Rebecca Wattam01-24-20050.83The methods of cell invasion and viral multiplication within vertebrate cells are described in general for the Flaviviruses, specifically for Yellow Fever virus, St. Louis encephalitis virus, Louping ill virus, and Powassan virus. These viruses belong to different subgroups within the Flaviviridae; Yellow fever virus belongs to the yellow fever group, St. Louis encephalitis virus belongs to the Japanese encephalitis group, and Louping ill and Powassan virus both belong to the tick-borne encephalitis group of the Flaviviridae.wattam@vbi.vt.eduE proteinTEXTExtracellularLigand binding or carrierThe E protein is the major surface protein of the viral particle, probably interacts with viral receptors, and mediates virus-cell membrane fusion (Lindenbach and Rice, 2001). The flavivirus major envelope glycoprotein has a dual function: as a receptor binding protein, it is the primary determinant of host range, cell tropism, virulence and is a major antigen in eliciting neutralizing antibodies during the immune response (Chu et al., 2005). Based on the structure of the soluble fragment of the tick-borne encephalitis (TBE) virus E protein, domain III has been implicated in binding to cellular receptors. Consequently, some of the virulence determinants that affect the pathogenesis of flaviviruses in animal models have been mapped to the lateral edge of this domain, and these act presumably through effects on virus attachment, post-receptor-binding events associated with virus entry, or both (Vlaycheva et al., 2004).Cell receptor(s)TEXTCell membraneLigand binding or carrierIt is thought that flaviviruses attach to the surface of host cells through an interaction of the E protein with one or more receptors,and many E-reactive antibodies have been shown to neutralize virus infectivity by interfering with virus binding (Lindenbach and Rice, 2001). Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells (Thepparit and Smith, 2004). Our data implicate the {alpha}v{beta}3 integrin, a prominent endothelial cell receptor, as the functional receptor and the associated signaling pathway necessary for WNV entry into vertebrate cells (Chu et al., 2004).Attached virionTEXTCell membraneOtherIt is thought that flaviviruses attach to the surface of host cells through an interaction of the E protein with one or more receptors,and many E-reactive antibodies have been shown to neutralize virus infectivity by interfering with virus binding (Lindenbach and Rice, 2001).Virion in cytoplasmTEXTCytoplasmOtherAfter binding, it is generally believed that virions are taken up by receptor-mediated endocytosis, although direct fusion at the plasma membrane has also been observed (Lindenbach and Rice, 2001). The entry modes of Japanese encephalitis (JE) and dengue-2 (DEN-2) viruses into C6/36 mosquito cells and of DEN-2 virus into human peripheral blood monocytes in vitro were studied. Inoculation of either JE or DEN-2 virions into C6/36 cells resulted in direct penetration of the virions into the cytoplasm at the cell surface in 3 stages. At stage 1, virions attached to the plasma membrane of host cells by their envelope spikes; at stage 2, the virion envelopes approximated to and eventually overlapped the host plasma membrane, and in the process the plasma membrane at the attachment sites dissolved; and, at stage 3, virions penetrated into the cytoplasm through the plasma-membrane disruptions created at the adsorption sites. Virions themselves apparently disintegrated at or near the penetration sites, for no virions were seen in the deeper cytoplasm (Hase et al., 1989).Virion in PhagolysosomeTEXTPhagolysosomeOtherVirions are internalized into clathrin-coated pits via receptor-mediated endocytosis. It is thought that virions are brought into a prelysosomal endocytic compartment where low pH induces fusion between the virus and host cell membranes to release the virus nucleocapsid (Lindenbach and Rice, 2003). Fused virionTEXTPhagolysosomeOtherVirions are later found in uncoated pre-lysosomal vesicles, where an acid-catalyzed membrane fusion is thought to release the nucleocapsid into the cytoplasm. Consistent with this, a conformational change in the viral E proteins, which probably exposes a fusogenic domain, occurs at low pH. Acid pH can promote fusion of virions with liposomal membranes in vitro or at the plasma membrane of cultured cells, although in the latter case this mode of entry does not lead to productive infection (Lindenbach and Rice, 2001). In vitro fusion experiments with pyrene phospholipid-labeled TBEV and artificial liposomes have revealed that the fusion reaction of this virus is a very facile process, occurring faster than that of any other enveloped virus analyzed to date. Exposure of the virus to acidic pH causes dramatic structural changes in the viral fusion protein E that also leads to a quantitative oligomeric switch from dimers to trimers. These changes are irreversible and apparently drive the fusion process (Heinz et al., 2004).NucleocapsidTEXTCytoplasmOtherThe isometric nucleocapsid is composed of a single capsid protein (C) and contains the plus-stranded RNA genome of approximately 11,000 nucleotides (Heinz et al., 2004). Following