Genetic and phenotypic changes to Venezuelan equine encephalitis virus following treatment with β-D-N4-hydroxycytidine, an RNA mutagen

Antiviral effect of NHC against VEEV TC-83

To study lethal mutagenesis, we first tested the antiviral effect of two well-known RNA mutagens, ribavirin and NHC in Vero 76 cells (Fig. 1), against VEEV TC-83. The antiviral effect of ribavirin was minimally detected as described by others²⁸ (Fig. 1B); NHC showed a dose-dependent antiviral response in our assay with an EC₅₀ of 0.86 µM and CC₅₀ of 26.2 µM (Fig. 1C), which confirms data published by others .

Collapse of the infectious population by NHC

To see if NHC induces a collapse of the infectious population as its antiviral mechanism, we passaged VEEV TC-83 in the presence of NHC. As a control, brequinar was used, which shows antiviral effect (EC₅₀ = 0.16 µM, See Supplementary data for detail) by interfering with host metabolism (i.e., the pyrimidine synthesis pathway)²⁹. We found that NHC-treated population experienced a collapse after 4 passages in the presence of 10 µM NHC (Fig. 2A). However, virus population exposed to 1 µM NHC or brequinar did not experience such a collapse at the end of the experiment (10 passages). Our results indicate that NHC can induce a population crash; however, a higher concentration than its EC₅₀ might be needed to achieve this effect.

NHC requires multiple rounds of replication for its efficacy

Next, we further sought to understand how induction of mutations by NHC results in replication incompetent progeny virus, ultimately leading to the NHC-mediated population crash. To see if NHC’s antiviral effect requires successive rounds of viral replication, as the mechanism suggests, we tested two MOIs, MOI = 10 for single round of replication and MOI = 0.1 for multiple rounds of replication. We used EGFP expression from a recombinant VEEV TC-83 double reporter strain²⁷ (rVEEV TC-83-dR) as a surrogate for virus replication to evaluate the antiviral effect of NHC following treatment. The EC₅₀ of NHC for rVEEV TC-83-dR, using EGFP expression as the readout, was determined to be 0.52 µM, which is similar to that for non-reporter VEEV TC-83. We found that while 10 µM NHC showed only a 43.0% reduction in reporter signal compared to the control in the single round of replication condition (i.e., high MOI.), the antiviral effect for the second round of replication observed in the low MOI condition was significantly higher (97.9% reduction, n = 6, Fig. 2B). This phenomenon was not detected with ML336, a direct antiviral against VEEV viral RNA synthesis (structure is shown Fig. 1A)³⁰. This result implies that the primary antiviral activity of NHC on VEEV impacts progeny virus more in subsequent rounds of replication than during the replication of the first exposed population, which is consistent with the concept of lethal mutagenesis.

NHC shows a two-phase dose-responsive virus yield reduction efficacy, leaving a replication-competent progeny virus population at a high concentration

Next, we measured the antiviral activity of NHC in a virus yield reduction assay with a broad range of NHC concentrations, ranging from 1 to 50 µM, against TC-83 virus in Vero 76 cells. Overall, NHC treatment yielded a significant reduction in virus titer compared to the mock-treated group, (Fig. 2C) as previously reported by others²¹. However, interestingly, we found that the antiviral effect did not further increase at concentrations greater than 10 µM, resulting in a similar amount of replication-competent progeny virus titer even at 50 µM. In other words, the antiviral effect of NHC was saturated at approximately 10 µM and some fractions of the populations were still able to maintain their infectivity when exposed to higher NHC concentrations. We detected a similar effect when we determined the relative infectivity (RNA copy number / pfu). RNA copy/pfu ratio of virus produced in the presence of NHC increased marginally above 10 µM (Fig. 2D).

This result indicates that NHC has a potent anti-VEEV effect, and the overall quality of viral genomic RNA decreases. However, NHC treatment during a single passage was not able to abrogate replication-competent progeny virus populations even at high concentrations. NHC might need multiple rounds of replication cycles to achieve its full antiviral effect.

