Human Kaposi Research Articles

Immune evasion strategies of the human gamma‐herpesviruses: Implications for viral tumorigenesis

Two human gamma-herpesviruses, Epstein-Barr virus and Kaposi's sarcoma associated herpesvirus/human herpesvirus 8 display oncogenic potential, causing benign and malignant lymphoproliferative disorders in genetically susceptible or immunosuppressed individuals. As a family of viruses that establish persistent life-long infections, herpesviruses have evolved strategies to limit innate antiviral responses and evade host immune surveillance. Herpesviruses have developed mechanisms to disrupt antigen presentation, pirate the production of immune regulating cytokines, and inhibit pro-apoptotic signaling pathways. Although these strategies are designed to facilitate the long-term persistence of herpesviruses, in certain circumstances they can contribute to viral-driven carcinogenesis.

Characterization of Kaposi's Sarcoma-Associated Herpesvirus-Related Lymphomas by DNA Microarray Analysis

Among herpesviruses, γ-herpesviruses are supposed to have typical oncogenic activities. Two human γ-herpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are putative etiologic agents for Burkitt lymphoma, nasopharyngeal carcinoma, and some cases of gastric cancers, and Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma (PEL) especially in AIDS setting for the latter case, respectively. Since such two viruses mentioned above are highly species specific, it has been quite difficult to prove their oncogenic activities in animal models. Nevertheless, the viral oncogenesis is epidemiologically and/or in vitro experimentally evident. This time, we investigated gene expression profiles of KSHV-oriented lymphoma cell lines, EBV-oriented lymphoma cell lines, and T-cell leukemia cell lines. Both KSHV and EBV cause a B-cell-originated lymphoma, but the gene expression profiles were typically classified. Furthermore, KSHV could govern gene expression profiles, although PELs are usually coinfected with KSHV and EBV.

Dissecting host-virus interaction in lytic replication of a model herpesvirus.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection. Murine γHV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus. γHV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in γHV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during γHV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during γHV68 lytic replication ex vivo. Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant γHV68 carrying these mutations can be used to recapitulate the phenotypes of γHV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo. Recently, we have discovered that γHV68 usurps an innate immune signaling pathway to promote viral lytic replication. Specifically, γHV68 de novo infection activates the immune kinase IKKβ and activated IKKβ phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, γHV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection. Murine γHV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. γHV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in γHV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during γHV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during γHV68 lytic replication ex vivo. Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant γHV68 carrying these mutations can be used to recapitulate the phenotypes of γHV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo. Recently, we have discovered that γHV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, γHV68 de novo infection activates the immune kinase IKKβ and activated IKKβ phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, γHV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

Sequencing of bovine herpesvirus 4 v.test strain reveals important genome features

BackgroundBovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC), the lack of a complete genome sequence for this strain limits its experimental use.MethodsIn this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach.ResultsThe long unique coding region (LUR) consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA). As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G) differs from the other prDNA units (prDNA-inner). Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions.ConclusionsIn this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses.

Rhesus Monkey Rhadinovirus ORF57 Induces gH and gL Glycoprotein Expression through Posttranscriptional Accumulation of Target mRNAs

Open reading frame 57 (ORF57) of gamma-2 herpesviruses is a key regulator of viral gene expression. It has been reported to enhance the expression of viral genes by transcriptional, posttranscriptional, or translational activation mechanisms. Previously we have shown that the expression of gH and gL of rhesus monkey rhadinovirus (RRV), a close relative of the human Kaposi's sarcoma-associated herpesvirus (KSHV), could be dramatically rescued by codon optimization as well as by ORF57 coexpression (J. P. Bilello, J. S. Morgan, and R. C. Desrosiers, J. Virol. 82:7231-7237, 2008). We show here that ORF57 coexpression and codon optimization had similar effects, except that the rescue of expression by codon optimization was temporally delayed relative to that of ORF57 coexpression. The transfection of gL mRNA directly into cells with or without ORF57 coexpression and with or without codon optimization recapitulated the effects of these modes of induction on transfected DNA. These findings suggested an important role for the enhancement of mRNA stability and/or the translation of mRNA for these very different modes of induced expression. This conclusion was confirmed by several different measures of gH and gL mRNA stability and accumulation with or without ORF57 coexpression and with or without codon optimization. Our results indicate that RRV gH and gL expression is severely limited by the stability of the mRNA and that ORF57 coexpression and codon optimization independently induce gH and gL expression principally by allowing accumulation and translation of these mRNAs.