entry and fusion, nucleocapsids are presumably disassembled, genomic RNA is translated, and RNA is initiated (Lindenbach and Rice, 2001). After fusion has occurred, the NC is released into the cytoplasm, the capsid protein and RNA dissociate, and replication of the RNA genome and particle assembly is initiated (Mukhopadhyay et al., 2005).Capsid ProteinTEXTOtherOtherAfter fusion has occurred, the NC is released into the cytoplasm, the capsid protein and RNA dissociate, and replication of the RNA genome and particle assembly is initiated (Mukhopadhyay et al., 2005).Genomic RNATEXTOtherOtherFollowing entry and fusion, nucleocapsids are presumably disassembled, genomic RNA is translated, and RNA is initiated (Lindenbach and Rice, 2001).Antigenomic RNATEXTCytoplasmOtherThe 5' noncoding region (NCR) sequence is poorly conserved between flaviviruses, but it appears to contain common secondary structural elements that influence the translation of flavivirus genomes. However, the most significant function of the 5' NCR probably resides in its reverse complement, the 3' NCR of the viral minus strands, which forms the site for initiation of plus-strand synthesis (Lindenbach and Rice, 2001). Replication begins with the synthesis of a genome-length minus-strand RNA, which then serves as a template for the synthesis of additional plus-strand RNAs. The first round of minus-strand accumulation has been detected in just over 3 hours after infection. Viral RNA synthesis appears to be asymmetric in vivo, with a plus-strand accumulation more than 10 times greater than that of minus strands. Virus minus strands appear to accumulate even late after infection and have been isolated exclusively in double-stranded forms (Lindenbach and Rice, 2001). This mode of replication, with minus strands serving as templates for the production of multiple plus strands, can be described as semiconservative and asymmetric (Lindenbach and Rice, 2001).Genomic RNATEXTCytoplasmOtherA single input genome can give rise to multiple daughter genomes (Lindenbach and Rice, 2003). RNA synthesis is asymmetric, leading to a 10- to 100-fold excess of positive strands over negative strands (Lindenbach and Rice, 2003).NucleocapsidTEXTCytoplasmOtherThe nucleocapsid core of the mature virion consists of the genomic RNA surrounded by multiple copies of the capsid protein C (Zhang et al., 2003). One of the earliest events in the flavivirus assembly is the formation of the NC, which consists of one copy of the genomic RNA and multiple copies of the capsid protein. Unlike other enveloped viruses, NCs are rarely found in flavivirus-infected cells (although they can be assembled in vitro) which indicates that particle formation is a coordinated process between the membrane associated capsid protein and the prM-E heterodimers in the ER (Mukhopadhyay et al., 2005).Enveloped NucleocapsidTEXTEROtherThis nucleocapsid core is envelped by a 40 A thick lipid bilayer derived from the endoplasmic reticulum of the host cell. Outside the membrane envelope is a layer of 180 copies of the E glycoprotein organized into a herringbone pattern plus 180 copies of the M protein. Both the E and M proteins are anchored in the membrane by their C-terminal domains (Zhang et al., 2003). Because virion particles first become visible in the lumen of the rough endoplasmic reticulum (RER), it is generally believed that particle formation occurs by a still undefined assembly and budding process at the RER membrane (Heinz and Allison, 2003). NCs are rarely found in flavivirus-infected cells (although they can be assembled in vitro) which indicates that particle formation is a coordinated process between the membrane associated capsid protein and the prM-E heterodimers in the ER (Mukhopadhyay et al., 2005).Enveloped NucleocapsidTEXTGolgiOtherCryoimmunoelectron microscopy studies carried out with the Kunjin virus using drugs that inhibit intracellular protein and/or membrane transport support a maturation model that involves virion assembly in the ER, transport of individual particles in transport vesicles to the Golgi apparatus, movement through the individual stacks into the trans-Golgi region, accumulation within large vesicles, and release by exocytosis (Heinz and Allison, 2003).Mature VirionTEXTGolgiOtherThe apparently final maturation step for producing fully infectious virions is the proteolytic cleavage of the prM protein immediately after the sequence Arg-X-Arg/Lys-Arg, which corresponds to the consensus sequence for the cellular protease furin, and enzyme that is concentrated in the trans golgi network but cycles between endosomes and the plasma membrane as well (Heinz and Allison, 2003).Mature VirionTEXTExtracellularOtherAccording to our best structural model, virions contain 180 copies of the E protein, an unknown amount of prM and C, and a single copy of the viral genome (Lindenbach and Rice, 2003).PolyproteinTEXTCytoplasmOtherGenomic RNA is the messenger RNA for translation of a single long open reading frame RNA for translation of a single long open reading frame (ORF) as a large polyprotein (Lindenbach and Rice, 2001). The flavivirus genome is translated as a large polyprotein that is processed co- and posttranslationally by cellular