FHL1 is a key player of chikungunya virus tropism and pathogenesis

. Chikungunya is an infectious disease caused by the chikungunya virus (CHIKV), an al-phavirus transmitted to humans by Aedes mosquitoes, and for which there is no licensed vaccine nor antiviral treatments. By using a loss-of-function genetic screen, we have recently identiﬁed the FHL1 protein as an essential host factor for CHIKV tropism and pathogenesis. FHL1 is highly expressed in muscles cells and ﬁbroblasts, the main CHIKV-target cells. FHL1 interacts with the viral protein nsP3 and plays a critical role in CHIKV genome ampliﬁcation. Experiments in vivo performed in FHL1-deﬁcient mice have shown that these animals are resistant to infection and do not develop muscular lesions. Altogether these observations, published in the journal Nature [1], show that FHL1 is a key host factor for CHIKV pathogenesis and identify the interaction between FHL1 and nsP3 as a promising target for the development of new antiviral strategies.

Abstract. Chikungunya is an infectious disease caused by the chikungunya virus (CHIKV), an alphavirus transmitted to humans by Aedes mosquitoes, and for which there is no licensed vaccine nor antiviral treatments. By using a loss-of-function genetic screen, we have recently identified the FHL1 protein as an essential host factor for CHIKV tropism and pathogenesis. FHL1 is highly expressed in muscles cells and fibroblasts, the main CHIKV-target cells. FHL1 interacts with the viral protein nsP3 and plays a critical role in CHIKV genome amplification. Experiments in vivo performed in FHL1deficient mice have shown that these animals are resistant to infection and do not develop muscular lesions. Altogether these observations, published in the journal Nature [1], show that FHL1 is a key host factor for CHIKV pathogenesis and identify the interaction between FHL1 and nsP3 as a promising target for the development of new antiviral strategies.

Introduction
Chikungunya virus (CHIKV) is an arbovirus (Arthropod-borne virus) belonging to the genus Alphavirus of the Togaviviradae family. This pathogen, isolated in 1952 during an outbreak in present-day Tanzania [2], has reemerged in 2000 and spread to new worldwide areas causing millions of infection [3]. CHIKV is the etiological agent of the chikungunya disease, a word from Makonde language that translates as "that which bends up", referring to the severe muscular and joint pains that impede the mobility and daily activities of infected patients. CHIKV is transmitted to humans through the bite of infected mosquitoes of the Aedes aegypti and albopictus species. Following inoculation in the skin, CHIKV infects dermal cells, mainly resident fibroblasts, then disseminates in the organ- ism to reach preferential sites of replication such as peripheral joints and musculoskeletal tissue [4]. Although it could be asymptomatic, CHIKV infection frequently progresses to high fever and debilitating arthralgia and myalgia that can persist for months. Intensive CHIKV replication in muscle and joints triggers a strong immune response leading to pro-inflammatory chemokines and cytokines release, most certainly responsible for the onset of the disease. Whereas the clinical aspects of the disease are well described, the molecular determinants of the viral tropism and pathogenesis are poorly understood. Like other viruses, CHIKV is an obligate intracellular parasite which relies on host cellular functions to accomplish its replication cycle. Several studies have already identified many host factors exploited by the virus to accomplish its infectious cycle. However, none of them accounts for the preferential tropism of the virus for muscle and joint tissues, nor the clinical onset. This report consists of an overview of the study done by our team that has led to the identification of the protein Four and Half LIM Domain (FHL1) as a crucial host factor for CHIKV infection and pathogenesis. This work was performed in collaboration with teams from the Pasteur Institute and the Hospital La Pitié Salpetrière. It has been recently published in the journal Nature [1].

Identification of FHL1 as a critical cellular factor for CHIKV infection cycle
To uncover host factors important for CHIKV replication, we performed a systematic genome-wide screen using the CRISPR-Cas9 technology ( Figure 1A). This Since CHIKV infection is cytopathic, only cells that lost a gene essential for viral replication can survive and proliferate. It was then possible to retrieve, by combining next-generation sequencing technology and bioinformatic analysis, the sgRNAs responsible for the "resistant" phenotype, and therefore the identity of the invalidated genes. This experimental approach allowed us to identify the gene FHL1 (Four and a Half LIM domains 1), which has the most enriched targeting-sgRNAs during the phenotypic selection ( Figure 1B). This gene localizes on the X chromosome and encodes the FHL1 protein for which three isoforms exist (A, B et C). Isoform FHL1A is predominantly expressed in striated muscle and fibroblasts [5], the preferential cellular targets of CHIKV. FHL1A contains 4 and a half LIM domains that are two tandemly arranged zinc finger domains ( Figure 1C) [6]. FHL1A is cytoplasmic protein that plays an important role in muscle development and assembly of large structural complexes. Mutations in the FHL1 gene leading to the absence of FHL1 protein have been described in X-linked myopathies such as the Emery-Dreifuss muscular dystrophy (EDMD) [7].

