FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Flaviviridae :T he Viruses and Their Replication

The crystallographically determined structure of a soluble fragment from the major envelope protein of a flavivirus reveals an unusual architecture. The flat, elongated dimer extends in a direction that would be parallel to the viral membrane. Residues that influence binding of monoclonal antibodies lie on the outward-facing surface of the protein. The clustering of mutations that affect virulence in various flaviviruses indicates a possible receptor binding site and, together with other mutational and biochemical data, suggests a picture for the fusion-activating, conformational change triggered by low pH.

The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution

Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three ‘fusion loops’ at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.

Structure of the dengue virus envelope protein after membrane fusion

Publisher Summary This chapter summarizes the current understanding of the molecular biology of flaviviruses and points out promising avenues for future work. The molecular biology of flaviviruses is best understood in the context of the viral life cycle, which provides a framework for the organization of this chapter. Flavivirus particles bind to cells via interactions between the viral surface glycoprotein and cellular receptors. Several cell surface proteins have been described as putative receptors. In addition, opsonization with immunoglobulins enhances virus particle binding and infection of cells expressing immunoglobulin Fc receptors. Virions 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 among the viruses and host cell membranes to release the virus nucleocapsid. The viral genome is released into the host cytoplasm by the process of nucleocapsid uncoating, which is not yet fully understood. Translation of the viral genome produces proteins that lead to replication of the viral genome and assembly of new virus particles. Flavivirus infection induces rearrangement of cytoplasmic membranes in the perinuclear region. Virus particles are thought to assemble by budding into the endoplasmic reticulum. A few studies have shown evidence for budding at the plasma membrane. Based on trans-complementation studies, it appears that genome packaging is coupled to RNA replication. Nascent virus particles pass through the host secretory pathway, where virion maturation occurs, and are released.

Molecular biology of flaviviruses.

Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide.

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.

Yellow Fever/Japanese Encephalitis Chimeric Viruses: Construction and Biological Properties

Host cell selection of murray valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence

Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication, and evidence for alternative processing of the NS2A protein.

Serial passage of yellow fever virus (YF17D) in mouse brain enhances neurovirulence, causing a reduction in survival time after intracerebral inoculation of adult mice. To study the biological and genetic basis for this phenomenon, we compared neurovirulence properties of the neuroadapted Porterfield strain (PYF) to a YF17D strain generated from a full-length YF cDNA template (YF5.2iv). Adult mice were infected by olfactory bulb inoculation, which results in widespread distribution of virus throughout the central nervous system. Although PYF and YF5.2iv spread rapidly throughout the neuraxis, maximal titres of PYF in the brain and spinal cord were 1000- to 10000-fold higher than those of YF5.2iv. Paralysis and death occurred earlier with the PYF strain. Several cDNA clones of the E/NS1 region of the PYF strain were sequenced. Three predicted amino acid changes were consistently observed in the envelope protein of the PYF strain compared to YF5.2iv. Common substitutions were also identified in NS1 and NS2A. The potential contribution of these genetic differences to neurovirulence was evaluated by generating recombinant, intertypic PYF/YF5.2iv viruses. Physical signs of disease and mean spinal cord titres after inoculation of one recombinant were not different from the YF5.2iv parent. Our data indicate that PYF and YF5.2iv differ significantly in their virulence properties, however, common amino acid substitutions in the E/NS1 region of the PYF strain do not determine its enhanced neurovirulence. Other regions of the viral genome may contribute dominant effects on the virulence properties of the PYF strain.

Replication of yellow fever virus in the mouse central nervous system: comparison of neuroadapted and non-neuroadapted virus and partial sequence analysis of the neuroadapted strain

Ann Nestorowicz

Papers

Yellow Fever/Japanese Encephalitis Chimeric Viruses: Construction and Biological Properties

Host cell selection of murray valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence

Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication, and evidence for alternative processing of the NS2A protein.

Replication of yellow fever virus in the mouse central nervous system: comparison of neuroadapted and non-neuroadapted virus and partial sequence analysis of the neuroadapted strain

Direct sequence analysis of amplified dengue virus genomic RNA from cultured cells, mosquitoes and mouse brain.