proteases and a virally encoded serine protease into at least 10 discrete products. The N-terminal one quarter of the polyprotein encodes the structural proteins, and the remainder encodes the nonstructural (NS) proteins in the following order: C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Lindenbach and Rice, 2001).anch CTEXTEROtherThe nascent C protein (anchC) contains a C-terminal hydrophobic domain that acts as a signal sequence for translocation of prM into the lumen of the endoplasmic reticulum (Lindenbach and Rice, 2001).Capsid ProteinTEXTCytoplasmOtherC protein (about 11 kD) is highly basic, consistent with its proposed role in forming a ribonucleoprotein complex with packaged genomic RNA. Basic residues are concentrated at the N- and C-termini of C, and they probably act cooperatively to specifically bind genomic RNA. The central portion of C contains a hydrophobic domain that interacts with cellular membranes and may play a role in virion assembly (Lindenbach and Rice, 2001). Mature C protein is generated by a serine protease cleavage at a site upstream of this hydrophobic domain (Lindenbach and Rice, 2001).NS4A-NS4BTEXTEROtherUnprocessed NS3-4A and NS4A-4B have been detected in flavivirus-infected cells, suggesting that polyprotein cleavage in this region may be inefficient or controlled in additionally ways (Lindenbach and Rice, 2001).NS3-NS4ATEXTEROtherUnprocessed NS3-4A and NS4A-4B have been detected in flavivirus-infected cells, suggesting that polyprotein cleavage in this region may be inefficient or controlled in additionally ways (Lindenbach and Rice, 2001).NS4BTEXTEROtherNS4A and NS4B are relatively small (about 16 kd and 27 kd) hydrophobic proteins that are membrane associated (Lindenbach and Rice, 2001). NS4B also localized to presumed sites of RNA replication, but it also appears to be dispersed throughout cytoplasmic membranes and possibly the nucleus (Lindenbach and Rice, 2001). NS4B is posttranslationally modified to a form that appears to be about 2 kd smaller than the nascent protein, although the nature of this modification is unknown (Lindenbach and Rice, 2001).NS4BTEXTNucleusOtherNS4B also localizes to presumed sites of RNA replication, but it also appears to be dispersed throughout cytoplasmic membranes, and possibly the nucleus (Lindenbach and Rice, 2001).NS5TEXTCytoplasmOther NS5, the largest (about 103 kd) and most conserved flavivirus protein, contains sequence homology to RdRPs of other positive-strand RNA viruses, including the invariant Gly-Asp-Asp (GDD) motif common to these enzymes (Lindenbach and Rice, 2001). The NS5 gene of flaviviruses and pestiviruses has been proposed to encode the RNA-dependent RNA polymerase and, in the case of flaviviruses, also to encode a methyltransferase domain. NS5 of DEN-2 was shown to exist in different phosphorylated forms in nuclear and cytoplasmic fractions (Bartholomeusz and Thompson, 1999). The conservation of NS3 and NS5 among flaviviruses and their homology with helicases and polymerases, respectively, suggest enzymatic roles in flavivirus RNA replication. Although both proteins are present in subcellular fractions containing polymerase activity, most of the West Nile NS5 can be removed without significant loss of activity. These investigators pointed out the possibility that only small quantities of replicase-associated NS5 are necessary for the observed polymerase activity (Chambers et al., 1990).NS5TEXTNucleusOtherPrevious studies have reported that NS5 of two other flaviviruses is localized in the nucleus and cytoplasm of infected cells . This study, as shown by immunofluorescence, subcellular fractionation, and immunoprecipitation methods, shows that there are two forms of NS5 present in the cytoplasmic fraction that are separable by SDS-PAGE. Only the form with the slower mobility is predominantly located in the nuclear fraction. The multiple forms of NS5 are due to differential phosphorylation, and the hyperphosphorylated form is located predominantly in the nucleus (Kapoor et al., 1995). prMTEXTEROtherThe nascent C protein (anchC) contains a C-terminal hydrophobic domain that acts as a signal sequence for translocation of prM into the lumen of the endoplasmic reticulum (Lindenbach and Rice, 2001). The N-terminus of prM (about 26 kd) is generated in the ER by host signal peptidase. This processing event appears to require prior processing of anchC by the cytoplasmic viral serine protease, although the temporal order of these cleavages has been questioned (Lindenbach and Rice, 2001). The prM protein has two transmembrane-spanning domains, which contain a stop transfer sequence and a signal sequence (Mukhopadhyay et al., 2005). prM is able to acquire its completely folded structure independently of E and appears to play a chaperone-like role for the folding of E (Heinz and Allison, 2003).ETEXTOtherOtherE protein (about 50 kd) is a type I membrane protein,containing adjacent transmembrane domains in the C-terminus that serve to anchor this protein to the membrane and as the signal sequence for NS1 translocation (Lindenbach and Rice, 2001). E protein homodimers disassociate at low pH, and each monomer reassociates with two adjacent E proteins, perhaps around