The comprehension of the molecular mechanisms that contribute to the establishment then maintenance of a persistent virus in its lethargic state is essential to understand the reasons for the resurgence of the associated disease once the virus reactivates. The VIRUCEPTION project is designed to understand one of key aspects of the interaction between a persistent virus, the herpes simplex virus 1 (HSV-1), and the host cell, i.e. the antiviral role of nuclear bodies.
Promyelocytic Leukemia nuclear bodies (PML-NBs) are major nuclear domains implicated in the nuclear innate/intrinsic antiviral response against viruses. The VIRUCEPTION project aims to study in vivo in a mouse animal model and in vitro in primary cell cultures, molecular mechanisms driving PML-NBs activity during the process of establishment of HSV-1 latency. We focus on the role of PML-NBs-associated proteins in the interception and clustering of the incoming viral genomes. We recently published a study that shows that, in a mouse model, the positioning and the distribution of viral genomes in the nucleus of infected neurons are not random (Catez et al, 2012). During the acute infection process (about 6 days post infection (d.p.i.) of mice) incoming viral genomes form nuclear clusters that are selectively associated with the PML-NBs (namely, DNA-containing PML-NBs or DCP-NBs). During latency (28 d.p.i.) in mice) the DCP-NBs persist however other viral genome patterns are also detected. DCP-NBs prevent the transcription of the virus, but we do not know what molecular mechanisms are implicated in the DCP-NBs formation, neither how they evolved during the whole process of latency establishment (from 6 to 28 d.p.i. in mice). The VIRUCEPTION project is designed to study both virus and cell features that lead to the formation of the DCP-NBs. We investigate if prior transcription of viral loci and/or replication of the incoming viral genomes are required. Concomitantly to the use of our well established in vivo mouse model of infection, we develop two in vitro systems reproducing features of latency, (i) a primary mouse neuron culture to analyze the role of PML-NBs-associated proteins in the formation of the DCP-NBs in cells that support the latent infection; (ii) a primary human fibroblasts culture that recapitulates some aspects of latency among other the formation of DCP-NBs and which will be an excellent complement to the primary neurons.
The originality of the VIRUCEPTION study is to use fluorescent in situ hybridization (FISH) combined to immunofluorescence to study at the single cell (neuron, fibroblast) level the role of PML-NBs as nuclear effector of the antiviral response against incoming viral genomes. This approach is complementary to the biochemical approaches used so far to study HSV-1 latency. Indeed, HSV-1 latency is heterogeneous from the point of view of several molecular features such as, expression of the LAT, chromatin marks, genome patterns and association with nuclear domains… Therefore, it is essential for a better understanding of the HSV-1 latency process to study the behavior of the viral genomes and cellular proteins at the single cell level. Beyond this, the VIRUCEPTION project will provide essential data concerning the probable role of PML-NBs in the control of the biology of other persistent viruses with nuclear tropism.
The novelty of the VIRUCEPTION project is to combined for the first time the use of in vivo and in vitro models of HSV-1 latency to analyze, using genetically modified HSV-1 and by FISH approaches, the molecular mechanism of DCP-NBs formation and the involvement of viral and cellular factors in the triggering of PML-NBs-dependent nuclear antiviral response.
At the in vivo level we have been able to dissect in a mouse model, the entire process of latency establishment of HSV-1 from the point of view of the interactions of the viral genome with the host cell nuclear environment and more particularly PML-NBs. We found that the viral genome patterns inside the infected neuron nuclei are changing dramatically during the 10 first days of the infection of the TG neurons. Then, the virus seems stabilized from the point of view of its genome nuclear distribution and interactions with nuclear domains, which is concomitant to the acquisition of stable latency features.
The relatively rapid set up of the mouse TG neurons primary cultures enabled us to understand quickly which viral factors are key players in the balance between latency and lytic cycle, especially at the level of the viral genome nuclear patterns associated with either process. In addition, we also found some cellular features that could drive the distribution of the viral genomes in the nucleus of the infected neurons, and as such, participate to the choice between latency and lytic cycle.
The second cell culture model that we developed is based on human primary fibroblasts infected with non-replicative virus, which mimics one important aspect of latency from the point of view of viral genome nuclear distribution and interaction with nuclear domains, i.e., the formation of DCP-NBs. We found several cellular components of the PML-NBs associated within the DCP-NBs but we also found specific chromatin-assembly factors that let us think that a process of chromatinization could take place within the DCP-NBs. This led to the development of an entire new aspect of research initially not planned in the project.