Pathogenesis and Host Control of Gammaherpesviruses: Lessons from the Mouse

Gammaherpesviruses are lymphotropic viruses that are associated with the development of lymphoproliferative diseases, lymphomas, as well as other nonlymphoid cancers. Most known gammaherpesviruses establish latency in B lymphocytes. Research on Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68/γHV68/MHV4) has revealed a complex relationship between virus latency and the stage of B cell differentiation. Available data support a model in which gammaherpesvirus infection drives B cell proliferation and differentiation. In general, the characterized gammaherpesviruses exhibit a very narrow host tropism, which has severely limited studies on the human gammaherpesviruses EBV and Kaposi's sarcoma-associated herpesvirus. As such, there has been significant interest in developing animal models in which the pathogenesis of gammaherpesviruses can be characterized. MHV68 represents a unique model to define the effects of chronic viral infection on the antiviral immune response.

Infectious agents in human cancers: Lessons in immunity and immunomodulation from gammaherpesviruses EBV and KSHV

Members of the herpesvirus family have evolved the ability to persist in their hosts by establishing a reservoir of latently infected cells each carrying the viral genome with reduced levels of viral protein synthesis. In order to spread within and between hosts, in some cells, the quiescent virus will reactivate and enter lytic cycle replication to generate and release new infectious virus particles. To allow the efficient generation of progeny viruses, all herpesviruses have evolved a wide variety of immunomodulatory mechanisms to limit the exposure of cells undergoing lytic cycle replication to the immune system. Here we have focused on the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) that, uniquely among the eight human herpesviruses identified to date, have growth transforming potential. Most people infected with these viruses will not develop cancer, viral growth-transforming activity being kept under control by the host's antigen-specific immune responses. Nonetheless, EBV and KSHV are associated with several malignancies in which various viral proteins, either predominantly or exclusively latency-associated, are expressed; at least some of these proteins also have immunomodulatory activities. Of these malignancies, some are the result of a disrupted virus/immune balance through genetic, infectious or iatrogenic immune suppression. Others develop in people that are not overtly immune suppressed and likely modulate the immunological response. This latter aspect of immune modulation by EBV and KSHV forms the basis of this review.

Pilot study evaluating the interaction between paclitaxel and protease inhibitors in patients with human immunodeficiency virus-associated Kaposi’s sarcoma: an Eastern Cooperative Oncology Group (ECOG) and AIDS Malignancy Consortium (AMC) trial

Paclitaxel, a cytotoxic agent metabolized by cytochrome P450 hepatic enzymes, is active for the treatment of human immunodeficiency (HIV) associated Kaposi's sarcoma. Protease inhibitors are commonly used to treat HIV infection and are known to inhibit cytochrome P450. We sought to determine whether protease inhibitors alter the pharmacokinetics of paclitaxel. Patients with advanced HIV-associated KS received paclitaxel (100mg/m(2)) by intravenous infusion over 3h, and plasma samples were collected to measure paclitaxel concentration. The area under the curve (AUC) was calculated using a combination of the log and linear trapezoidal rule, and clearance was calculated as the dose/AUC. Pharmacokinetics were compared with respect to antiretroviral therapy and toxicity, Thirty-four patients received paclitaxel, of whom 20 had no prior paclitaxel therapy and were assessable for response. Twenty-seven had pharmacokinetic studies performed. Paclitaxel exposure was higher in patients taking protease inhibitors compared to those who were not taking protease inhibitors. The increased exposure did not correlate with efficacy or toxicity. Of the 20 patients assessable for response, 6 (30%) had an objective response and median progression-free survival was 7.8months (95% confidence interval, 5.6, 21.0months). Despite higher exposure to paclitaxel, patients on protease inhibitors did not experience enhanced toxicity or efficacy.

Murine Gamma-Herpesvirus 68 Hijacks MAVS and IKKβ to Initiate Lytic Replication

Upon viral infection, the mitochondrial antiviral signaling (MAVS)-IKKβ pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKβ impaired the lytic replication of gamma-herpesvirus 68 (γHV68), a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. γHV68 infection activated IKKβ in a MAVS-dependent manner; however, IKKβ phosphorylated and promoted the transcriptional activation of the γHV68 replication and transcription activator (RTA). Mutational analyses identified IKKβ phosphorylation sites, through which RTA-mediated transcription was increased by IKKβ, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant γHV68 carrying mutations within the IKKβ phosphorylation sites was greatly impaired. These findings support the conclusion that γHV68 hijacks the antiviral MAVS-IKKβ pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation.