a three-fold axis of symmetry at the stalk, to form trimeric complexes. E trimers are thought to extend outward from the virion surface, presumably exposing the hydrophobic fusogenic domains. The stem-anchor region of E, for which structure was not determined, contains determinants for E trimer formation and for stabilization of prM-E heterodimers (Lindenbach and Rice, 2001).prMETEXTEROtherNewly synthesized prM and E proteins associate together to form heterodimers (Heinz and Allison, 2003). On the lumenal side of the ER, the prM and E proteins form a stable heterodimer within a few minutes of translation (Mukhopadhyay et al., 2005).Subviral particleTEXTEROtherA common characteristic of the flavivirus surface proteins prM/M and E is their ability to assemble into subviral particles, which are formed in variable amounts as a by product of flavivirus infection. These subviral particles are smaller than infectious virions and do not contain a nucleocapsid (Gehrke et al., 2003). Subviral particles are also produced in the ER, but only contain the glycoproteins and membrane, and lack capsid protein and genomic RNA, making these particles non-infectious (Mukhopadhyay et al., 2005). Initially, immature particles are formed in the lumen of the endoplasmic reticulum. These particles, which contain E and prM proteins, lipid membrane, and NC, cannot induce host-cell fusion, making them non-infectious. (Mukhopadhyay et al., 2005).Subviral particleTEXTGolgiOtherInitially, immature particles are formed in the lumen of the endoplasmic reticulum. These particles, which contain E and prM proteins, lipid membrane, and NC, cannot induce host-cell fusion, making them non-infectious (Mukhopadhyay et al., 2005). The resultant non-infectious, immature viral and subviral particles are transported through the trans-Golgi network (Mukhopadhyay et al., 2005).Subviral particleTEXTExtracellularOtherIn addition to mature virions, smaller noninfectious particles are released from flavivirus-infected cells. These particles are termed slowly sedimenting (70S) hemagglutinin (SHA) because like virions, they can agglutinate red blood cells at low pH. SHA are smaller than virions (approximately 14 nm diameter). These particles contain E and M proteins, but lack nucleocapsids (Lindenbach and Rice, 2003). The released particles only contain the E and M proteins and a lipid membrane (Mukhopadhyay et al., 2005).prTEXTGolgiOtherDuring the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal 'pr' segment (Lindenbach and Rice, 2001).prTEXTExtracellularOtherDuring the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal 'pr' segment, which is secreted into the extracellular medium (Lindenbach and Rice, 2001). Recombinant Subviral ParticleTEXTExtracellularOtherA related or identical type of particle, recombinant subviral particles (RSP) can be produced by cells expressing prM and E alone. RSPs are 30 nm in diameter, less dense than viral particles, and can undergo acid-catalyzed fusion similar to virions (Lindenbach and Rice, 2003). Recombinant subviral particles (RSPs) can also be generated by coexpression of proteins prM/M and E in the absence of protein C and have been shown to resemble whole virions with respect to their structural and functional organization of surface proteins (Gehrke et al., 2003).NS1TEXTEROtherThe NS1 glycoprotein (about 46kd) exists in cell-associated, cell-surface, or extracellular nonvirion forms. NS1 is translocated into the ER lumen and released from the C-terminus of E by signal peptidase (Lindenbach and Rice, 2001). NS1 is cleaved from its downstream neighbor, NS2A, about 10 minutes after synthesis by and unknown, membrane-bound, ER-resident host protease (Lindenbach and Rice, 2001). Several lines of evidence implicate NS1 in the process of RNA replication (Lindenbach and Rice, 2001).NS1TEXTExtracellularOtherNS1 is slowly secreted from mammalian cells and is not secreted from mosquito cells. During secretion, one of the N-linked glycans is modified to contain complex sugars, and three NS1 dimers come together into a soluble hexameric form (Lindenbach and Rice, 2001). It is now understood that the extracellular forms of NS1 strongly elicit humoral immune responses, and immunization with purified or recombinant NS1 can be protective. Furthermore, protective immunity can be passively transferred with antibodies against NS1, apparently by their ability to direct complement-mediated lysis of infected cells via interaction with the cell-surface-associated form of NS1. The secretion of a viral NS protein that elicits protective immune responses is one of the more curious aspects of flavivirus biology that await further inquiry (Lindenbach and Rice, 2001).NS2ATEXTOtherOtherNS2A is a relatively small (about 22 kd), hydrophobic protein of unknown function (Lindenbach and Rice, 2001).NS2A-alphaTEXTOtherOtherIn Yellow fever-infected cells, 22- and 20-kDa forms of NS2A have been identified and both of these forms possess the same N-terminal sequence. The 20-kDa form (called NS2Aalpha) is believed to result from an additional internal cleavage by the NS2B-3 serine protease (Kummerer and Rice, 2002). An alternative cleavage within Yellow Fever NS2a can also be utilized by the