We have now three models (one in vivo and two in vitro) to study the latency of HSV-1 from the point of view of the interaction between the viral genomes and the nuclear environment. We will now use genetically modified viruses and cells to understand the role of viral and cellular factors in the interaction between the viral genomes and nuclear domains. Because our published and unpublished data revealed that viral genome nuclear distribution is a hallmark of latency establishment, we will pursue our investigation in order to decipher the molecular mechanisms that drive the interaction between the viral genomes and the nuclear environment.
1.Cavallero S., Huot N., Francelle L., Lomonte P., Naas T., Labetoulle M. 2014. Biological features of herpes simplex virus type 1 latency in mice according to experimental conditions and type of neurones. Invest Ophthalmol Vis Sci. 14-14673.
2. Ramakrishna C, Ferraioli A, Calle A, Nguyen TK, Openshaw H, Lundberg PS, Lomonte P, Cantin EM. Establishment of HSV1 Latency in Immunodeficient Mice Facilitates Efficient In Vivo Reactivation. PLoS Pathog. 2015 Mar 11;11(3):e1004730.
The comprehension of the molecular mechanisms that contribute to the establishment then maintenance of a persistent virus in its lethargic state is essential to understand the reasons for the resurgence of the associated disease once the virus reactivates. The VIRUCEPTION project is designed to understand one of the key aspects of the interaction between a persistent virus, the herpes simplex virus type 1 (HSV-1), and the host cell, which is the role of promyelocytic leukemia nuclear bodies (PML-NBs) as nuclear relays of the intrinsic antiviral response.
HSV-1 is a common human pathogen, which stays a major public health problem. HSV1 induces recurrent infections, mainly in the face and particularly at the level of the eyes, which results in severe ocular pathologies. Primary infection occurs in oral mucosa, and is followed by a secondary infection called latency, which affects the peripheral nervous system. Following various triggering factors, HSV1 reactivates, and each episode of ocular herpes may affect the visual prognosis. Indeed, corneal herpes infections are still today the first infectious cause of blindness in western countries. The comprehension of the molecular mechanisms that regulates the balance between latency and reactivation of HSV-1 is crucial for the understanding of the biology of the virus. This is also a major challenge for the design of therapeutic approaches based on preventing the virus to reactivate rather than stopping the lytic cycle. Indeed, antiviral therapies are administrated at a time often so late that the virus has already caused irreversible damage. This is particularly true when reactivation affects noble tissues (eye or brain), or in the cases of reactivations provoked by severe immunodeficiencies like in patients co-infected by other viruses such as HIV.
The study of the latency/reactivation process at the level of individual neurones within neuronal tissues has been hampered for more than a decade by the lack of efficient and accurate detection of latent viral genomes by fluorescent-based techniques. We recently developed a Fluorescent In Situ Hybridization (FISH) methods specifically adapted for the detection of latent viral genomes. We showed that the positioning and the distribution of the latent HSV-1 genomes in the nucleus of infected neurones are not random. The viral genomes are forming clusters that are preferentially associated with PML-NBs (namely, DNA-containing PML-NBs or DCP-NBs). Using PML KO mice we demonstrated that PML/PML-NBs were directly implicated in the acquisition of the virus patterns, especially the DCP-NBs, and in the control of the non-coding viral RNA, called LAT (for Latency Associated Transcript), which is synthesized during the latency process. As a consequence, all DCP-NBs-associated viruses are transcriptionally silent. The analysis of the first steps of establishment of latency, enabled us to demonstrate that the formation of DCP-NBs starts very early during the infectious process.
Our published data ask numerous questions regarding to the role of PML-NBs during HSV-1 latency establishment, i.e., which viral features are responsible for the association of PML-NBs with the viral genome? Is transcription of the LAT nucleating the formation of DCP-NBs? Is replication of the virus during acute infection required for the formation of the DCP-NBs? Which PML-NBs-associated protein(s) is (are) responsible for the recognition of incoming viral genomes and for the formation of DCP-NBs?... The VIRUCEPTION project aims to answer those questions using in vivo and in vitro models of infection as well as genetically modified viruses. Therefore, in addition to the use of our well-characterized mouse in vivo model, we will develop in vitro primary cell culture systems in which PML-NBs-associated proteins will be inactivated to analyse the role of selective proteins in the recognition of incoming viral genomes and in the formation of the DCP-NBs.
Monsieur Patrick Lomonte (Centre de Génétique et de Physiologie Moléculaire et Cellulaire)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UPR3296 Laboratoire de Virologie Moléculaire et Strucutrale
UMR5534 Centre de Génétique et de Physiologie Moléculaire et Cellulaire
Help of the ANR 400,000 euros
Beginning and duration of the scientific project: December 2013 - 48 Months