# Advancements in Research on Duck Tembusu Virus Infections
## 2 Genomic Characteristics
### Non-Structural Proteins  
DTMUV NS1 has a molecular weight of approximately 40 to 50 kDa. It is a highly conserved glycoprotein that exists on the cell surface or in association with cell-related proteins [57]. Despite the absence of a transmembrane domain, NS1 forms hydrophobic dimers on the cell membrane once it reaches the cell surface [58]. NS1 is one of the few NS genes capable of conducting retrograde complementation in stable expression replicase complexes [59]. NS1 plays a pivotal role in viral RNA replication, antiviral immunity, and host recognition of virus-associated molecular patterns [60; 61]. DTMUV NS2A and NS2B are crucial in constructing the replicase complex and play pivotal roles in virus replication. NS2A has a molecular weight of approximately 25 kDa and is a small transmembrane protein containing eight transmembrane domains located on the endoplasmic reticulum, contributing to endoplasmic reticulum reorganization [62]. NS2A interacts with the 3' UTR of viral RNA and other components of the replicase complex, indicating its involvement in viral RNA replication [63]. Additionally, the flavivirus NS2A protein is implicated in regulating host antiviral interferon responses [64]. Apart from being a component of the replicase complex, some mutational studies suggest that flavivirus NS2A participates in virus particle assembly/secretion and virus-induced cytopathic effects [65; 66; 67]. NS2B has a molecular weight of about 14 kDa. It is a transmembrane protein whose connection with NS3 is autocatalytically cleaved by the viral protease, resulting in NS3 conformational changes, converting NS3 into the more active and stable NS2B/3 protease. This complex is responsible for cleaving various viral proteins [68; 69]. Through comparison with yellow fever virus NS2B, conserved amino acids were site-directed mutagenized, revealing that the mutations P32A, G36A, and G37A completely blocked viral RNA synthesis. The mutations D25A, A43Y, V44A, G92A, and P112A led to minor defects in virus replication. Only the G92A mutation affected virus proliferation by blocking virus release, without affecting viral RNA synthesis, indicating the involvement of G92 in the assembly of infectious TMUV particles [70]. DTMUV NS3 has a molecular weight of approximately 68 kDa and contains an N-terminal domain with a serine protease (peptide_S7), while its C-terminal region harbors NT-Pase and RNA helicase activities (designated as Flavi_DEAD and HELICc, respectively) [71]. The NS3 N-terminal domain peptidase_S7 associates with the cofactor NS2B to form the NS2B-NS3 complex. NS3's C-terminus encompasses three distinct enzyme activities: RTPage, NTPase, and helicase [72]. RTPage cleaves the phosphate bond of 5'-phosphorylated RNA and is considered the first step in the three consecutive enzymatic reactions required for the formation of RNA 5' caps, crucial for viral translation and RNA stability [73]. The RTPage cleaves the phosphodiester bonds of 5'-triphosphorylated RNA, typically considered the first step of the three consecutive enzyme-catalyzed reactions for RNA 5'-end capping, which is crucial for viral translation and RNA stability [74; 75]. The hydrolysis of NTPases provides energy, facilitating the viral unwinding and translocation processes [76]. Current research suggests that the NS3 HELICc domain of flaviviruses can establish an ATP-dependent stable equilibrium between RNA unwinding and annealing, allowing the enzyme's two opposing activities to be regulated by ATP concentration [51; 77; 78; 79; 80]. DTMUV NS4A has a molecular weight of around 14 kDa and is a membrane-associated protein featuring four transmembrane helices and a cytoplasmic N-terminal region. It associates with the viral replicase complex, inducing endoplasmic reticulum (ER) membrane rearrangements. Once the virus genome is translated within the ER, NS4A is cleaved by the NS2B-3 viral protease complex in the cytoplasm [81]. The transmembrane domain of NS4A is cleaved by ER luminal host signalases [82].