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tease. This is the only example known of a metalloprotease in viruses.

Flaviviruses have capped genomes whose translation is cap-dependent. In contrast, the hepacivirus and pestivirus genomes are not capped and have an IRES in the 5' nontrans-lated region. Members of Flaviviridae do not have a poly(A) tail at the 3' end of the RNA. A stable stem-loop structure present at the 3' end of the Flavivirus genome is illustrated in Fig. 3.20. This structure is required for replication of the genomic RNA and for its stability. No nucleotide or amino acid sequence identity can be detected between members of different genera except for isolated motifs that are signatures of various enzymatic functions.

Viruses in the family are enveloped. They mature at intra-cytoplasmic membranes rather than at the plasma membrane.

Genus Flavivirus

There are about 70 known flaviviruses, of which a representative sample is listed in Tables 3.9 and 3.10. All members of the genus are closely related, as illustrated by the dendrogram in Fig. 3.21. They share significant amino acid sequence identity in their proteins, which results in serolog-

ical cross reactivity. Historically, members of this genus were assigned to it on the basis of these cross reactions. Most are arthropod-borne, and they were once referred to as Group B arboviruses. They can be divided into three major groups based on the vector utilized: the mosquito-borne group (which includes yellow fever, the dengue complex, and the Japanese encephalitis complex), the tick-borne encephalitis group (the TBE complex), and a group that lacks an arthropod vector. The last are of limited medical importance and will not be considered here. Notice that in the phylogenetic tree in Fig. 3.21, the tick-borne viruses and the mosquito-borne viruses belong to different lineages. Thus, the viruses are adapted to a tick vector or to a mosquito vector, and interchange of vectors does not occur.

Expression of the Viral Genome

The genome organization of a typical flavivirus is illustrated in Fig. 3.19. The processing of the long polyprotein produced from the genome is complicated and is illustrated in Fig. 3.22 as an example of complex processing events that can occur in viral polyproteins associated with lipid bilayers. The nucleocapsid protein is 5' terminal in the genome and is removed from the precursor polyprotein by

Viruses

■ Tick-borne encephalitis

Arthropod Vectors

Langat bat virus

Kunjin

West Nile

Japanese encephalitis

Dengue 2

Vellow Fever

Dengue 4

Cell fusing agent

None

FIGURE 3.21 Phylogenetic tree of the flaviviruses. The dendrogram illustrates the relationships among flaviviruses, based on the nucleotide sequences encoding the NS5 protein. The cell-fusing agent is a distantly related flavivirus that was found as a contaminant in mosquito cell cultures. Its natural host range and distribution are unknown. [From Marin etal. (1995) reported in Strauss and Strauss (1996, p. 115).]

the viral NS2B/NS3 protease. Two envelope proteins, prM (precursor to M) and E (envelope), follow. Both are anchored in the endoplasmic reticulum by C-terminal membrane-spanning domains and are usually, but not always, glycoproteins. A series of internal signal sequences is responsible for the multiple insertion events required to insert prM, E, and the following protein, NS1 into the endoplasmic reticulum. After separation of these three proteins by signalase, prM and E form a heterodimer. prM is cleaved to M by furin during transport of the heterodimer or during virus assembly. After cleavage, E forms a homod-imer. These events are shown schematically in Fig. 3.23. If cleavage of prM does not occur, an immature form of the virion is produced that is not infectious.

Following the E protein is NS1 (NS for nonstructural). NS1 is a glycoprotein and is required for RNA replication (how is an interesting question since it is external to the cell). Cleavage at its C terminus is by an unknown cellular protease. Although lacking a C-terminal anchor, some fraction of it remains cell associated. Next are two hydrophobic polypeptides called NS2A and NS2B. These proteins are cleaved by the viral NS2B/NS3 protease. They are associated with membranes and may serve to anchor parts of the replication machinery to internal membranes in the cell. NS2B forms a complex with NS3 that activates the serine protease, which cleaves many bonds in the polyprotein. It also has at least two other activities—the middle domain of NS3 is a helicase, required for RNA replication, and the C-terminal domain has RNA triphosphatase activity, an activity that is required for the capping of the viral genome.

NS4A and NS4B are hydrophobic polypeptides that are associated with membranes. They may function in assembly of the viral replicase on intracellular membranes. Both the viral NS2B/NS3 protease and cellular signalase are required to produce the final cleaved products.

NS5 is the viral RNA polymerase. It also has methyl-transferase activity required for capping of the viral genome. Thus, the capping activity is divided between proteins NS3 and NS5. NS5 appears to be a soluble cytoplas-mic protein that associates with membranes through association with other viral peptides.

RNA replication is associated with the nuclear membrane. The composition of the replicase complex is not understood but is assumed to consist of many (most?) of the viral nonstructural proteins with associated cellular proteins.

Structure of the Virion

The nucleocapsid is thought to be icosahedral in symmetry, perhaps having a triangulation number of 3. The structure of E has been solved to atomic resolution for tickborne encephalitis virus (Fig. 3.24). Unlike the glycopro-

Proteolytic Cleavages

■<5= WS2B-3 (alternative) •<" unknown protease <2= Signal as e

FIGURE 3.22 Schematic illustration of the processing of the flavivirus polyprotein precursor into the structural proteins (blue) and the nonstructural proteins (green). As described in the text, some of the cleavages are cotranslational, while others are delayed. The proteases responsible for the various cleavages are colored as shown in the key and the amino termini of the major proteins are labeled "N." The cellular enzyme that cleaves following NS1, the first nonstructural protein, has not been identified. An alternative site of cleavage within NS2A is shown that might lead to an anchored form of NS1. NS2B and NS3 form a complex that involves the central 40 amino acids of NS2B and is required for expression of the proteolytic activity of NS3. This interaction also ties NS3 to the membrane. The orientation of NS4A and NS4B within the membrane has not been determined, but this model is consistent with the sequences of these peptides. [Redrawn from Strauss and Strauss (1996).]

Structural proteins

Intramembrane domain

Nonstructural proteins S Stop transfer signal

Signal sequence teins of other enveloped viruses, the homodimer lies flat along the membrane rather than projecting upward as a spike. Thus, the surface of the flavivirus lacks projecting spikes and is relatively smooth (Fig. 2.5). This also has the effect that the diameter of the flavivirion (about 50 nm) is less than that of many enveloped viruses. For example, the alphaviruses, which are otherwise fairly comparable to flaviviruses, have a diameter of 70 nm). Very recent studies have shown that E of flaviviruses and E1 of alphaviruses have the same structure, and thus were derived from the same ancestral protein. Although the protein structures are the same, they are used in somewhat different ways to construct the protein shell external to the lipid bilayer, as is clear from a comparison of the cryoelectron reconstructions of alphaviruses and flaviviruses in Fig. 2.5.

Flaviviruses mature at intracellular membranes. Budding figures have been described only rarely and assembly may be associated with the complex processing of the viral polyprotein.

Diseases Caused by Flaviviruses

Many flaviviruses are important pathogens of humans. Different viruses may cause encephalitis, hemorrhagic fever with shock, fulminant liver failure, or disease characterized by fever and rash. Several important viruses and their diseases are listed in Tables 3.9 and 3.10. Many of these viruses are individually described below.

Yellow Fever Virus

The type flavivirus is yellow fever virus (YFV), once greatly feared and still capable of causing large epidemics. The virus is viscerotropic in primates, the only natural hosts for it. The growth of the virus in the liver, a major target

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