viral protease, leading to a c-terminally truncated form of this proteins that is about 2 kd smaller in mass (Lindenbach and Rice, 2001).NS2BTEXTOtherOtherNS2B is a small (about 14 kd) membrane-associated protein containing two hydrophobic domains surrounding a conserved hydrophilic region. It forms a complex with NS3 and is a required cofactor for the serine protease function of NS3 (Lindenbach and Rice, 2001).NS3TEXTCytoplasmOtherNS3 is a large (about 70 kd) cytoplasmic protein that associates with membranes via its interaction with NS2B. NS3 contains several enzymatic activities that implicate this protein in polyprotein processing and RNA replication (Lindenbach and Rice, 2001).NS4ATEXTOtherOtherNS4A and NS4B are relatively small (about 16 kd and 27 kd) hydrophobic proteins that are membrane associated. Based on its subcellular distribution and interaction with NS1, NS4A appears to function in RNA replication, perhaps by anchoring replicase components to cellular membranes (Lindenbach and Rice, 2001).NS2B-3TEXTOtherEnzymeThe flavivirus protease is a complesx of NS2B and NS3 (Wu et al., 2003). The NS2B-3 protease cleaves in both cis and trans configurations, and it mediates cleavages at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 junctions, as well as cleavages that generate the C-termini of mature C and NS4A (Lindenbach and Rice, 2001).NS2B-3TEXTOtherEnzymeThe flavivirus protease is a complesx of NS2B and NS3 (Wu et al., 2003). The NS2B-3 protease cleaves in both cis and trans configurations, and it mediates cleavages at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 junctions, as well as cleavages that generate the C-termini of mature C and NS4A (Lindenbach and Rice, 2001).NS2B-3TEXTOtherEnzymeThe flavivirus protease is a complesx of NS2B and NS3 (Wu et al., 2003). The NS2B-3 protease cleaves in both cis and trans configurations, and it mediates cleavages at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 junctions, as well as cleavages that generate the C-termini of mature C and NS4A (Lindenbach and Rice, 2001).Replication ComplexTEXTOtherNucleic acid bindingTo date, the flavivirus proteins: NS1, the NS3 helicase and the NS5 polymerase have been shown to be directly involved in flaviviral RNA replication and, in addition, NS2B and NS4B are co-localized with the RNA replication complex (Bartholomeusz and Thompson, 1999). Model for the RNA replication cycle of the Flavivirus replication complex. During translation of the ns proteins in cis from genomic RNA, NS3 is assumed to bind to the terminal product NS5 at one or more conserved regions in the N-terminal domain while other polyprotein cleavage products (NS1, NS2, and NS4A) move . On completion of translation, assembly of the RC commences on the 3'UTR via binding of NS2A, probably at the conserved 3'- terminal stem loop at which the NS3 and NS5 components also bind. The location of the complex shown on the loop is arbitrary. The complex still attached to the RNA (+) stand is transported to the membrane site of replication by affinity of the hydrophobic regions of NS2A interacting with those of NS4A, which in turn is bound by its hydrophilic extensions into the lumen between transmembrane domains to dimeric NS1 in the lumen. The RC is now complete and may undergo rearrangement as the RdRp domains of NS5 bind to the template, which circularizes (Westaway et al., 2003).Replication ComplexTEXTOtherNucleic acid bindingTo date, the flavivirus proteins: NS1, the NS3 helicase and the NS5 polymerase have been shown to be directly involved in flaviviral RNA replication and, in addition, NS2B and NS4B are co-localized with the RNA replication complex (Bartholomeusz and Thompson, 1999). Model for the RNA replication cycle of the Flavivirus replication complex. During translation of the ns proteins in cis from genomic RNA, NS3 is assumed to bind to the terminal product NS5 at one or more conserved regions in the N-terminal domain while other polyprotein cleavage products (NS1, NS2, and NS4A) move . On completion of translation, assembly of the RC commences on the 3'UTR via binding of NS2A, probably at the conserved 3'- terminal stem loop at which the NS3 and NS5 components also bind. The location of the complex shown on the loop is arbitrary. The complex still attached to the RNA (+) stand is transported to the membrane site of replication by affinity of the hydrophobic regions of NS2A interacting with those of NS4A, which in turn is bound by its hydrophilic extensions into the lumen between transmembrane domains to dimeric NS1 in the lumen. The RC is now complete and may undergo rearrangement as the RdRp domains of NS5 bind to the template, which circularizes (Westaway et al., 2003).Replication ComplexTEXTOtherNucleic acid bindingTo date, the flavivirus proteins: NS1, the NS3 helicase and the NS5 polymerase have been shown to be directly involved in flaviviral RNA replication and, in addition, NS2B and NS4B are co-localized with the RNA replication complex (Bartholomeusz and Thompson, 1999). Model for the RNA replication cycle of the Flavivirus replication complex. During translation of the ns proteins in cis from genomic RNA, NS3 is assumed to bind to the terminal product NS5 at one