The sequence of the entire RNA genome of the type flavivirus, yellow fever virus, has been obtained. Inspection of this sequence reveals a single long open reading frame of 10,233 nucleotides, which could encode a polypeptide of 3411 amino acids. The structural proteins are found within the amino-terminal 780 residues of this polyprotein; the remainder of the open reading frame consists of nonstructural viral polypeptides. This genome organization implies that mature viral proteins are produced by posttranslational cleavage of a polyprotein precursor and has implications for flavivirus RNA replication and for the evolutionary relation of this virus family to other RNA viruses.

The first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal beta barrels of domain II of the glycoprotein E are inserted into the cellular membrane.

SummaryThe first structure of a flavivirus has been determined by using a comThe first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal β barrels of domain II of the glycoprotein E are inserted into the cellular membrane.

We present fine mapping of a cis-acting nucleotide sequence found in the 5 region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3 region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5 region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquitoborne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.Flaviviruses are small enveloped viruses. The virion contains a positive-strand RNA genome of about 10.7 kb encapsidated by 180 copies of the capsid (C) protein in a nucleocapsid core, which is enveloped by a lipid bilayer in which 180 copies of each of two transmembrane proteins, envelope (E) and membrane (M), are anchored (2, 9). The flaviviruses include a large number of serious human pathogens such as yellow fever virus (YFV), four serotypes of dengue virus (DENV), Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus. Although good vaccines have been developed against some flaviviruses (YFV, Japanese encephalitis virus, and tickborne encephalitis virus), they remain a serious health risk around the world.The flavivirus RNA contains one large open reading frame flanked by 5Ј and 3Ј nontranslated regions that are required for replication and translation of the RNA (11). Analysis of the sequences of several flavivirus RNAs showed that short sequences close to the 5Ј and 3Ј ends are complementary, and we proposed that these sequences might function to cyclize the RNA during replication (5). The cyclization sequence at the 5Ј end is located within the region encoding the N-terminal region of the capsid protein. Its 3Ј counterpart is located in the 3Ј nontranslated region. A core region of 8 nucleotides within the cyclization domain is conserved among all mosquito-borne flaviviruses (5). Khromykh et al. (7,8) examined the importance of the cyclization sequences by analyzing the ability of truncated and mutated Kunjin virus (KUNV) RNA molecules to replicate in transfected cells. They found that deletion mutants lacking the sequence encoding the first 20 amino acids of KUNV capsid protein are unable to replicate (8). They further showed that a mutant RNA that had five changes within the 8-nucleotide core conserved sequence was unable to replicate. Compensating changes in the 3Ј region that restored the complementarity of the cyclization sequence restored the ability of the RNA to replicate, althoug...

Three Aedes albopictus (mosquito) cell lines persistently infected with Sindbis virus excluded the replication of both homologous (various strains of Sindbis) and heterologous (Aura, Semliki Forest, and Ross River) alphaviruses. In contrast, an unrelated flavivirus, yellow fever virus, replicated equally well in uninfected and persistently infected cells of each line. Sindbis virus and Semliki Forest virus are among the most distantly related alphaviruses, and our results thus indicate that mosquito cells persistently infected with Sindbis virus are broadly able to exclude other alphaviruses but that exclusion is restricted to members of the alphavirus genus. Superinfection exclusion occurred to the same extent in three biologically distinct cell clones, indicating that the expression of superinfection exclusion is conserved among A. albopictus cell types. Superinfection of persistently infected C7-10 cells, which show a severe cytopathic effect during primary Sindbis virus infection, by homologous virus does not produce cytopathology, consistent with the idea that cytopathology requires significant levels of viral replication. A possible model for the molecular basis of superinfection exclusion,which suggests a central role for the alphavirus trans-acting protease that processes the nonstructural proteins, is discussed in light of these results.