Biochemistry and Molecular Biology of Flaviviruses

Abstract

Flaviviruses, such as dengue, Japanese encephalitis, tick-borne encephalitis, West Nile, yellow fever, and Zika viruses, are critically important human pathogens that sicken a staggeringly high number of humans every year. Most of these pathogens are transmitted by mosquitos, and not surprisingly, as the earth warms and human populations grow and move, their geographic reach is increasing. Flaviviruses are simple RNA-protein machines that carry out protein synthesis, genome replication, and virion packaging in close association with cellular lipid membranes. In this review, we examine the molecular biology of flaviviruses touching on the structure and function of viral components and how these interact with host factors. The latter are functionally divided into pro-viral and antiviral factors, both of which, not surprisingly, include many RNA binding proteins. In the interface between the virus and the hosts we highlight the role of a noncoding RNA produced by flaviviruses to impair antiviral host immune responses. Throughout the review, we highlight areas of intense investigation, or a need for it, and potential targets and tools to consider in the important battle against pathogenic flaviviruses.

Figure 1

Secondary and tertiary structures within the DENV2 (New Guinea C) genome. (Top) Structures within the 5′UTR and proximal capsid-coding region are shown. 5′cap structure (m7G) and AUG start codon are indicated in black. Sequences that participate in long-range tertiary interactions with sequences in the 3′UTR are highlighted with colored lines. (Bottom) Known 3′UTR structures are shown. Arrows indicate 5′ ends of identified subgenomic flavivirus (sf)RNAs.

Figure 2

Flaviviral genome and polyprotein. (A) Flaviviral genome. Flaviviruses have a single-stranded (+) RNA genome of approximately 11 kb. Genome is capped but not polyadenylated. It encodes three structural (blue) and seven nonstructural (red) proteins which are translated from a single ORF. In between NS4A and NS4B, the genome also encodes a small peptide of 2 kDa (2K peptide). 5′ and 3′ UTRs are known to have complex structure, with several hairpins, which are important for translation, RNA synthesis, and sfRNA formation. (B) Flaviviral polyprotein topology and predicted transmembrane domains. Flavivirus polyprotein is integrated into the ER membrane. Viral proteins prM, E, and NS1 are mainly on the luminal side and C, NS3, and NS5 on the cytoplasmic side. Proteins NS2A, NS2B, NS4A, and NS4B have several transmembrane domains spanning across the ER, and thus, large parts of these proteins are on each side and on the ER membrane. 2K peptide is entirely inserted in the ER membrane. Polyprotein is cleaved co- and post-translationally at multiple sites. Cleavages on the cytoplasmic side are done by the viral protease NS3 and its cofactor NS2B, and cleavages on the ER lumen side are done by the signal peptidase complex. Polyprotein also has an additional furin protease cleavage in prM that gives rise to the mature M protein in the Golgi and one additional site between NS1 and NS2A that is cleaved by an unknown enzyme.

Figure 3

Multiple roles of the flavivirus genome. Viral genomes are translated, used as templates for negative strand synthesis, packaged into virions, and partially degraded to form subgenomic flaviviral RNAs (sfRNA). First three of these roles take place in association with the ER.

Figure 4

sfRNA can impact many components of the immune system in mosquitos and humans. sfRNA sequesters Xrn1, leading to changes in the cellular transcriptome, binds to TRIM25, dampening IFN production, soaks up RBPs that are required for efficient ISG mRNA translation, and inhibits RNAi (see text for references).