Early Intervention in Herpes Simplex Virus-1 Replication in Vitro with Allenic Macrolide Archangiumide

Abstract

Archangiumide is a unique macrolide natural product that features an endocyclic allene functionality, rendering it a prototype of a new class of secondary metabolites of microbial origin. However, its biological and/or pharmaceutical roles remain obscure. In this study, we have unveiled an antiviral potency of archangiumide that was effective against herpes simplex virus (HSV-1) replication. We found that archangiumide did not affect host cell viability, nor pathogen infectivity, but suppressed HSV-1 early replication, in terms of early replication genes, such as ICP0, ICP4, etc. Further scrutinizing the underlined master regulator, we found that HSV-1 VP16 protein expression was inhibited by archangiumide, as well as VP16 nuclear translocation. As VP16 is a coactivator of transcription, archangiumide harnessed the master regulator of HSV-1 early replication. Together, here we provide evidence that allene macrolide archangiumide possesses robust antiviral functions that may be valuable for a novel viral infection intervention, as macrolides are generally safe drugs for prolonged treatments.

Keywords: HSV-1; antiviral; archangiumide.

Figure 1

Archangiumide suppressed HSV-1 replication. (a,b) Archangiumide was added to HSV-1-eGFP virus-infected HeLa cells (MOI = 0.5) and cultured for 24 h. Sham infection was performed as control (Mock). GFP signal was captured (a). Fluorescent images were in 200× magnification. HSV-1 titer (TCID50/mL) was analyzed in culture supernatant after 24 h (b). Statistics were analyzed with non-parametric one-way ANOVA (Kruskl–Wallis test). ***, p < 0.001. n = 3. (c) Archangiumide was added to infected cells (MOI = 0.1). After 24 h culture, genomice DNA was extracted and the HSV-1 genome was analyzed by qPCR. Kruskl–Wallis one-way ANOVA. ****, p < 0.0001. n = 3. (d) gD transcription (Us6 mRNA) was analyzed by qPCR under high and low dose infections (MOI = 1 and MOI = 0.1). Kruskl–Wallis one-way ANOVA for each MOI group. ****, p < 0.0001. n = 3. (e) Western of gD and VP14 protein under archangiumide. β-actin as control. (f) Quantification of e.****, p < 0.0001. n = 3. (g) Cell survival rate under different doses of archangiumide. Dashed line represents CC50. n = 3. Data shown represent three independent experiments.

Figure 2

Archangiumide showed no effect on virus inactivation. Archangiumide was pre-incubated with HSV-1-eGFP virus stock for 2 h, then the stock was diluted into MOI = 0.1 to infect cells. (a) GFP signal was captured at 24 h. Fluorescent images were in 200× magnification. (b) HSV-1 titer (TCID50/mL) was analyzed in culture supernatant after 24 h. Kruskl–Wallis one-way ANOVA. n = 3. (c) gD transcription (Us6 mRNA) was analyzed by qPCR after 24 h. Kruskl–Wallis one-way ANOVA. n = 3. Data shown represent three independent experiments.

Figure 3

Archangiumide showed no effects on viral attachment and penetration. (a–c) With or without archangiumide, HSV-1 (MOI = 0.01) was loaded onto cells at 4 °C for 2 h for attachment, then removed. Infected cells were cultured in fresh medium and the GFP signal was captured at 24 h (a). Fluorescent images were in 200× magnification. (b) HSV-1 titer (TCID50/mL) was analyzed in culture supernatant after 24 h. Kruskl–Wallis one-way ANOVA. n = 3. (c) gD transcription (Us6 mRNA) was analyzed by qPCR after 24 h. Kruskl–Wallis one-way ANOVA. n = 3. (d–f) HSV-1 (MOI = 0.01) was loaded onto cells at 4 °C for 2 h for attachment, then removed. With or without archangiumide, attached HSV-1 was stimulated to penetrate at 37 °C for 15 min. After using an acidic PBS wash (pH = 3) to remove non-penetrated virus then neutralizing cells with a basal PBS wash (pH = 11), infected cells were cultured 24 h for analysis. (d) GFP signal was captured at 24 h. Fluorescent images were in 200× magnification. (e) HSV-1 titer (TCID50/mL) was analyzed in culture supernatant after 24 h. Kruskl–Wallis one-way ANOVA. n = 3. (f) gD transcription (Us6 mRNA) was analyzed by qPCR after 24 h. Kruskl–Wallis one-way ANOVA. n = 3. Data shown represent three independent experiments.

Figure 4

Archangiumide suppressed early viral replication. At different times after HSV-1 infection (MOI = 0.1), 0.4 mM archangiumide was added to culture systems to suppress replication. (a) GFP signal was captured at 24 h. Fluorescent images were in 200× magnification. (b) gD transcription (Us6 mRNA) was analyzed by qPCR after 24 h. Unpaired test, two-tailed. **, p < 0.01, n = 3. (c) Early-replication-related gene transcriptions (ICP0, ICP4, ICP22, ICP27, UL29 mRNA) were analyzed by qPCR before 6 h post infection. Kruskl–Wallis one-way ANOVA for each gene. n = 3. Data shown represent three independent experiments.

Figure 5

Archangiumide down-regulated VP16 expression and blocked its nuclear translocation. 0.4 mM archangiumide was added to HSV-1-infected cells (MOI = 4). (a) At 3 h and 6 h, VP16 (UL48 mRNA) was analyzed by qPCR. Kruskl–Wallis one-way ANOVA for each timepoint. ***, p < 0.001, **, p < 0.01, n = 3. (b) Western blot of VP16 protein under archangiumide. β-actin as control. (c) Intracellular distribution of VP16. DAPI as nuclear indicator. Bars indicate 10 μm. Data shown represent three independent experiments.