Flaviviridae

1. Flaviviridae Positive stranded RNA viruses

3. Flaviviridae • Enveloped virions made up of a lipid bilayer with two or more types of envelope (E) glycoproteins surrounding a nucleocapsid. • Single-stranded, positive-sense genome RNA complexed with multiple copies of a small, basic capsid (C) protein.

4. Life Cycle • Binding and uptake involve receptor-mediated endocytosis via cellular receptors specific for viral envelope proteins. • Low pH of the endosomal pathway induces fusion of the virion envelope with cellular membranes. • Following uncoating of the nucleocapsid, the RNA genome is release into the cytoplasm. • The viral genome serves three discrete roles within the life cycle: • mRNA for translation of all viral proteins, • template during RNA replication, and • genetic material packaged within new virus particles. • Viral proteins are produced as part of a single long polyprotein of more than 3,000 amino acids that is cleaved by a combination of host and viral proteases. • The structural proteins are encoded in the N-terminal portion of the polyprotein with the nonstructural (NS) proteins in the remainder.

5. RNAreplication occurs in cytoplasmicreplication complexes via synthesis of a genome-length minus strand RNA intermediate. • Progeny virions are thought to assemble by budding into an intracellular membrane compartment, most likely the endoplasmic reticulum (ER), then transited through the host secretory pathway and released at the cell surface.

7. Examples • Dengue virus • Hepatitis C Virus

12. Attachment • Flaviviruses can utilize multiple receptors for different cell types and in different host species. • Infection of dendritic cells (DC) is particularly important because these intradermal cells can be primary targets early in infection. • Other proteins identified as receptors include: • αvβ3 integrin, • GRP78 (BiP), • and CD14 • In addition, highly sulfatedglycosaminoglycans (e.g., heparansulfate) have been shown to play an important role in the initial attachment of several flaviviruses to target cells. • Virus particles opsonized with immunoglobulins show enhanced binding and infection of cells expressing immunoglobulin Fcreceptors. It is widely suspected that antibody-enhanced infection may be relevant to the pathogenesis of DF and DHF, which occur more frequently in people previously exposed to other DENV serotypes.

13. Entry • Internalized via clathrin-coated pits and trafficked to a prelysosomalendocyticcompartment where low pH induces fusion between the virus and host cell membranes to release the virus nucleocapsid • It appears that viral genomes are directly accessible for translation after membrane fusion.

14. Dengue Virus • Genomes consist of a single, positive-strand RNA of ≈11 kb (sedimentation, 42S) • 5 ‘cap, m7GpppAmpN2 • Additional methylation of the N2 residue (type II cap) has also been detected in RNA from infected cells. • Unlike cellular messenger RNA (mRNA), flavivirusgenomes lack a 3 polyadenylatetail. • Genomes encode a single long open reading frame (ORF) flanked by 5 and 3 noncoding regions (NCR) of ≈100 nucleotide (nt) and 400 to 700 nt, respectively.

16. RNA Replication • 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 RNA. • Minus strand RNA has been detected as early as 3 hours after infection

18. Hepatitis C Virus (HCV) • The HCV genome is an uncapped, • 9.6-kb RNA containing highly structured 5 and 3 ends. • The 5 NCR is a well-conserved, 341-nt sequence element that folds into a complex structure consisting of four major domains and a pseudoknot. • The first 120 nt serves as a minimal replication element, although nearly the entire 5 NCR is needed for efficient RNA replication.

20. RNA Replication • Translation and RNA replication are mutually exclusive processes, because they proceed in opposite directions on a given RNA template. • Positive-strand RNA viruses regulate the rate of genome translation versus replication. • Translation of the HCV genome occurs more frequently than replication. • One way this could be regulated is via crosstalk between the determinants that control translation and genome replication. • E.g the cellular PTB protein binds to the HCV 5 NCR and core coding region where it may modulate IRES activity and to the 3 end where it may repress replication.

21. It has been suggested that HCV translation can be autoregulated through product inhibition. • Low levels of HCV core protein can enhance HCV IRES mediated translation, whereas high concentrations inhibits HCV translation • Polycytidine-binding protein 2 (PCBP-2) binds to the HCV 5 NCR also noted in polioviruses that PCBP binds 5’ NCR and RdRP of poliovirus, to control the switch between translation and replication.

22. Location of Replication • The HCV genome is recruited out of translation and into a membrane associated replication complex, or replicase. • Specifically, NS proteins and HCV RNA associate with a dense perinuclear matrix of≈85-nm vesicles termed the membranous web.

23. HCV RNA replication is stimulated by increased availability of saturated and monounsaturated fatty acids, and inhibited by polyunsaturated fatty acids or inhibitors of fatty acid synthesis. • HCV RNA is protected from nuclease degradation by a detergent-sensitive membrane suggesting that RNA synthesis may be enclosed within the membranous web. • Similarly, replicase activity is insensitive to protease digestion unless solubilized by detergent, whereas most of the NS proteins are digested by this treatment . • These data support the hypothesis that active replicase is bound by a limiting membrane and demonstrate that a vast excess of NS proteins are produced.