or more conserved regions in the N-terminal domain while other polyprotein cleavage products (NS1, NS2, and NS4A) move . On completion of translation, assembly of the RC commences on the 3'UTR via binding of NS2A, probably at the conserved 3'- terminal stem loop at which the NS3 and NS5 components also bind. The location of the complex shown on the loop is arbitrary. The complex still attached to the RNA (+) stand is transported to the membrane site of replication by affinity of the hydrophobic regions of NS2A interacting with those of NS4A, which in turn is bound by its hydrophilic extensions into the lumen between transmembrane domains to dimeric NS1 in the lumen. The RC is now complete and may undergo rearrangement as the RdRp domains of NS5 bind to the template, which circularizes (Westaway et al., 2003).prTEXTGolgiOtherA pH-induced irreversible conformational change involving the prM and E proteins occurs in the trans-Golgi network, which is followed by cleavage of prM by furin (Mukhopadhyay et al., 2005).prTEXTExtracellularOtherDuring the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal 'pr' segment, which is secreted into the extracellular medium (Lindenbach and Rice, 2001).FurinTEXTGolgiEnzymeDuring the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal "pr" segment (Lindenbach and Rice, 2001).FurinTEXTGolgiEnzymeDuring the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal "pr" segment (Lindenbach and Rice, 2001).TEXTFlavivirus particles bind to cells via interactions between the viral surface glycoprotein and cellular receptors (Lindenbach and Rice, 2003). The E glycoprotein is the major antigenic determinant on virus particles and mediates binding and fusion during virus entry (Lindenbach and Rice, 2003).TEXTFlaviviruses enter host cells by receptor-mediated endocytosis (Mukhopadhyay et al., 2005). Virions penetrated into the cytoplasm through the plasma-membrane disruptions created at the adsorption sites. Virions themselves apparently disintegrated at or near the penetration sites, for no virions were seen in the deeper cytoplasm (Hase et al., 1989).TEXTThe acidic environment of the endosome triggers an irreversible trimerization of the E protein that results in fusion of the viral and cell membranes (Mukhopadhyay et al., 2005). At low pH, the E protein undergoes a conformational change involving dissociation of the E dimer, thereby exposing a hidden fusion peptide followed by reorganization of E into a trimer, in which the fusion peptide is brought close to the membrane-anchoring carboxy terminus. Remarkably similar structural features and conformational rearrangements have been noted between the flavivirus E protein and the alphavirus E1, suggesting a common evolutionary origin for these two virion surface proteins (Anderson, 2003).TEXTAfter fusion has occurred, the NC is released into the cytoplasm (Mukhopadhyay et al., 2005).TEXTAfter fusion has occurred, the NC is released into the cytoplasm, the capsid protein and RNA dissociate, and replication of the RNA genome and particle assembly is initiated (Mukhopadhyay et al., 2005).TEXTFlavivirus replicaiton is entirely cytoplasmic and budding in general occurs into the luemn of the rough endoplasmic reticulum (RER) cisternae (Galler et al., 1997). According to our best structural model,virions contain 180 copies of the E protein, an unknown amount of prM and C, and a single copy of the viral genome. Thus, translation of the viral genome must occur at least 180 times for every nascent genome that is produced and packaged (Lindenbach and Rice, 2003). A single input genome can give rise to multiple daughter genomes (Lindenbach and Rice, 2003). A number of polymerase assays have been developed to investigate RNA synthesis in flavivirus-infected cells. Chu and Westaway proposed, from Kunjin virus RNA-labeling studies, that flavivirus RNA synthesis occurred through a semiconservative and asymmetric replication cycle. In their model, positive strands were preferentially synthesized from the replicative-form dsRNA template. Their model is supported by the data that purified radiolabelled replicative-form Dengue-2 RNA could be utilized as a template when added to polymerase assays containing Dengue-2-infected cell lysate (Bartholomeusz and Thompson, 1999). Utilization of the flavivirus genome as a template for translation is probably more efficient than its use as a template for replication (Lindenbach and Rice, 2003). Genomic RNA is the messenger RNA for translation of a single long open reading frame RNA for translation of a single long open reading frame (ORF) as a large polyprotein (Lindenbach and Rice, 2001). During translation of the ns proteins in cis from the genomic RNA, NS3 is assumed to bind to the terminal product NS5 at one or more conserved regions in the N-terminal domain while other polyprotein cleavage products (NS1, NS2A, and NS4) move to the sites as indicated (Westaway et al., 2003).TEXTFlavivirus RNA replication has been studied in infected cells and also by the use of polymerase assays on extracts from infected cells. Flavivirus RNA synthesis occurs in the cytoplasm in membrane-induced vesicles. There is co-localization of a number of the non-structural viral