Lethal mutagenesis of VEEV TC-83 by NHC. (A) Passage of TC-83 in the presence of compound. Vero76 cells were infected with TC-83 at 0.2 MOI initially and the progeny virus was passaged subsequently until passage 10. Two replicates per treatment and each line of passages is depicted in a connected line. LOD: limit of detection (B) Vero 76 cells grown in 96-well plates were infected with rVEEV TC-83-dR at two MOIs (10 MOI for a single cycle and 0.1 MOI for multi cycle replication) and treated with 50 nM of ML336 or 10 µM of NHC. The expression of EGFP signal was measured at the noted timepoints and normalized to the mean of the DMSO control (0.25%). Each dot represents independent wells. P value from Student’s t-test with n  = 6). (C) Virus yield reduction by NHC. Virus titers of TC-83 cultured in the presence of NHC at the denoted concentrations for 18 h were determined (n  = 3). (D) RNA copy numbers were determined by the real-time PCR method specific to nsP3 region with copy number standards. P  

Concentration-dependent increase of mutations in NHC-treated virus population

To understand how NHC-induced mutations may lead to decreased viral titers in NHC-treated viral populations, we first determined the genomic sequences of the viral genome from the total population using a long-read bulk sequencing of PCR-amplicons covering nsP2-4 region, approximately 6 kb or 50% of the total genome (Fig. 3A. See Methods section for detail). This method allows us to analyze genomic sequences at a single molecule level with greater accuracy (Q43, > 99.99%) and phasing than conventional short-read bulk sequencing (e.g., Illumina-based sequencing) without additional methodological needs.

The analysis showed a significant increase in mutation frequency due to NHC treatment. The mock-treated group (DMSO-treated) was determined to have a mutation-frequency of 1.30 × 10⁻⁴ s/n (n = 3). The increase in mutation frequency due to NHC treatment was concentration dependent. The mutation frequencies increased to 5.60 and 26.06 (10⁻⁴ s/n) for 1 and 10 µM NHC-treated populations, respectively, which is an approximately 4 and 20-fold increase compared to the DMSO control. The mutation frequency increased further in the 50 µM NHC treated population with 32.65 (10⁻⁴ s/n, or 32.65-fold increase compared to the DMSO control (Fig. 3B). Our data showed that NHC is an effective RNA mutagen for VEEV and induces ~ 8-fold increase during a single passage at a concentration that induced a population collapse in subsequent passages (i.e., 10 µM).

NHC revealed a mutational threshold for the replication-competent VEEV population

Interestingly, the virus yield reduction experiment (Figs. 2C and 3C) indicated the presence of a residual population in the progeny virus that is replication-competent (evidenced by plaque formation) after a single passage. We sought to better characterize the genetic landscape within this replication-competent population, apart from the total population, as the replication-competent population establishes the progeny virus in the next round of replication under the treatment of NHC. To address this, we developed a consensus sequence-based sequencing approach using a limiting dilution of virus in cell plates (Single Infectious Unit or SIU sequencing hereafter). Our SIU approach only determines viral sequences of populations that can replicate at the individual infectious unit level (Fig. 3A). Because the final sequences are deduced from the consensus sequences in the clone, errors during sequencing processes can be minimized for analysis.

We found that the mutation frequency profile of replication-competent populations (Fig. 3B) differed from that of the total population. The maximum mutation frequency of the replication-competent populations reached approximately 6.3 × 10⁻⁴ s/n, plateauing at around 10 µM of NHC, without a significant increase beyond 20 µM. This is a striking difference from the total population, which displayed a concentration-dependent increase in mutation frequency. (p −4 s/n, and this threshold can be reached following a single round of replication with 10 µM NHC treatment.

Mutations induced by NHC. (A) The overall pipeline to detect mutations in total and replication-competent single infectious units. (B) Mutation frequency of the total (blue) and infectious (red) progeny virus of VEEV TC-83 treated with NHC in Vero 76 cells. Each circle represents one biological sample ( n  = 3 ~ 6). (C) Analysis between the mutation frequency and their titers of progeny virus cultured in the presence of various concentrations of NHC. Each circle and error bar represents means ± stdev from three replicates and the line represents the medians.

Characterization of mutations induced by NHC in the replication-competent population

To understand more precisely how the mutations are distributed within the replication-competent population, we analyzed the mutations in each isolate (Fig. 4, and Table 1). The median number of mutations within the 5.9 kb amplicon examined from the 1 µM NHC-treated infectious population increased to 2, compared to 0 for the DMSO-treated group. When treated with at least 10 µM of NHC, the change in the distribution of the number of mutations in the amplicon was more obvious, especially for the strands with higher number of mutations within the amplicon. For example, while only 6.8% of the 1 µM-NHC treated SIUs had more than 5 mutations, more than 30% of the 10 µM NHC-treated SIUs had more than 5 mutations in the same region.

