Dengue virus type 2 (DEN-2), a member of the family Flaviviridae, contains a
single-stranded RNA genome of 10,723 nucleotides (New Guinea C strain), having a type 1 cap at the 5' end, but lacking a poly(A) tract at the 3' end [Westaway et al., 1985; Rice et al., 1986;
Brinton, 1986.; Westaway, 1987; Chambers et al., 1990]. The genomic RNA is of positive-strand polarity, having a single open reading frame that encodes a polyprotein of 3,391 amino acids, which is processed into three structural and at least seven nonstructural proteins so far identified.
The gene order is 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4BNS5-3', where C, prM, and E are the structural proteins and NS1 through NS5 represent the nonstructural proteins. The processing of the polyprotein precursor occurs cotranslationally as well as posttranslationally and is performed by either the host signalase in association with the membranes of the endoplasmic reticulum or the viral protease(s) [Chambers et al., 1990].
Figure 1.4 The genome organization of dengue virus. (Gubler, D. J., Centers for Disease Control and Prevention, Fort Collins, Colorado, USA)
1.5.1 Capsid (C) protein
The dimeric capsid or core protein is one of DEN structural proteins, it is essential in virus
assembly to ensure specific encapsidation of the viral genome. The mature form of DEN C protein is a highly basic protein of 12 kDa after removal of the C-terminal hydrophobic signal sequence. When the polyprotein is processed, the function of C-terminal region for capsid protein is serving as a signal sequence, the capsid protein then anchors into the ER membrane and thus translocates prM into the lumen of the endoplasmatic reticulum. Subsequently, this signal sequence is cleaved by the host cell signalases liberating the N-terminus of prM whereas C remains closely associated with the ER membrane promoting viral assembly. The membrane-associated capsid protein mediates the viral assembly by coordinated interaction with the E-prM heterodimer in the ER. The immune viral particles containing C protein and genomic RNA that forms as the nucleocapsid (NC) are then budding into the lumen of the ER [Wang et al., 2002]. It was reported that the capsid protein is also found in the nucleus and can possibly interact with hnRNP K, suggesting that C protein may play a role in regulation of the dengue lifecycle by controlling apoptosis [Chang et al., 2001].
1.5.2 Envelope (E) protein
The virus attachment and entry are mainly dependent on the envelope (E) protein, the major glycoprotein on the flavivirus particle [Chambers et al., 1990; Monath and Henize, 1996]. The E protein forms an oligomer with the small membrane (M) protein and constitutes most of the accessible virion surface [Lee et al., 2000]. This reflects that the E protein is essential for membrane fusion and mediates the binding to host, it is also the primary antigen that inducing protective immunity and the major antigen for virus neutralization [Rice et al., 1996; Roehrig, 1997]. Therefore, the protein directly affects the host range, cellular tropism and in part, the virulence of DEN virus [Monath and Henize, 1996].
Based on the crystallography data of the tick-borne encephalitis flavivirus E protein, Rey et al. [Rey et al., 1995] noted that each E-protein monomer is folded into three distinct structural domains, domain I, II, and III. The domain I in the central structure of E protein is the antigenic
domain that carries the N glycosylation site. Structural domain II of the E protein is suggested to be responsible for pH-dependent fusion of the viral E protein and the endosomal membrane during uncoating. Structural domain III is reported to play the important role for flavivirus binding to host cells [Rey et al., 1995]. The structural domain III of E protein contains an immunoglobulin-like constant domain and is postulated to form the receptor-binding site for the virus particles.
1.5.3 Membrane (M) and pre-membrane (prM) protein
The flavivirus particle consists of a nucleocapsid core, which is surrounded by an ER-derived lipid bilayer containing E and prM/M, the structural proteins that were synthesized as a polyprotein [Mukhopadhyay et al., 2005]. The prM will be processed to the mature M protein late in secretion in the trans Golgi compartment by furin [Stadler et al., 1997]. Maturation of processed from prM to M protein is necessary to expose the E receptor binding domain and thus for virus infectivity [Heinz and Allison, 2003]. The prM is suggested to protect the E protein from pH-induced reorganization and premature fusion during the secretion of E proein [Guirakhoo et al., 1991; Guirakhoo et al., 1992; Zhang et al., 2003], and the prM protein is possibly to serve as a chaperone for proper E folding and assembly [Heinz and Allison, 2003]. It was reported that regulated efficiency of cleavage of prM/M is important for viral replication [Keelapang et al., 2004].