proteins with dsRNA inside these vesicles, and polymerase activity was associated with membrane fractions (Bartholomeusz and Thompson, 1999). Genome replication occurs on intracellular membranes (Mukhopadhyay et al., 2005). A number of polymerase assays have been developed to investigate RNA synthesis in flavivirus-infected cells. Chu and Westaway proposed, from Kunjin virus RNA-labeling studies, that flavivirus RNA synthesis occurred through a semiconservative and asymmetric replication cycle. In their model, positive strands were preferentially synthesized from the replicative-form dsRNA template. Their model is supported by the data that purified radiolabelled replicative-form Dengue-2 RNA could be utilized as a template when added to polymerase assays containing Dengue-2-infected cell lysate (Bartholomeusz and Thompson, 1999).TEXTDuring translation of the polyprotein, the structural proteins are translocated and anchored in the ER by various signal sequences and membrane anchor domains (Mukhopadhyay et al., 2005). Host signal peptidase is responsible for cleavage among C-prM, prM-E, E-NS1, and near the C terminus of NS4A (Lindenbach and Rice, 2003). The C protein at the NH2-terminal end of the flavivirus polyprotein is separated from the prM protein by an internal signal sequence which directs translocation of prM, a type I transmembrane protein. The viral NS2B-3 protease catalyzes the cytoplasmic cleavage at the COOH terminus of C; interestingly, in the absence of C protein cleavage, ER luminal signalase cleavage of prM is inefficient (Lobigs and Lee, 2004). The NS2B-3 protease cleaves at a site within NS4A, just upstream of this signal sequence and mutations that block this cleavage also block subsequent signal peptidase cleavage in the ER lumen to generate the N-terminus of NS4B. (Lindenbach and Rice, 2001). The NS2B-3 protease cleaves in both cis and trans configurations, and it mediates cleavages at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 junctions, as well as cleavages that generate the C-termini of mature C and NS4A (Lindenbach and Rice, 2001). The enzyme responsible for NS1-2A cleavage is presently unknown (Lindenbach and Rice, 2003).TEXTThe nascent C protein (anchC) contains a C-terminal hydrophobic domain that acts as a signal sequence for translocation of prM into the lumen of the endoplasmic reticulum. Mature C protein is generated by viral serine protease cleavage at a site upstream of this hydrophobic domain (Lindenbach and Rice, 2001). The NS2B-3 protease cleaves in both cis and trans configurations, and it mediates cleavages at the NS2A/NS2B, NS2B,NS3, NS3/NS4A, and NS4B/NS5 junctions, as well as cleavages that generate the C-termini of mature C and NS4A (Lindenbach and Rice, 2001).TEXTOne of the earliest events in the flavivirus assembly is the formation of the NC, which consists of one copy of the genomic RNA and multiple copies of the capsid protein. (Mukhopadhyay et al., 2005).TEXTInitially, immature particles are formed in the lumen of the endoplasmic reticulum (ER). These particles, which contain E and prM proteins, lipid membrane and NC, cannot induce host-cell fusion, making them noninfectious (Mukhopadhyay et al., 2005). Virus assembly occurs on the surface of the endoplasmic reticulum when the structural proteins and newly synthesized RNA buds into the lumen of the ER (Mukhopadhyay et al., 2005). TEXTVirus assembly occurs on the surface of the endoplasmic reticulum when the structural proteins and newly synthesized RNA buds into the lumen of the ER. The resultant non-infectious, immature viral and subviral particles are transported through the trans-Golgi network (Mukhopadhyay et al., 2005).TEXTCleavage of prM occurs in the trans-Golgi network, which creates mature, infectious particles (Mukhopadhyay et al., 2005). The immature virion particles are cleaved by the host protease furin, resulting in mature infectious particles (Mukhopadhyay et al., 2005). During the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal 'pr' segment (Lindenbach and Rice, 2001). prM is cleaved into a soluble peptide and a virion-associated M protein (approximately 8 to 8.5 kDa by trans-Golgi resident furin, resulting in two different forms of virion: the intracellular , E- and prM- containing virion, and the extracellular form, E- and M- cotatining virions (Keelapang et al., 2004).TEXTMature virus and subviral particles are released from the host cell by exocytosis (Mukhopadhyay et al., 2005).TEXTDuring the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal 'pr' segment, which is secreted into the extracellular medium (Lindenbach and Rice, 2001).TEXTNewly synthesized prM and E proteins associate together to form heterodimers (Heinz and Allison, 2003). On the lumenal side of the ER, the prM and E proteins form a stable heterodimer within a few minutes of translation (Mukhopadhyay et al., 2005). Cosynthesis of E and prM is necessary for proper folding of E (Lindenbach and Rice, 2001).TEXTSubviral particles are also produced in the ER, but only contain the glycoproteins and membrane, and lack capsid protein and genomic RNA, making these particle non-infectious (Mukhopadhyay et al., 2005).TEXTA related or identical type of