Discovery and preliminary structure-activity relationship of the marine natural product manzamines as herpes simplex virus type-1 inhibitors.

Herpes simplex virus type-1 (HSV-1) is a member of alpha-herpesviridae family and is known to cause contagious human infections. The marine habitat is a rich source of structurally unique bioactive secondary metabolites. A small library of marine natural product classes 1-10 has been screened to discover a new hit entity active against HSV-1. Manzamine A showed potent activity against HSV-1 via targeting the viral gene ICP0. Manzamine A is a ß-carboline alkaloid isolated from the Indo-Pacific sponge Acanthostrongylophora species. Currently, acyclovir is the drug of choice for HSV-1 infections. Compared with 50 µM acyclovir, manzamine A at 1 µM concentration produced potent repressive effects on viral replication and release of infectious viruses in SIRC cells in recent studies. The potent anti-HSV-1 activity of manzamine A prompted a preliminary structure-activity relationship study by testing targeted manzamines. These included 8-hydroxymanzamine A (11), to test the effect of the C-8 hydroxy substitution at the ß-carboline moiety; manzamine E (12), to assess the importance of substitution at the azacyclooctane ring; and ircinal A (13), to determine whether the ß-carboline ring is required for the activity. Manzamine A was chemically transformed to its salt forms, manzamine A monohydrochloride (14) and manzamine A monotartrate (15), to test whether improving water solubility and hydrophilicity will positively affect the activity. Compounds were tested for activity against HSV-1 using fluorescent microscopy and plaque assay. The results showed the reduced anti-HSV-1 activity of 11, suggesting that C-8 hydroxy substitution might adversely affect the activity. Similarly, manzamines 12 and 13 showed no activity against HSV-1, indicating the preference of the unsubstituted azacylcooctane and ß-carboline rings to the activity. Anti-HSV-1 activity was significantly improved for the manzamine A salts 14 and 15, suggesting that improving the overall water solubility as salt forms can significantly enhance the activity. Manzamines have significant potential for future development as anti-HSV-1 entity.

Overexpression of thyroid hormone receptor ß1 altered thyroid hormone-mediated regulation of herpes simplex virus-1 replication in differentiated cells.

Thyroid hormone (T3) has been suggested to play a role in herpes simplex virus 1 (HSV-1) replication. It was previously reported that HSV-1 replication was suppressed by T3 in mouse neuroblastoma cells overexpressing thyroid hormone receptor ß1 (TRß1). Using a human neuro-endocrine cells LNCaP differentiated by androgen deprivation, HSV-1 replication was active but decreased by T3 at very low moi, probably due to low copy of TRß1. In this study, a recombinant HSV-1 was constructed expressing TRß1 (HSV-1/TRß1). Infection of Vero cells (very little TRß1 expression) with HSV-1/TRß1 exhibited increased replication in the presence of T3 compared to the counterpart without TRß1 overexpression. Interestingly, HSV-1/TRß1 infection of differentiated LNCaP cells showed strong suppression of viral replication by T3 and the removal of hormone did not fully reversed the suppression as was observed in parent virus. Quantitative analyses indicated that ICP0 expression was blocked using HSV-1/TRß1 for infection during T3 washout, suggesting that overexpression of TRß1 is likely to delay its inhibitory effect on viral gene expression. Together these results emphasized the importance of TRß1 in the regulation of HSV-1 replication in differentiated environment with neuronal phenotype.

Lytic HSV-1 infection induces the multifunctional transcription factor Early Growth Response-1 (EGR-1) in rabbit corneal cells.

BACKGROUND: Herpes simplex virus type-1 (HSV-1) infections can cause a number of diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells, critical for viral replication, is being extensively investigated by many laboratories. In this study, we tested the hypothesis that HSV-1 lytic infection triggers the expression of important multi-functional transcription factor Egr1. The mechanisms of induction are mediated, at least in part, by signaling pathways such as NFκB and CREB. METHODS: SIRC, VERO, and 293HEK cell lines were infected with HSV-1, and the Egr-1 transcript and protein were detected by RT-PCR and Western blot, respectively. The localization and expression profile of Egr-1 were investigated further by immunofluorescence microscopy analyses. The recruitment of transcription factors to the Egr-1 promoter during infection was studied by chromatin immunoprecipitation (ChIP). Various inhibitors and dominant-negative mutant were used to assess the mechanisms of Egr-1 induction and their effects were addressed by immunofluorescence microscopy. RESULTS: Western blot analyses showed that Egr-1 was absent in uninfected cells; however, the protein was detected 24-72 hours post treatment, and the response was directly proportional to the titer of the virus used for infection. Using recombinant HSV-1 expressing EGFP, Egr-1 was detected only in the infected cells. ChIP assays demonstrated that NFкB and cAMP response element binding protein (CREB) were recruited to the Egr-1 promoter upon infection. Additional studies showed that inhibitors of NFкB and dominant-negative CREB repressed the Egr-1 induction by HSV-1 infection. CONCLUSION: Collectively, these results demonstrate that Egr-1 is expressed rapidly upon HSV-1 infection and that this novel induction could be due to the NFкB/CREB-mediated transactivation. Egr-1 induction might play a key role in the viral gene expression, replication, inflammation, and the disease progression.

