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. 2014 Dec 3;6(265):265ra169.
doi: 10.1126/scitranslmed.3010643.

Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes

James M Hill  1 , Debra C Quenelle  2 , Rhonda D Cardin  3 , Jodi L Vogel  4 , Christian Clement  1 , Fernando J Bravo  3 , Timothy P Foster  1 , Marta Bosch-Marce  5 , Priya Raja  6 , Jennifer S Lee  7 , David I Bernstein  3 , Philip R Krause  5 , David M Knipe  7 , Thomas M Kristie  8
Affiliations

Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes

James M Hill et al. Sci Transl Med. .

Abstract

Herpesviruses are highly prevalent and maintain lifelong latent reservoirs, thus posing challenges to the control of herpetic disease despite the availability of antiviral pharmaceuticals that target viral DNA replication. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 (lysine-specific demethylase 1) and a member of the JMJD2 family (Jumonji C domain-containing protein 2). Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and blocks infection and reactivation in vitro. We demonstrate that viral infection can be epigenetically suppressed in three animal models of herpes simplex virus infection and disease. Treating animals with the monoamine oxidase inhibitor tranylcypromine to inhibit LSD1 suppressed viral lytic infection, subclinical shedding, and reactivation from latency in vivo. This phenotypic suppression was correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Therefore, epi-pharmaceuticals may represent a promising approach to treat herpetic diseases.

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Conflict of interest statement