particle, recombinant subviral particles (RSP) can be produced by cells expressing prM and E alone. RSPs are 30 nm in diameter, less dense than viral particles, and can undergo acid-catalyzed fusion similar to virions (Lindenbach and Rice, 2003).TEXTVirus assembly occurs on the surface of the endoplasmic reticulum when the structural proteins and newly synthesized RNA buds into the lumen of the ER. The resultant non-infectious, immature viral and subviral particles are transported through the trans-Golgi network (Mukhopadhyay et al., 2005). Subviral particles are also cleaved by furin (Mukhopadhyay et al., 2005). During the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal 'pr' segment (Lindenbach and Rice, 2001).TEXTMature virus and subviral particles are released from the host cell by exocytosis (Mukhopadhyay et al., 2005).TEXTDuring the egress of virions through the secretory pathway, prM is cleaved by the trans-Golgi resident enzyme furin, to form the structural protein M (about 8 kd) and the N-terminal 'pr' segment which is secreted into the extracellular medium (Lindenbach and Rice, 2001).TEXTNS1 is slowly secreted from mammalian cells and is not secreted from mosquito cells. During secretion, one of the N-linked glycans is modified to contain complex sugars, and three NS1 dimers come together into a soluble hexameric form (Lindenbach and Rice, 2001).TEXTAn alternative cleavage within Yellow Fever NS2a can also be utilized by the viral protease, leading to a c-terminally truncated form of this proteins that is about 2 kd smaller in mass (Lindenbach and Rice, 2001).TEXTThe template is then copied into RNA (-) by the polymerase activity of NS5 as part of the newly formed replication complex with the consensus composition NS5, NS3, NS1, NS2A, and NS4A (Westaway et al., 2003).TEXTExpression of the N-terminal 167 to 181 residues of NS3, together with NS2B, is sufficient to form the active protease. The NS2B-3 protease cleaves in both cis and trans configurations, and it mediates cleavages at the NS2A/NS2B, NS2B,NS3, NS3/NS4A, and NS4B/NS5 junctions, as well as cleavages that generate the C-termini of mature C and NS4A (Lindenbach and Rice, 2001).TEXTNS4B also localizes to presumed sites of RNA replication, but it also appears to be dispersed throughout cytoplasmic membranes, and possibly the nucleus (Lindenbach and Rice, 2001).TEXT>Previous studies have reported that NS5 of two other flaviviruses is localized in the nucleus and cytoplasm of infected cells . This study, as shown by immunofluorescence, subcellular fractionation, and immunoprecipitation methods, shows that there are two forms of NS5 present in the cytoplasmic fraction that are separable by SDS-PAGE. Only the form with the slower mobility is predominantly located in the nuclear fraction. The multiple forms of NS5 are due to differential phosphorylation, and the hyperphosphorylated form is located predominantly in the nucleus (Kapoor et al., 1995).TEXTTEXTTEXTMembers of the family Flaviviridae are small, isocahedral, enveloped viruses that contain a positive-sense RNA genome. The family consists of three genera: Flavivirus, Pestivirus and Hepacivirus, which includes hepatitis C virus (HCV). Many of the viruses belonging to this family are important human pathogens, including Japanese encephalitis virus (JEV), yellow fever virus (YFV), the four dengue virus serotypes (DEN-1, DEN-2, DEN-3, DEN-4) and HCV, and animal pathogens including bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV) and border disease virus (BDV) from sheep (Bartholomeusz and Thompson, 1999).Hase T, Summers PL, Eckels KH.Arch Virol.19891041-2129143Chu JJ, Rajamanonmani R, Li J, Bhuvanakantham R, Lescar J, Ng ML. J Gen Virol200586Pt 2405412Vlaycheva L, Nickells M, Droll DA, Chambers TJ.Virology20043271419Thepparit C, Smith DR. J Virol.200478221264712656Chu JJ, Ng ML. J Biol Chem.2004279525453354541Lindenbach BD, Rice CM.Adv Virus Res.2003592361Anderson R.Adv Virus Res.200359229274Heinz FX, Stiasny K, Allison SL.Arch Virol Suppl200418133137Bartholomeusz A, Thompson P.J Viral Hepat199964261270Mukhopadhyay S, Kuhn RJ, Rossmann MG.Nat Rev Microbiol2005311322Lobigs M, Lee E.J Virol.2004781178186Westaway EG, Mackenzie JM, Khromykh AA.Adv Virus Res20035999140Kapoor M, Zhang L, Ramachandra M, Kusukawa J, Ebner KE, Padmanabhan R.J Biol Chem1995270321910019106Heinz FX, Stiasny K, Allison SL.Arch Virol Suppl200418133137 Gehrke R, Ecker M, Aberle SW, Allison SL, Heinz FX, Mandl CW.J Virol.2003771689248933Galler R, Freire MS, Jabor AV, Mann GF.Braz J Med Biol Res.1997302157168Keelapang P, Sriburi R, Supasa S, Panyadee N, Songjaeng A, Jairungsri A, Puttikhunt C, Kasinrerk W, Malasit P, Sittisombut N.J Virol.200478523672381Kummerer BM, Rice CM.J Virol2002761047734784Zhang Y, Corver J, Chipman PR, Zhang W, Pletnev SV, Sedlak D, Baker TS, Strauss JH, Kuhn RJ, Rossmann MG.EMBO J2003221126042613Wu CF, Wang SH, Sun CM, Hu ST, Syu WJ.J Virol Methods.200311414554TEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXTLindenbach BD and Rice CMFlaviviridae: The Viruses and Their ReplicationKnipe DM and Howley PM.2001991-1041Lippincott Williams and WilkinsTEXTTEXTTEXTTEXTTEXTTEXTTEXTTEXT

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