Analysis of rhesus rhadinovirus microRNAs expressed in virus-induced tumors from infected rhesus macaques

Rhesus rhadinovirus (RRV), a primate gamma-herpesvirus related to human Kaposi's sarcoma-associated herpesvirus (KSHV), causes a similar pattern of pathogenesis. Previously, RRV was shown to express 7 pre-microRNAs (pre-miRNAs) in latently infected cells. Using deep sequencing, we analyzed the pattern of small RNA expression in vivo using latently RRV-infected B-cell lymphoma and retroperitoneal fibromatosis tissues. We identified 15 virally encoded pre-miRNAs in both tumors, including all previously reported RRV pre-miRNAs. Although all 15 RRV pre-miRNAs, like all 12 KSHV pre-miRNAs, are located 3' to the conserved viral ORF71 gene and in the same transcriptional orientation, only one RRV miRNA is homologous to a KSHV miRNA. One previously identified RRV miRNA, miR-rR1-3, is actually a miRNA offset RNA (moRNA) derived from sequences located adjacent to pre-miR-rR1-3. Several other RRV-derived moRNAs were obtained, including one recovered >600 times. Together, this research provides a comprehensive list of the miRNAs and moRNAs encoded by RRV.

MiR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator

MicroRNAs are small, non-coding RNAs that negatively regulate gene expression. It has been proposed that microRNAs could function in the regulation of innate immunity, but this has not been demonstrated for viral infection. Here we test this hypothesis using the human pathogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) and one of its putative natural cellular targets, primary lymphatic endothelial cells (LECs). We show that an early antiviral microRNA response (6 h post-infection) includes expression of microRNAs that enhance viral gene expression. In particular, the CREB-induced miR-132 microRNA is highly upregulated after infection and has a negative effect on the expression of interferon-stimulated genes, facilitating viral replication. We show a similar function for miR-132 during infection of monocytes with herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). miR-132 regulates innate antiviral immunity by inhibiting expression of the p300 transcriptional co-activator. p300 is downregulated early after KSHV infection, and inhibition of miR-132 induction restores p300 expression. Furthermore, p300 regulates miR-132 levels, revealing a dynamic equilibrium between miR-132 and p300. By targeting p300, rather than a transcription factor or signalling protein, miR-132 has a broad role in the regulation of antiviral immunity.

The RING-CH Ligase K5 Antagonizes Restriction of KSHV and HIV-1 Particle Release by Mediating Ubiquitin-Dependent Endosomal Degradation of Tetherin

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.

A Human Herpesvirus MicroRNA Inhibits p21 Expression and Attenuates p21-Mediated Cell Cycle Arrest

The oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) expresses 12 viral microRNAs (miRNAs) in latently infected cells. Here, we report that cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key inducer of cell cycle arrest, are direct targets for KSHV miR-K1. Ectopically expressed KSHV miR-K1 specifically inhibited the expression of endogenous p21 in KSHV-negative cells and strongly attenuated the cell cycle arrest that normally occurs upon p53 activation, yet miR-K1 did not prevent the induction of other p53-responsive genes. Stable knockdown of miR-K1 in latently KSHV-infected human primary effusion lymphoma (PEL) B cells revealed a derepression of p21 expression and enhanced cell cycle arrest following activation of p53. Our data demonstrate that miR-K1 represses the expression of p21, a protein with known tumor suppressor functions, and suggest that this KSHV miRNA is likely to contribute to the oncogenic potential of this opportunistic viral pathogen.

Immune evasion by gammaherpesvirus genome maintenance proteins

Viruses that establish lifelong latent infections must ensure that the viral genome is maintained within the latently infected cell throughout the life of the host, yet at the same time must also be capable of avoiding elimination by the immune surveillance system. Gammaherpesviruses, which include the human viruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, establish latent infections in lymphocytes. Infection of this dynamic host-cell population requires that the viruses have appropriate strategies for enabling the viral genome to persist while these cells go through rounds of mitosis, but at the same time must avoid detection by host CD8(+) cytotoxic T lymphocytes (CTLs). The majority of gammaherpesviruses studied have been found to encode a specific protein that is critical for maintenance of the viral genome within latently infected cells. This protein is termed the genome maintenance protein (GMP). Due to its vital role in long-term latency, this offers the immune system a crucial target for detection and elimination of virus-infected cells. GMPs from different gammaherpesviruses have evolved related strategies that allow the protein to be present within latently infected cells, but to remain effectively hidden from circulating CD8(+) CTLs. In this review, I will summarize the role of the GMPs and highlight the available data describing the immune-evasion properties of these proteins.

Latent Murine Herpesvirus-4 Infection Arms NK Cells.

