# New Insights into the Biology of the Emerging Tembusu Virus
## 2 Tembusu Virus  
### Genomic Organization and Replication  
As with other flaviviruses, viruses from the TMUV group have a ~11 kb positive strand RNA genome, which is composed of a single open reading frame (ORF) flanked by 5'terminal and 3'terminal non-coding regions (UTR) (Figure 1). The 5' end is capped with an m7 GppAmp structure (type 1 cap), and the 3' end is free from a poly A tail. The ORF is predicted to be translated into a single polyprotein that is subsequently cleaved by host and viral proteases in three structural proteins: envelope (E), membrane precursor (prM) and capsid (C), as well as seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Viral proteins are necessary for viral replication and assembly [6]. The virion has an icosahedral capsid enclosed by a lipid envelope with a diameter of 30 to 60 nm [4; 5; 7]. Despite a lack of information on TMUV replication, this process is thought to be more or less identical to that of other members of the Flavivirus genus [6]. Prior to cell internalization, flaviviruses bind to a set of the cellular surface factor via cellular receptors. The recognition and internalization process involves different types of cell surface receptors and interaction with external viral proteins such as E and M proteins [8]. The E protein is involved in surface receptor binding and facilitates viral entry and the subsequent fusion steps between the viral envelope and the intracellular membranes [9]. At the cell surface membrane, the heparan sulfate receptor family, C-type lectin receptors or phosphatidylserine receptors facilitate flavivirus entry, although their exact role remains poorly understood [8]. Given its recent identification, TMUV cellular receptors are less known than for other members of the Flavivirus genus. Experiments with mammalian (BHK-21) and avian (DEF) cells using different drug treatments targeting surface receptors revealed the implication of the heparan sulfate molecule in recognition and cell attachment [10]. This precursor study has paved the way to a better understanding of TMUV attachment and entry mechanisms. A range of endocytic pathways are usually used by flaviviruses to penetrate into the cell [11]. [PERSON] et al., using the hamster kidney BHK-21 cell line, reported the involvement of three different endocytosis pathways in the entry process of TMUV. The authors showed an impact on virus infection when they antagonized clathrin-dependent endocytosis by chlorpromazine and dynasore treatment or after depletion of cholesterol, suggesting involvement of clathrin- and cholesterol-dependent endocytosis in viral replication. In contrast, treatment of cells using siRNA targeting CAV1 or the genistein inhibitor shows that the caveolin pathway is not required for TMUV entry [12]. These results confirmed the first observations reported by [PERSON] et al. on the role of the clathrin endocytosis pathway  
Figure 1: Structure of TMUV genome. The RNA genome is similar to other flaviviruses, with an open reading frame encoding three structural proteins, C, prM and E, and five nonstructural proteins NS1 to NS5. The structural and nonstructural proteins are generated after proteolytic cleavage of the polyprotein. Untranslated regions are shown schematically. in TMUV entry [13]. In this study, using chemical treatment and knockdown experiments, the authors also showed the involvement of the proteasome and low-pH endosome in the internalization process of TMUV. Indeed, the ubiquitin-proteasome pathway represents an important cellular proteolytic mechanism mediating a range of cellular processes including virus entry. In this respect, TMUV was reported to take advantage of this mechanism for cell entry [13].