Coupling of replication and assembly in flaviviruses

Abstract

Flaviviruses affect hundreds of millions of people each year causing tremendous morbidity and mortality worldwide. This genus includes significant human pathogens such as dengue, West Nile, yellow fever, tick-borne encephalitis and Japanese encephalitis virus among many others. The disease caused by these viruses can range from febrile illness to hemorrhagic fever and encephalitis. A deeper understanding of the virus life cycle is required to foster development of antivirals and vaccines, which are an urgent need for many flaviviruses, especially dengue. The focus of this review is to summarize our current knowledge of flaviviral replication and assembly, the proteins and lipids involved therein, and how these processes are coordinated for efficient virus production.

Figure 1. Flavivirus polyprotein processing

Translation of the viral genome results in a polyprotein, which is cleaved by cellular and viral proteases. The proposed topologies of the viral proteins with respect to the ER lumen and cytoplasm and the proteases responsible for their cleavages are indicated. The membrane bilayer is shown in pink, transmembrane domains for individual proteins are shown as cylinders spanning the membrane and connecting loops are shown as lines. Black, blue and red arrow / arrowheads represent signal peptidase, NS2B-3 protease and furin protease cleavage sites respectively. Open arrow represents a protease cleavage site that required additional characterization.

Figure 2. Flavivirus genome replication and assembly

A) Spatial model for RC on Ve membranes. Surface representation of DENV NS1 dimer (PDB: 4O6B) [23] located in the ER bound to the Ve membrane through its “GF” loops. Dipeptide RQ10 residues of NS1 potentially interact with the NS4B dimer. NS1 also interacts with NS4A. NS2A is represented as a 5 membrane-pass protein interacting with NS3. NS4A and NS4B are also transmembrane proteins interacting with NS3 on the cytoplasmic side. Surface representations of DENV NS3 (PDB: 2VBC) [68], NS5 Methyltransferase domain (PDB: 1L9K) [69] and RdRp domain (PDB: 2J7W) [70] are shown towards the cytoplasmic side of the membrane. The transmembrane domains of membrane proteins are shown as cylinders and the connecting loops are shown as black lines. This model does not depict the exact topology or oligomeric state of the RC proteins and their interactions with host factors. B) Coupling of Flavivirus replication and assembly. Genome replication takes place on virus-induced Ve membranes that have opening towards the cytoplasm. The replication complex is composed of NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 along with genomic RNA. The newly synthesized RNA (shown in red) exits the pore and is captured by C (shown as green ovals) attached to LDs or localized on the cytoplasmic side of the ER in close vicinity of the Ve pore to form nucleocapsid. The nucleocapsid complex acquires the viral glycoproteins expressed on the ER membrane by budding into the ER lumen. The spiky immature particles (shown in blue) undergo maturation as they traffic through the TGN and become smooth mature particles (shown in gray) by conformational changes in E protein and furin cleavage of prM protein. C: Capsid, CM: Convoluted Membranes, GF: Greasy Finger, LD: Lipid Droplets, ER: Endoplasmic Reticulum, PM: Plasma membrane, RC: Replication complex, tGN: Trans-Golgi Network, Vp: Vesicle packets, Ve: membrane vesicles.

Figure 2. Flavivirus genome replication and assembly

A) Spatial model for RC on Ve membranes. Surface representation of DENV NS1 dimer (PDB: 4O6B) [23] located in the ER bound to the Ve membrane through its “GF” loops. Dipeptide RQ10 residues of NS1 potentially interact with the NS4B dimer. NS1 also interacts with NS4A. NS2A is represented as a 5 membrane-pass protein interacting with NS3. NS4A and NS4B are also transmembrane proteins interacting with NS3 on the cytoplasmic side. Surface representations of DENV NS3 (PDB: 2VBC) [68], NS5 Methyltransferase domain (PDB: 1L9K) [69] and RdRp domain (PDB: 2J7W) [70] are shown towards the cytoplasmic side of the membrane. The transmembrane domains of membrane proteins are shown as cylinders and the connecting loops are shown as black lines. This model does not depict the exact topology or oligomeric state of the RC proteins and their interactions with host factors. B) Coupling of Flavivirus replication and assembly. Genome replication takes place on virus-induced Ve membranes that have opening towards the cytoplasm. The replication complex is composed of NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 along with genomic RNA. The newly synthesized RNA (shown in red) exits the pore and is captured by C (shown as green ovals) attached to LDs or localized on the cytoplasmic side of the ER in close vicinity of the Ve pore to form nucleocapsid. The nucleocapsid complex acquires the viral glycoproteins expressed on the ER membrane by budding into the ER lumen. The spiky immature particles (shown in blue) undergo maturation as they traffic through the TGN and become smooth mature particles (shown in gray) by conformational changes in E protein and furin cleavage of prM protein. C: Capsid, CM: Convoluted Membranes, GF: Greasy Finger, LD: Lipid Droplets, ER: Endoplasmic Reticulum, PM: Plasma membrane, RC: Replication complex, tGN: Trans-Golgi Network, Vp: Vesicle packets, Ve: membrane vesicles.