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2.5.2.1. Non-Structural Protein 1
The NS1 protein is a variably glycosylated, ≈46kDa, protein that is translocated into the ER during translation of the viral mRNA17,21. NS1 folds into its mature structure and
forms highly stable dimers with affinity for membrane interaction109,110.
NS1 is mainly intracellular, but can also follow the secretory pathway and can localize to the cell membrane or be secreted17,110.
Although its exact involvement remains unclear, studies have shown that intracellular NS1 plays an essential role as a cofactor for viral RNA replication111,112. The finding of
NS1 co-localising at sites of RNA replication and with dsRNA intermediates, have suggested a structural role within the replication complex in close interaction with NS4A111,113,114.
Extracellular NS1 can be found as a secreted hexamer or as a membrane-bound dimer115,116. The secreted soluble forms have been implicated in viral pathogenesis117-119, evasion of immune response120,121 and induction of autoimmunity122,123, while the
membrane-anchored NS1 has been linked with a signal transduction capacity that might affect viral replication inside infected cells124.
More recently, a series of studies have successfully connected NS1 from dengue virus to disruption of the endothelial cell integrity by inducing degradation of proteoglycans present on the cell surface125 and by inducing a strong pro-inflammatory response via
TLR4 signalling, leading to endothelium hyper-permeability126,127.
2.5.2.2. Non-Structural Protein 2A
NS2A is a ≈22kDa, hydrophobic protein (Fig. 5) that is not well conserved among flaviviruses but seems to share similar structural features, the most important being the presence of an internal hydrophobic domain that suggests a transmembrane topology17,128. Regarding its functions, several studies have demonstrated a role for NS2A
in viral assembly129,130, particularly in the process of generating viral-induced membrane
structures which seem to be pivotal for the assembling process130. Additionally, NS2A not
only co-localises with the double-stranded RNA (a replication intermediate) foci within infected cells, but also binds strongly to the 3’UTR region of the flaviviral genome, NS3
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and NS5131. Taken together these results suggest that NS2A might also have a role in
RNA replication with a function in coordinating the transition between RNA replication and viral assembly that leads to the generation of new viral particles86,131.
Recently, NS2A has also been linked to a role in modulation of the host immune response by inhibiting interferon signals in infected cells132,133.
2.5.2.3. Non-Structural Protein2B
NS2B is a (≈14kDa) membrane-associated protein that associates with NS3 to activate the serine protease activity of NS3. In this sense NS2B serves as a cofactor for the viral protease134,135. Structurally, NS2B is formed by a hydrophilic central domain
between two hydrophobic regions at the N- and C-terminus17. Residues 67-80 within the
hydrophilic domain are critical for the cofactor activity of NS2B and had been shown to change the structure of NS3 activating its proteolytic activity136,137. The hydrophobic
domains of NS2B also mediate the association of the NS2B-NS3 complex to the ER membrane138,139.
2.5.2.4. Non-Structural Protein 3
NS3 (≈70kDa) is a multifunctional protein that contains several enzymatic activities involved in the processing of viral proteins and replication of viral genome86,140.
The N-terminal third of NS3 bears the chymotrypsin-like, serine protease catalytic domain involved in post-translational processing of the viral polyprotein17,134,141. The
enzyme is formed by two -barrels (each one consisting of 6 -strands) with the catalytic triad (His51, Asp75, Ser135) located between them86,142; binding of NS2B stabilises NS3
structure activating the substrate binding site and the protease activity136,143. The NS3
cleavage site requires two basic aminoacids (dibasic (Arg/Lys)-Arg motif) followed by a small, un-branched aminoacid at P1’ position144.
Following a poorly conserved inter-domain linker, the C-terminal portion of NS3 carries domains with several functions associated with RNA replication including: sites for RNA binding, RNA helicase, RNA-stimulated nucleoside triphosphatase (NTPase) and RNA triphosphatase (RTPase)17,140. The helicase structure comprises 3 subdomains: the
helicase activity is contained within a complex of -sheets and -helices and has been proven to be essential for viral replication142,145; the subdomain III is unique to the
flaviviruses and contains a small region of highly basic aminoacids that may serve as site for protein and RNA binding, specifically to the 3’SL and the NS5 protein, during viral replication146,147. In addition to these proposed functions, subdomain III also encodes for
the RTPase activity involved in dephosphorylating the 5’ end of the genomic RNA for the subsequent addition of the cap148,149.
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NS3 has also been involved in other functions regarding virus assembly150 and
modulation of intracellular environment151-153. In particular, the NS2B/NS3 protease
complex is directly involved in cleaving the human mediator of interferon regulatory factor 3 activator (STING) which down-regulates the intracellular defence against viral infections154
2.5.2.5. Non-Structural Protein 4A
NS4A is a small, hydrophobic protein (≈16kDa) that consists of an initial cytoplasmic N-terminal sequence, a central hydrophobic region formed by three different domains that are tightly associated with the ER membrane, and a final C-terminal sequence that acts as a signal for the translocation of NS4B into the ER155,156. Among its many proposed
functions, NS4A is reported to be a cofactor for the NS3 helicase157-159; additionally, the
membrane-interacting domains of the protein have been reported to participate in inducing membrane rearrangements for the viral replication site as well as in anchoring the replication complex157,160-162.
Additional functions seem to involve modulation of the cellular environment163 and
inhibition of immune response by blocking IFN signalling133,164.
2.5.2.6. Non-Structural Protein 4B
NS4B is a small (27kDa) hydrophobic protein closely associated with the ER membrane through at least 4 transmembrane domains, although its complete structure and topology is not fully understood156,165,166. Little is known about the actual role of NS4B
during viral infection but it has been proved to be essential for viral RNA replication167,168.
In particular, the protein co-localises to the RNA replication sites169, aids in the
disassociation of the NS3 and the replication intermediates168,170, and induces the
formation of specialized membrane compartments for replication168.
In addition to these functions, NS4B has also been described as suppressor of cellular interference RNA and immune responses171-173.
2.5.2.7. Non-Structural Protein 5
Located at the C-terminus of the viral polyprotein, NS5 is the largest (≈105kDa) and most conserved of the flaviviral proteins with two main functions: it serves as the viral RdRp and as a methyltransferase (MTase) involved in cap-processing of the RNA17,174.
The N-terminal region of NS5 carries the MTase function with close homology to S- adenosyl-methionine (SAM) dependent methyltranferases motifs175,176. The enzymatic
domain is conserved, both in structure and function, among all flaviviruses, with data showing its capacity to sequentially catalyse a guanine N-7 and a ribose2’-O-
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methylation174,177. Its involvement in capping of the viral genome was further supported by
studies demonstrating the lethal effect of mutations disrupting the MTase activity178,179.
The C-terminus of NS5 contains the viral RdRp enzyme, which has significant homology to other single-strand, positive-sense RNA viruses’ polymerases180,181.
The central region between the MTase and the RdRp domains has nuclear localization signals that effectively guide a fraction of the viral protein to the nucleus of infected cells suggesting additional roles for NS5 probably involving suppression of cellular immune responses although this has not yet been confirmed182,183. Finally, this
interdomain region also mediates the interactions with the 3’SL element of the viral genome146 and with NS3, which regulates its NTPase-RTPase activities184.
Moreover, in the case of dengue, NS5 mediates STAT2 degradation by interaction with the host UBR-4 (ubiquitin protein ligase E3 component N-recognin 4) protein, thus interfering with the IFN signalling pathway185.