1.5.4 Non-structural 1(NS1) protein
NS1 is the first non-structural protein in the DEN polyprotein, following E protein and preceding the NS2A protein. It is a 46 kD glycoprotein, with two glycosylated asparagines, and 12 cysteines that form 6 disulfide bridges. Although it does not contain a hydrophobic membrane spanning region, the NS1 is translocated into the lumen of the ER during translation, where it dimerizes and stays membrane associated. The double stranded RNA corroborating evidence
showed that NS1 is essential for viral replication by an unknown mechanism [Muylaert et al., 1997; Flamand et al., 1999; Lindenbach and Rice, 1999]. Additionally, when the NS1 is exported through the Golgi excretory pathway, NS1 can be detected both outside the plasma membrane of infected cells and anchored into the membrane by a glycosyl-phosphatidylinositol (GPI). It has been suggested that the antibodies against NS1 in a DEN infected patient may bind to the NS1 protein both on the cell surface and activate GPI-mediated signaling in the infected cell. The binding of anti-NS1 antibodies is possibly enhancing viral replication or disease pathology [Jacobs et al., 2000]. NS1 is also excreting in a soluble hexameric form from mammalian cells but not from mosquito cells [Flamand et al., 1999]. Soluble NS1 is found in the blood of DEN infected patients [Young et al., 2000; Alcon et al., 2002] and the NS1 blood levels is reported to correlate with disease severity [Libraty et al., 2002; Avirutnan et al., 2006]. Soluble NS1 in the blood is suggested to contribute to DEN pathology by activating complement [Avirutnan et al., 2006], inducing auto-immune antibodies [Lin et al., 2003] or accumulating in hepatocytes in the liver [Alcon-LePoder et al., 2005]. Vaccination studies used to target and reduce free NS1 circulating in the blood have shown different results, some studies showed protection against an intracerebral challenge with DEN [Costa et al., 2005], but other studies did not show protection [Timofeef et al., 2004; Calvert et al., 2006].
1.5.5 NS3 protein
NS3 is a 67 to 70-kDa protein of 618 to 623 amino acids that is highly conserved among flaviviruses. The NS3 is proposed to have two functions in viral replication: serine protease and helicase. A region near the N-terminus of NS3 exhibits sequence and structural homology to the active domain of trypsin related serine protease [Bazan and Fletterick, 1989]. In combination with NA2B, it is required for proteolytic processing at the dibasic site of many viral proteins.
The C-terminus of flaviviruses NS3 is suggested to be involved in several functions including RNA helicase [Gorbalenya et al., 1989], RNA-stimulated NTPase activity [Wengler and Wengler,
1991; Wallner et al., 1993], and the capping and methylation [Wengler and Wengler, 1993].
1.5.6 NS2A, NS2B, NS4A, and NS4B protein
NS2A, NS2B, NA4A, and NS4B are small non-structural proteins. All four proteins are poorly conserved in sequence but exhibit conserved hydrophobicity profiles among flaviviruses.
The evidence suggesting that they are membrane-associated proteins (Chambers T. J., 1990). The functions of these four proteins remain largely undefined. NS2A (18 to 22-kDa of 218 to 231 amino acids) is reported to be required for the C-terminal processing of NS1 [Flagout and Lai, 1989]. NS2B (13 to 15-kDa of 130 to 132 amino acids) is suggested to be involved in the protease function of the NS2B-NS3 complex and essential for protease activity of the complex [Flagout et al., 1993]. The functions for NS4A (16.0 to 16.4-kDa of 149 to 150 amino acids) and NS4B (27 to 28-kDa of 248 to 256 amino acids) have not yet been identified. They may be involved in membrane localization of NS3-NS5 replication complex via protein-protein interaction, since the NS3-NS5 complex is reported to weakly associate with the membrane in spite of its hydrophilic characteristic [Chambers et al., 1990; Wengler et al., 1990].
1.5.7 NS5 protein
The NS5 protein consists of at least three important enzymatic functions which are essential for viral propagation in flaviviruses [Khromykh et al., 1998; Hanley et al., 2002]. The N-terminal region of NS5 protein represents the active domain of S-adenosyl-L-methionine dependent methyltransferase (SAM)(amino acids 1-320), which possess the methyl transferase and guanylyl transferase activities responsible for capping and methylating at the positive strand genomic RNA on its 5’ terminus [Egloff et al., 2002]. The C-terminal domain encodes the RNA dependent RNA polymerase (residues 420-900) responsible for synthesizing the double stranded replicative intermediate RNA template and also plus(+) strand RNA genomic RNA [Bartholomeusz and Thompson, 1999; Egloff et al., 2002; Nomaguchi et al., 2003]. The RNA dependent RNA
polymerase activity of this domain has been demonstrated for several other flaviviruses including West Nile virus, Kunjin virus, Hepatitis C viruses (HCV) and BVDV [Tan et al., 1996;
Khromykh et al., 1998; Steffens et al., 1999; Guyatt et al., 2001]. The RNA dependent RNA polymerase has an essential GDD motif. In Flavivirus, it is shown that the mutations on this motif would result the virus non-replicative [Khromykh et al., 1998; Ranjith-Kumar et al., 2001].