. Results of a one-way ANOVA to compare each sample to the untreated control are shown.

Fig. 5.. Validation of AVN-944 efficacy during in vitro infection with Tacaribe virus.

(A) Assessment of AVN-944 inhibition at various doses. Vero76 cells were infected with Tacaribe virus for 1 h at an MOI of 0.01 after which they were treated with medium containing increasing doses of AVN-944, as indicated, or DMSO (as a control). After 48 h supernatants were harvested for titer determination by TCID50 and cells were analyzed for cytotoxicity based on cellular ATP levels. Titers were calculated using the Spearman-Kärber method and data are shown as the means and standard deviations of 9 biological replicates from 3 independent experiments. Results of a one-way ANOVA to compare each sample to the DMSO control are shown when statistically significant (B) Comparison of AVN-944 efficacy and cytotoxicity to Ribavirin. Vero76 cells were infected as described in (A) before being treated with DMSO (0.33%), AVN-944 (1.6 μM or 7.5 μM) or Ribavirin (400 μM), as indicated. After 48 h supernatants were harvested for titer determination by TCID50 and cells were analyzed for cytotoxicity, also as described in (A). Data are shown as the means and standard deviations of at least 12 biological replicates from at least 4 independent experiments. Results of a one-way ANOVA to compare each sample to the DMSO control are shown when statistically significant.