Abstract Abstract 3678 Poster Board III-614 Natural killer (NK) cells are lymphocytes originally identified by their ability to kill target cells without prior sensitization. However, murine NK cells require an antecedent ‘arming’ event to translate cytotoxic effector proteins (perforin and granzymes) resulting in potent cytotoxic capacity. In contrast to mice, most NK cells (CD56dim) from the peripheral blood of healthy humans are armed with pre-formed perforin and granzyme B proteins and mediate killing of NK sensitive targets directly ex vivo. This suggests that NK cells from specific pathogen free (SPF) laboratory mice are lacking a critical arming event present in healthy humans, and provides an experimental system to elucidate the events that result in NK cell arming in vivo. Since latent herpesviruses are highly prevalent and alter multiple aspects of host immunity, we hypothesized that the immune environment created by a latent herpesvirus infection arms NK cells. NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4), a virus closely related to the human viruses Kaposi's sarcoma associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), were armed as evidenced by increased granzyme B protein expression, cytotoxicity, and interferon-gamma production. Adoptive transfer experiments with naïve NK cells indicated that NK arming occurred rapidly (≤ 72 hours) in the latently infected host and did not require acute infection. In addition, experiments utilizing a genetic variant of MuHV-4 that acutely infects mice similar to wild-type MuHV-4, but does not establish latency, confirmed that acute infection was not responsible for this NK cell modulation. Finally, NK cells armed by latent infection protected the host against a lethal RMA-S lymphoma challenge. Thus, the immune environment created by a latent herpesvirus infection arms NK cell function in vivo. These results indicate that some of the differences between human and SPF laboratory-mouse NK cell function may reflect a disparity in immune activation induced by chronic virus infection, as opposed to an intrinsic difference in the NK cells themselves. Moreover, these findings indicate that the activation state of the innate immune system needs to be taken into account when using mice to model immune responses against pathogens and tumors. Disclosures: No relevant conflicts of interest to declare.

MHV68 complement regulatory protein facilitates MHV68 replication in primary macrophages in a complement independent manner

Murine gammaherpesvirus-68 (MHV68) is genetically related to human Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and provides a tractable model to study gammaherpesvirus-host interactions in vivo and in vitro. The MHV68-encoded v-RCA product inhibits murine complement activation and shares sequence homology with other virus and host regulators of complement activation. Here we show that v-RCA is required for efficient MHV68 replication in primary murine macrophages, but not in murine embryonic fibroblasts. v-RCA-deficient MHV68 mutant viruses display defects in viral DNA synthesis in infected macrophages. Importantly, attenuated growth of v-RCA mutant viruses is not rescued in macrophages lacking critical components of the complement system including C3, indicating that the macrophage-specific role of v-RCA in MHV68 replication is complement-independent. This contrasts with the situation in vivo in which attenuated neurovirulence of v-RCA mutant viruses is rescued in C3-deficient mice. This study shows a novel, complement independent cell-type-specific function of a gammaherpesvirus RCA protein.

Regulation of angiogenesis in malignancies associated with Epstein–Barr virus and Kaposi‘s sarcoma-associated herpes virus

Tumor angiogenesis is the process by which new blood vessels are formed within emerging or progressing malignancies. The human Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus critically contribute to the pathogenesis of selected tumor types, including nasopharyngeal carcinoma and Kaposi's sarcoma, respectively, where angiogenesis is robust and often disrupted. Lymphangiogenesis, the process by which new lymphatic vessels are formed, is also induced in Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus-associated malignancies and in some cases may contribute to metastasis. Recent studies have identified a number of molecules and signaling pathways that underlie angiogenesis and lymphangiogenesis, and clarified the pivotal role of the VEGF family of proteins and their receptors. New treatment modalities that target members of this family have gained approval for clinical use in cancer. Pathogenetic steps are often difficult to dissect in many cancer types, but virus-induced malignancies provide a unique opportunity for understanding the molecular regulation of cancer progression, including angiogenesis. Dissection of viral gene contribution to tumor angiogenesis could result in a better understanding of the angiogenic process, its contribution to cancer and help in the design of rational therapies that target tumor growth and vascularization.

Protease dimer formation disrupted

A small-molecule inhibitor of the obligate dimeric protease of human Kaposi's sarcoma-associated herpesvirus was identified. The agent functions by a "monomer trap" mechanism in which the compound binds to a partially unfolded monomer and disrupts the formation of the enzymatically active protease dimer.

Inhibition of a viral enzyme by a small-molecule dimer disruptor

Small molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi’s sarcoma-associated herpesvirus (KSHV) were identified by screening an α-helical mimetic library. Subsequently, a second generation of low micromolar inhibitors with improved potency and solubility was synthesized. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor. 1H-15N-HSQC titration studies mapped the inhibitor binding-site to the dimer interface, and mutagenesis studies targeting this region were consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of a dynamic intermediate. These results validate the interface of herpesvirus proteases and other similar oligomeric interactions as suitable targets for the development of small molecule inhibitors.