Flavivirus structure

Eor many fiaviviruses, a subviral particle is released from infected cells that contains the antigenic properties of native virus but lacks the genome RNA and core protein and is thus noninfectious. These subviral particles are two-thirds the size of the native particle and appear to undergo the same type of maturation process in which the prM protein is cleaved in a late compartment by furin. Several studies have demonstrated that similar subviral particles (RSPs) can be produced by means of coexpression of prM and E in eukaryotic cell culture (Schalich et al, 1996). Cryo-EM analysis of TBEV RSPs demonstrated that the particles were smooth on the outside as predicted from the earlier structural studies on the E protein 

Although the positions of all E dimers in the outer shell of the particle were known, a precise interpretation of the density contributed by the M protein was not possible. This was due to the lack of detailed information concerning the C-terminal 101 amino acids of the E protein that were missing from the crystal structure. These residues form the stalk region, the transmembrane domain, and the NSl signal sequence. Approximately 52 residues would compose the stalk and are found in a shell of density in which the short M protein (37 amino acids outside of the membrane) would also be predicted to be found. Together, the M and the E proteins completely cover the lipid bilayer so that there is no exposed membrane in the dengue particle. 

Unlike the nucleocapsid core found in the alphaviruses, the flavivirus core is an open structure with no well-defined subunit organization. At the current resolution of flavivirus cryo-EM reconstructions, little can be said about the transmembrane domains that cross the bilayer or the possible contacts the envelope proteins might make with the underlying core (Kuhn et al, 2002). 

Although the alphaviruses and flaviviruses share similarities in overall architecture as icosahedral enveloped viruses and exhibit striking structural similarities in their fusion proteins, several aspects of their 

Ear less is known about the process of flavivirus assembly. Electron microscopy has shown that immature virions can be found in the lumen of the endoplasmic reticulum (Murphy, 1980). The nucleocapsid core is not assembled free in the cytoplasm rather, its assembly appears to take place on the cytoplasmic face of membranes with which prM and E proteins are associated (Khromykh et al., 2001). The carboxy-terminal signal sequence of the precursor to the capsid protein is thought to anchor that protein to the membrane (Amberg et al., 1994). This should allow interactions to occur between the capsid protein and the envelope proteins, which are also anchored to the membrane but reside in the lumen of the endoplasmic reticulum or vesicles. The capsid protein also contains a conserved stretch of hydrophobic residues located roughly in the middle of the protein that has been suggested to serve as an additional or alternative membrane anchor (Markoff et al., 1997). 

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Although there are mechanistic differences between retroviruses, paramyxoviruses, and the orthomyxovirus influenza, the viruses discussed to this point have definite structural and functional similarities including spikelike, trimeric native structures and the presence of coiled coils in their fusion-active subunits. The flaviviruses and alphaviruses, however, appear to be another class of enveloped viruses entirely. Flaviviruses include yellow fever. West Nile virus. Dengue virus, and tick-borne encephalitis virus (TBEV). Alphaviruses, of the togavirus family, include... 

These structural studies on the alphavirus and flavivirus membrane fusion proteins strengthen the relationship between these virus families, but apparently distance them from the trimeric influenza and retrovirus envelope assemblies. However, low pH specifically triggers not only a conformational change in alphavirus and flavivirus surface proteins, but also an oligomerization switch to a trimeric state (Allison et ah, 1995). This... 

Fig. 13. Flavivirus and alphavirus envelope protein structures. (A) Side view of the TBEV E protein ectodomain (Rey et al, 1995). One promoter of the dimer is shaded and the other is white. The black curve below the structure represents the approximate curvature at the membrane of a virus with radius 250 A. (B) Top view of the TBEV E protein dimer with the same shading as in (A). (C) Ribbon trace of the SFVEl protein. [Figure courtesy of Felix Rey.]...

Stiasny et al, 1996). This region fell outside the proteolytically released ectodomain of the TBEV E protein and SFV El structures, and its conformation is not known in the native state. Although the trimeric form of an alphavirus El protein has been analyzed by proteolysis (Gibbons and Kielian, 2002), no high-resolution structures of the low-pH trimerized state are yet available for alphaviruses or flaviviruses, so a further structural comparison with flu cannot be made at this time. 

Unfortunately, there are no structures available for either the flaviviruses or alphaviruses under conditions approximating the fusion state. For both groups of viruses, entry is believed to occur following attachment of the virus to the cellular receptor and internalization of the particle into an endosome (Kielian, 1995 Heinz and Allison, 2001). Acidification of the endosome results in rearrangement of envelope proteins and subsequent insertion of the fusion peptide into the endosomal membrane (Levy-Mintz and Kielian, 1991 Allison et al., 2001). Ultimately this results in fusion of cellular and viral membranes and release of the nucleocapsid core and genome RNA into the cytoplasm of the infected cell. In vitro experiments... 

Endogenous transcription/exit pathways, of double-stranded RNA viruses, 231-232 Envelope protein structures in alphavirus, 369 in flavivirus, 369, 383, 384 of murine leukemia-related viral group (MuLV), 361... 

Mukhopadhyay, S., Kuhn, R.J., and Rossmann, M.C. (2005) A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology, 3, 13-22. 

Flaviviruses are small, enveloped viruses, with cubic symmetry, approximately 45 nm in diameter, which replicate in vertebrate and invertebrate cells. The nucleocapsid contains the single-stranded plus RNA associated with a nucleocapsid protein, C, which has a molecular weight of 13K. The viral envelope consists of one large glycoprotein, E, of molecular weight approximately 55K, and a small 8K membrane-associated protein, M, which is not glycosylated (Wes-taway, 1980 Matthews, 1982). The structural components of flaviviruses have been reviewed by Russell et al. (1980) and the replication strategy of these viruses reviewed by Westaway (1980). 

Westaway (1977) has proposed that three structural polypeptides, C, E, and M as well as five nonstructural polypeptides are separately initiated and terminated during translation, which would make the flavivirus mRNA unique since most other animal mRNAs studied to date have only a single translation initiation site, with a few exceptions (Kozak, 1981 Strauss and Strauss, 1983). On the other hand, Wengler et al. (1979) and Svitkin et al. (1981) reported that in an in vitro translation system only a single initiation site appeared to be used. The entire sequence of the flavivirus genome as well as amino acid sequence data for the polypeptides will be required to decide between these alternative translation strategies. 

Infection of A. albopictus cells with alphaviruses leads to the synthesis of maximum titers of infectious virus by approximately 24 hr postinfection (acute phase). At the acute phase, up to 85% of the cells released infectious virus (Davey and Dalgarno, 1974), and the rate of synthesis of intracellular 42 S and 26 S viral RNA and structural proteins is maximal. By 48 hr postinfection, viral 26 S RNA and protein syntheses are inhibited, and the proportion of cells releasing virus is dramatically reduced (Davey and Dalgarno, 1974 Eaton, 1979). Inhibition of 42 S RNA synthesis occurs at 3 days after infection (start of chronic phase see Fig. 2). The nature of the factor(s) responsible for inhibiting viral replication in infected mosquito cells at the chronic phase is not known. The fact that large amounts of 42 S RNA are made in infected mosquito cells 48 hr after infection, at a time when viral structural protein synthesis is inhibited, suggests that the replication inhibition factor may act initially at the level of viral protein synthesis (Eaton, 1979). Eaton demonstrated that viral structural protein synthesis is inhibited before a decrease in 26 S RNA synthesis is detected in SIN-infected A. albopictus cells. Thus, the biphasic nature of alphavirus infection is also observed in the case of flavivirus infection in mosquito cells (Paul et al., 1969). 

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