Competing interests: The NIH has the following patent applications: (i) Methods of preventing or treating viral infection or reactivation from latency in a host using inhibitors of the LSD1 protein (T.M.K. J.L.V., and Y.L.; U.S. patent application no. 61/083,304; International patent application no. PCT/US2009/051557); (ii) Method of preventing or treating viral infection via inhibition of the JMJD2 proteins (T.M.K., Y.L., and J.L.V.; U.S. patent application no. 61/366,563). All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Multiple chromatin modulation enzymes drive the initial chromatin state of the HSV genome. Model illustrating the initial heterochromatic chromatin state of viral genome during infection or latency and the coactivator complex required to modulate this to a euchromatic chromatin structure for productive infection or reactivation from latency. TCP inhibits the required activity of the histone demethylase LSD1.
Fig. 2
Fig. 2
Inhibition of LSD1 reduces primary acute infection in the mouse model system. (A-B) Ocular infection with 1×105 pfu HSV-1(F)/eye. (A) Survival of mice treated topically with Vehicle, ACV (5 mM) or TCP (5 mM). (B) Viral loads in ganglia at 10 dpi of mice treated IP with Vehicle, ACV (100 mg/kg) or TCP (5 or 10 mg/kg). (C-E) Intranasal infection with 1.1×104 pfu (one LD50) HSV-2(MS). Mice were treated orally with Vehicle, ACV (100 mg/kg) or TCP (15 mg/kg). (C) Survival chart. (D) Viral loads in ganglia at 5 and 10 dpi. (E) Viral loads in select organs at 5 dpi. %T, % of total mice positive. (F-H) Intranasal infection with 1.1×105 pfu (one LD90) HSV-2(MS). (F) Survival of mice treated orally with Vehicle, ACV, or TCP at the indicated levels. (G) Viral loads in ganglia at the indicated dpi in mice treated orally with Vehicle, ACV (100 mg/kg), or TCP (6 mg/kg). (H) Viral loads in ganglia of mice implanted with Vehicle or TCP time-release pellets (ACV, oral, 100 mg/kg). (I) Survival of mice infected intranasally with 1.1×105 pfu (one LD90) HSV-2(MS) and treated orally with Vehicle, ACV, TCP, or the combination of ACV and TCP. Data are means +/- s.e.m. *Statistically significant.
Fig. 3
Fig. 3
Inhibition of LSD1 reduces viral yields and ocular disease during acute infection of rabbit corneas. (A-G) Ocular infection with 2.5×105 pfu HSV-1(17sn+). Rabbits were treated orally with Vehicle, Valtrex (VCV, 30 mg/kg), or TCP (12 mg/kg) from -2 to 14 dpi. (A) Levels of infectious virus detected in eye swabs collected 3-8 dpi. (B-G) Parameters of ocular disease were assessed at 7-14 dpi. (B-F) Mean values of scores for Corneal Stroma, Ocular Neovascularization, Corneal Epithelium, Scleral Inflammation/Injection, and Slit-Lamp Examinations (SLE). (G) Cumulative scores of (B-F). Data are means +/- s.e.m. *Statistically significant.
Fig. 4
Fig. 4
Inhibition of LSD1 blocks viral reactivation and shedding in the rabbit eye model system. (A-J) Ocular infection with 3×105 HSV-1(17syn+). (A-B) Eye swabs, collected at 33-53 dpi, were assayed for the presence of infectious HSV-1 and viral DNA in animals treated orally with Vehicle, Valtrex (250 mg/kg) or TCP (12 mg/kg). (C-D) Latently infected rabbits were implanted with placebo (Vehicle) or TCP (9.5 mg/kg) time-release pellets at 48 dpi. Eye swabs collected at 52-68 dpi were assayed for the presence of infectious HSV-1 and represented as positive per group (C) and per rabbit (D). (E-F) Latently infected rabbits were treated with Placebo or Selegiline (EMSAM) time-release topical patch (5.5 mg/kg) at 17-47 dpi. Eye swabs collected at 20-47 dpi were assayed for infectious HSV-1 and represented as positive per treatment group (E) and per eye (F). (G) Viral loads in ganglia of rabbits treated with Vehicle and TCP time-release pellets at 68 dpi. (H) Viral loads in ganglia of rabbits treated with Vehicle or EMSAM transdermal patches at 47 dpi. *Statistically significant.
Fig. 5
Fig. 5
Inhibition of LSD1 results in enhanced epigenetic suppression of HSV-1. (A-B) ChIP assays showing increased levels of histone H3 and H3K9-methylation at viral genomes in ganglia of TCP-time release (A) and transdermal EMSAM treated rabbits (B). (C-E) ChIP assays showing (C) increased levels of histone H3 and H3K9-me3 and (D-E) decreased levels of H3K9-me2 and H3K27-me3 on the viral genome of latently infected mice treated with time-release TCP pellets. Data are means +/- s.e.m.
Fig. 6
Fig. 6
Inhibition of LSD1 reduces recurrent disease in the guinea pig model. (A-F) Vaginal infection with 1×106 pfu HSV-2(MS). Guinea pigs were treated orally with Vehicle, ACV (5 mg/ml ad libitum in water), or TCP (15 mg/kg/day) from 15-35 dpi. (A-C) Lesions were scored (26-52 dpi) and shown as (A) cumulative average disease and recurrent lesion scores per day as defined in S3A; (B) average lesion scores per day; and (C) cumulative lesion scores per guinea pig. (D-E) HSV DNA detected in vaginal swabs taken from 21-35 dpi. (F) Viral loads in DRGs and spinal cords at 52 dpi. (G-J) Vaginal infection with 2×105 pfu HSV-2(MS). Guinea pigs were treated IP with ACV (6 mg/kg/day), TCP (5 mg/kg/day), or the combination for 22-43 dpi. (G-I) Lesion recurrences were scored as defined in S3B and are shown as (G) cumulative average recurrences; (H) total recurrences per guinea pig; and (I) average recurrences per group per day. (J) Viral loads in DRGs at 43 dpi. (K-L) ChIP assays showing increased levels of histone H3 and H3K9-methylation at viral promoters in dorsal root ganglia of guinea pigs (K) treated orally with Vehicle or TCP (30 mg/kg/day) and (L) treated IP with ACV (6 mg/kg/day) or TCP (5 mg/kg/day). Data are means +/- s.e.m. *Statistically significant.
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