Positive-strand RNA virus genome replication organelles: structure, assembly, control

Figure 1.. Positive-strand (+)RNA virus genome replication cycle and associated membrane-associated replication organelles. (A) (+)RNA virus replication initiates when the infecting virion releases a messenger-sense RNA genome into the host cytoplasm. After translation of the RNA genome, viral replicase proteins repurpose it to template the synthesis of a complementary strand (−)RNA. The resulting double-stranded (ds)RNA replication intermediate subsequently becomes the template for production of progeny (+)RNA genomes, which re-enter translation to amplify the infection to produce thousands to millions of new genomes that are packaged into progeny virions and released from the cell. The RNA replication process is compartmentalized in either of two types of membrane-associated RNA replication complexes: (B) Spherular 50–100-nm-diameter RNA replication compartments are formed by, for example, alphavirus and flavivirus via invagination of intracellular organellar membranes. (C) Double-membrane 200–300-nm-diameter RNA replication compartment vesicles (DMVs) are derived from intracellular membranes in cells infected with, for example, coronaviruses and picornaviruses.

Figure 2.. Positive-strand (+)RNA virus genome organization. Selected genomes of the noda-, alpha-, flavi-, and coronaviruses presented at a common scale. Enlarged white boxes represent the nonstructural (i.e., nonvirion) replicase proteins. Light gray and darker gray boxes represent accessory proteins and structural (i.e., virion) proteins, respectively. Replicase protein enzymatic domains are colored to highlight 5′ RNA capping enzymes (yellow), RNA-dependent RNA polymerases (blue), NTPase/helicases (green), and viral proteases and their corresponding cleavage sites (pink and orange; cleavage sites shown as vertical arrowheads). Autocatalytic cleavage sites or those processed by host proteases are shown in gray and white, respectively. The central channel domain that is structurally conserved between nodaand alphaviruses (see main text) is indicated in red. Membrane-associated sequences are underlined in purple. Black dots represent 5′ RNA cap structures, An the 3′ RNA poly(A) tail. Abbreviations: gRNA, genomic RNA; NS/nsP/nsp, nonstructural protein; sgRNA, subgenomic RNA.

Figure 3.. Cryo-electron microscopy (cryo-EM) tomographic 3D reconstructions of RNA replication organelles. Images adapted with permission from [13,15,45,78–81]. (A) Flock House nodavirus spherules (white) on the mitochondrial outer membrane (blue). In the inset, RNA is colored red and the replication complex crown structure light blue. (B) Chikungunya alphavirus spherules invaginating from the plasma membrane (yellow) with RNA content (red). (C) Dengue virus and (D) Zika virus (flaviviruses) spherules invaginating into the endoplasmic reticulum (ER). Virus particles in close proximity can be seen in red and white, respectively. (E) Mouse hepatitis coronavirus ER-derived double-membrane vesicles (DMVs, yellow surrounding green), with RNA in red and DMV pore complexes in blue. An intracellularly budding new virus particle is seen in the center top of the image inside the tan-colored vesicle. (F) Middle East respiratory syndrome coronavirus DMV in yellow and purple. Other virus-induced membrane rearrangements are colored green and blue.

Figure 4.. Cryo-electron microscopy (cryo-EM) structures of RNA replication complex crowns. (A) Replication organelles (ROs) on mitochondria from Flock House virus (FHV)-infected cells. Upper panel: Proto-crown precursor assembly on the flat outer mitochondrial membrane (OMM). Docked at single-particle cryo-EM-derived atomic resolution, the RNA capping domains (yellow) and flanking central ring domains (red) of the N-terminal halves of 12 copies of protein A form a flat toroidal floor. Membrane-binding helices are shown in purple and C-terminal protein A polymerase domains atop in blue. Lower panel: Mature ROs are spherular membrane invaginations with larger crowns matured by addition of a second 12-mer ring of protein A atop the proto-crown. A long flexible linker (broken line) enables the top Pol ring to position its N-proximal RNA capping/membrane binding domains as outward extending ‘legs’. Teal-colored electron density in the bottom of the central channel could be a potential additional protein A Pol domain, either as a 25th protein A copy or, via the same flexible tether, released from the upper or lower polymerase ring. The width of the double-stranded RNA inside the spherule is…

Figure 5.. Nodavirus RNA replication complex assembly. Nodavirus replication organelle (RO) assembly and maturation involve multiple successive steps: First, replicase protein A multimerizes on the outer mitochondrial membrane (OMM) into a single-ring proto-crown precursor that recruits the viral genomic RNA template. Then, in close linkage, the proto-crown matures into a double-ring mature crown, (−)RNA is synthesized, and the resulting dsRNA intermediate is captured inside newly formed or forming spherular RNA replication vesicles. Finally, once formed, the mature RO switches modes to copy the dsRNA template to synthesize large numbers of 5′ capped (+) strands, which are released into the cytosol for translation and encapsidation. The width of double-stranded RNA is drawn to scale.

Figure 6.. Crown interactions with other viral and host proteins. As discussed further in the main text, during assembly and function of (+)RNA virus replication organelles (ROs), the viral RNA replication proteins interact with numerous host factors and pathways. Examples depicted in this generic diagram include the recruitment of lipid synthesis enzymes and incorporation of their lipid products in RO vesicles and membrane remodeling factors such as reticulons and ESCRT proteins to assist in building these vesicles. Viral proteins in the assembled RO can have additional functions to counteract actions of proteins involved in host defense systems, such as TLR3, MDA5, and STING. The nascent progeny (+)RNA genomes are released from ROs into the cytosol and, in at least some cases assisted by transitional interactions of relevant host or viral factors with crown proteins, enter any of several fates. These include interaction with host translation factors to become templates for viral protein synthesis, interaction with viral capsid proteins to become substrates for encapsidation, or interaction with viral RNA replication proteins to become templates for forming new ROs.