We also found that significant fractions of replication-competent viruses had a low number of mutations, even when treated with a high concentration of NHC. Within the 1, 10, and 50µM-treated groups, we found that 63.6%, 44.3%, and 45.5% of SIUs in those groups, respectively, contained fewer than 2 mutations within the sequenced amplicon. Particularly, more than 12.5% of the replication-competent population was mutation-free within the amplicon in all NHC-treated groups (Table 1). This may indicate that progeny virus with a lower number of mutations, albeit a small fraction of the population, can be produced even at a high NHC concentration, supporting the need for multiple rounds of replication to reach a population collapse (Fig. 2A).

An analysis of mutations identified in the infectious population (Fig. 4) showed all four types of mutations that can be induced by NHC²⁴, indicating the incorporation of NHC into viral RNA might have happened during both (-)-sense RNA and progeny genomic RNA synthesis. Unlike the study using MERS-CoV, which found an equal preference of A-to-G or U-to-C mutations incorporated into in the viral genome by NHC²⁴, the majority of mutations induced by NHC in VEEV were T-to-C (U-to-C in the viral genome), indicating the incorporation of the NHC occurred during the synthesis of genomic RNA across adenosine (Fig. 4B). We did not find any specific mutational hot spots within the amplicon (Fig. 4C), which indicates that (1) the incorporation of NHC occurred randomly, and (2) no NHC-resistant populations with mutations within nsP4, the viral RdRP gene, were selected during the treatment.

Effect of mutations induced by NHC on viral replication. (A) Number of mutations detected in nsP2-nsP4 amplicons (5.8 kb) of replication-competent progeny viruses. Replication competent progeny virus harvested from NHC treated cells was individually cultured and its nsP2-nsP4 sequences were determined through the S.I.U. procedure. Each circle represents a single infectious unit and thick lines represent the median from > 48 isolates (B) Analysis of mutation types detected in the infectious population treated with NHC. The blue color scale bar represents the frequency of substitutions per 10,000 n.t. ( n  = 3). (C) Frequency and location of mutations identified in NHC-conditioned virus isolates. N indicates number of replication competent isolates analyzed in the study.

NHC-treatment resulted in a population with a negatively-skewed, diversified fitness

Based on our finding that NHC-treated VEEV has a minor replication-competent population with increased sequence diversity, we sought to measure the extent of growth defects resulting from those mutations. To test this, we established a viral growth kinetic assay for single infectious units using rVEEV TC-83-dR, which, as stated previously, allows us to use the expression of the EGFP as a surrogate for viral replication. To evaluate growth variations, we determined the maximum growth rate (gr, the maximum slope of EGFP expression) and the Area Under the Curve (AUC) of the EGFP expression from individual wells (Fig. 5A) infected with virus at 0.5 TCID50/well. Under this condition, approximately 20–30% of wells were positive for viral infection, implying that most virus-positive wells began with a single infectious unit. We found that NHC-treated TC-83 virus population showed a significant decrease in viral replication with a negative skewing with respect to gr and AUC in the histogram analysis (Fig. 5B and C). For example, the growth rate and AUC of the NHC 10 µM-treated group was reduced by 20% compared to the mock group. Inversely, other time-associated kinetic parameters such as the lag time (the time intercept between the regression line associated with the gr calculation and the baseline defined by the first point in the calculation zone, defined as ‘LagC’) and time at maximum growth rate (t_gr) were increased, indicating a delayed replication of the NHC-treated populations compared to the mock-treated group (Supportive Fig. 2). At the individual level, however, approximately 44–55% of the single isolates from NHC-conditioned virus exhibited growth rates similar to the untreated group (mean ± 1 x S.D.).

These results demonstrate that even though NHC treatment did not result in an extinction of virus within a single round of treatment, the minor replication-competent population produced from treatment with NHC has a negatively skewed, yet diversified growth rate.

Phenotypic effect of mutations induced by NHC on viral replication. Cells plated in a 384-well plate were infected with rVEEV TC-83-dR at 0.5 TCID50/well and the expression of EGFP was monitored in a plate reader with a live culture mode. (A) A representative growth curve of rVEEV TC-83-dR shown for demonstration of growth parameters. Distribution of growth rate (gr) (B) and AUC (C) of mock- or NHC- treated infectious populations. P values were from one-way ANOVA test with Brown-Forsythe method. (n  = 63,39, and 22 for the mock, 10, and 50 µM NHC treated groups.) Dotted and solid lines represent the median and top and bottom 25% percentile in each group and the shaded areas indicate mean ± 1 x S.D from the mean of the mock-treated group.