Manzamine A as a novel inhibitor of herpes simplex virus type-1 replication in cultured corneal cells.

This study investigated the putative inhibitory effect of manzamine A on HSV-1 infection. Our results indicated that manzamine A effectively inhibited viral replication and infection in the cell line SIRC, a corneal cell line, at 1 µM. The existing anti-HSV-1 drug acyclovir was analyzed and showed a comparable activity at 50 µM. Plaque assays demonstrated that manzamine A reduced the release of infectious virus by 10 (11)-fold. RTPCR assays indicated that HSV-1 virion host shutoff (vhs) activity and ICP0 transcription were decreased by manzamine A treatment. These results bode well for the development of manzamines as potential leads to reduce viral infection in corneal cells and to prevent HSV-1-induced eye infections such as keratitis.

Thyroid hormone controls the gene expression of HSV-1 LAT and ICP0 in neuronal cells.

Various factors/pathways including hormonal regulation have been suggested to control herpes simplex virus type 1 (HSV-1) latency and reactivation. Our computer analysis identified a DNA repeat containing thyroid hormone-responsive elements (TRE) in the regulatory region of HSV-1 latency-associated transcript (LAT). Thyroid hormone (triiodothyronine, T(3)) functions via its receptor TR (thyroid hormone receptor), a transcription factor. Present study investigated the roles of TR and T(3) in HSV-1 gene expression using cultured neuoroblastoma cell lines. We demonstrated that liganded TR activated LAT transcription, but repressed infected cell protein no. 0 (ICP0) transcription in the presence of LAT TRE. Chromatin immunoprecipitation (ChIP) assays showed that TRs were recruited to LAT TREs independently of T(3) and hyperacetylated H4 was associated with the LAT promoter that was transcriptionally active. In addition, ChIP results showed that the chromatin insulator protein CCCTC-binding factor was enriched at the LAT TREs in the presence of TR and T(3). In addition, the BRG1 chromatin remodeling complex is found to participate in the T(3)/TR-mediated LAT activation since overexpression of BRG1 enhanced the LAT transcription and the dominant-negative mutant K785R abolished the activation. This is the first report revealing that TR elicits epigenetic regulation on HSV-1 ICP0 expression in neuronal cells and could have a role in the complex processes of HSV-1 latency/reactivation.

Regulation of herpes simplex virus type 1 thymidine kinase gene expression by thyroid hormone receptor in cultured neuronal cells.

Herpes simplex virus type 1 (HSV-1) undergoes acute infection in epithelial cells followed by establishment of latency in the neurons of trigeminal ganglia. The latent virus maintains a dormant state and can recurs spontaneously, suggesting transcriptional silencing and reactivation occur in neurons. Computer data mining identified a nuclear hormone response element (NRE), the binding site for the thyroid hormone receptor (TR) or other nuclear hormone receptor, in the promoter of HSV-1 thymidine kinase (TK). TRs are transcription factors whose activity is dependent on their ligand thyroid hormone (T(3); triiodothyronine). We hypothesize that TR and T(3) exert regulation on HSV-1 gene expression in neurons. A neuroblastoma cell line expressing the TR isoform beta (N2aTRbeta) was utilized for in vitro investigation. Results showed that liganded TR repressed TK promoter activity but unliganded TR relieved the inhibition. The mutagenesis study demonstrated that one nucleotide mutation at the NRE abolished the T(3)/TR-mediated regulation. N2aTRbeta cells treated with T(3) were suppressive to TK expression and virus release but the removal of T(3) de-repressed TK expression and increased virus release, confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and plaque assays, respectively. Chromatin immunoprecipitation (ChIP) assays showed that TRs were enriched at TK NRE in the presence of T(3). Additional results demonstrated that hyper acetylated histone H4 and monomethylated H3 modified at lysine 9 (H3K9me1) were enriched at transcriptionally active TK promoters but were dissociated from the NRE by T(3)/TR. These results suggest that T(3) could regulate HSV-1 gene expression through its receptor via histone modification in cultured neuronal cells.