FHL1 is a cellular factor essential for CHIKV replication
Since FHL1A expression correlates with the natural tropism of the virus and that this protein was never previously reported as a host factor involved in viral infection, we have focused our study on understanding its role during CHIKV infection. We have first assessed the susceptibility to CHIKV in different cellular models for which FHL1 expression was invalidated by CRISPR-Cas9. In these cells, FHL1 depletion greatly decreased infection by different primary strains of CHIKV responsible for outbreaks. Interestingly, FHL1 poorly affected a CHIKV strain maintained in a sylvatic cycle between Aedes mosquitoes and African monkeys. Secondly, we have assessed the importance of FHL1 on the infectious cycle of other viruses. We showed that FHL1 is also selectively used by O'nyong-nyong virus (ONNV), an alphavirus phylogenetically closely related to CHIKV. In contrast, FHL1 is dispensable for infection by other related alphavirus or other arborviruses such as Dengue or ZIKA virus. Finally, the importance of FHL1 in CHIKV biology was strengthened by our observations showing that the virus was unable to replicate in cells isolated from patients with a rare genetic disease, the Emery-Dreifuss muscular dystrophy (EDMD). In these patients, the muscular pathology results from mutations in the FHL1 gene responsible for FHL1 protein degradation. We have shown that primary myoblasts and fibroblasts from EDMD patients lack FHL1 expression and are naturally refractory to CHIKV infection (Figure 2A). In these cells, ectopic expression of FHL1 confers permissiveness to CHIKV, highlighting the essential role of FHL1 in CHIKV tropism ( Figure 2B).

FHL1 regulates the viral replication step by interacting with the viral protein nsP3
We have used several molecular virology and cellular biology tools to identify which step of CHIKV infectious cycle is impacted by FHL1. Our results revealed that FHL1 is critical for the replication of viral RNA. Indeed, in cells lacking FHL1 and infected by CHIKV, we have observed a lack of viral genome synthesis both at the level of neosynthesized genomic viral RNA (gvRNA) ( Figure 3A) and negative strand intermediates, which serve within the replication complex as matrix for gvRNA synthesis ( Figure 3B).
In line with these data, our electronic microscopy studies showed that FHL1 is involved in the formation of plasma-membrane-associated spherules as well as the cytoplasmic vacuolar membrane structures, which are CHIKV-induced viral replication platforms [8]. We have also shown that FHL1 is recruited at these structures by the viral protein nsP3, a key player in viral RNA synthesis. FHL1 interacts directly with nsP3 C-terminal hypervariable domain, an intrinsically disordered domain known to mediate recruitment of cellular proteins [9]. By comparing the HVD sequence of several alphaviruses, we have identified a common region between CHIKV and ONNV strains that is essential for the interaction with FHL1 ( Figure 3C). Deleting this region abolishes the interaction between FHL1 and nsP3, and viruses lacking the FHL1-interacting sequence showed a strong defect in viral replication when compared to their wildtype counterpart.

Role of FHL1 in pathophysiology of CHIKV in mice
To assess in vivo the role of FHL1 in CHIKV pathogenesis, Fhl1 heterozygous female mice were crossed with wild-type males carrying the same genetic background. FHL1-deficient male mice were identified and 9-day-old newborn mice were injected with CHIKV by intradermal route and then sacrificed 7 days after inoculation. Our results, obtained in collaboration with Marc Lecuit's team at Pasteur Institute, showed high viral loads in muscle (10 8 TCID 50/g) and joints (order of magnitude of 10 6 TCID 50/g) of wild-type littermates, whereas viral loads in FHL1-deficient mice remain below the threshold of detection of the technique ( Figure 4A). Moreover, histology and immunohistochemistry analyses showed necrotizing myositis with massive infiltrates and necrosis of the muscle fibers in skeletal muscle of WT littermates, while FHL1-null mouse muscle showed no detectable pathology ( Figure 4B). Altogether these results showed that FHL1-deficient mice are resistant to CHIKV and that FHL1 contributes to CHIKV pathogenesis in mice.

Conclusions and perspectives
Our study identifies for the first time a cellular factor specific of CHIKV and essential for its replication cycle. High FHL1 expression in muscle and fibroblasts suggests that FHL1 is a key player of CHIKV tropism and pathogenesis. In normal conditions, FHL1 participates in healthy muscle homeostasis. During infection, FHL1 is highjacked from its physiological function by the CHIKV-nsP3 protein, likely to build the replication complex essential for viral genome amplification. The molecular mechanisms by which FHL1 accomplishes its pro-viral function are currently unknown and remain to be elucidated.