During infection, RNA viruses dynamically interact with stress granules and P bodies, leading to varying stress granule phenotypes. Many virus have evolved mechanisms to antagonize the formation of stress granules, suggesting that stress granules are involved in restricting virus replication through RNA silencing. In contrast, other RNA viruses, such as respiratory suncytial virus, induce stress granule formation and take advantage of stress granule responses as part of the infectious cycle.
STRESS GRANULES AND PROCESSING BODIES IN REPLICATION-TRANSCRIPTION COMPLEX ACTIVITY
Stress granules and processing (P) bodies are cytoplasmic RNA granules that contain translationally silenced messenger ribonucleoproteins, contributing to translation regulation in cells. Whereas P bodies are constitutively expressed and include components involved in mRNA decay, stress granules are though to be sites of mRNA storage and triage formed in response to stress conditions. Stress granules represent an intermediate stage in the dynamic equilibrium between active translation on free polysomes and mRNA decay in P bodies.
The function and dynamics of DMVs and CMs and the precise localization of the sites of active viral RNA synthesis are still unresolved questions, and further studies are required. A possible model proposes that DMVs may be the initial sites of active RNA synthesis early in infection, whereas at later times, after membrane connections are lost, RNA synthesis shifts to the CMs, and DMVs become end-stage products that sequester nonfuctional dsRNAs to prevent the stimulation of the innate immune response.
The coexpression of the SARS-CoV transmembrane nonstructural proteins nsp3, nsp4 and nsp6 resulted in the formation of CMs and DMVs, suggesting a functionin the biogenesis of the membranous replicative structures, and also in the anchoring of the RTC.
In mouse hepatitis virus (MHV)-infected cells, newly synthesized RNA was detected in close proximity to DMVs and CMs, and viral RNA levels correlated with the number of DMVs. However, other data do not necessarily support the active contribution of DMVs to viral RNA synt hesized. Nascent MHV RNAs colocalize with dsRNA only at early times postinfection; at later times, the dsRNA distributed throughout the cell is apparently transcriptionally inactive. Furthermore, RdRp or nascent viral RNA has not been detected inside DMVs, and ultrastructural analysis could not confirm any connection between the DMV interior and cytoplasm, raising questions about the import and export of ribonucleotide precursors and produced RNAs exported from RNA synthesis areas.
Viral replicase subunits (nsp3, nsp5, and nsp8) localized to CMs, whereas dsRNA, presumably by the replicative intermediate, mainly localized to the DMV interior, supporting the concept that the membrane network would contribute to protecting replicating RNA from antiviral defense mechanisms.
ROLE OF DOUBLE-MEMBRANE VESICLES
Like that of other positive-strand RNA viruses, coronavirus RNA synthesis is associated with extensively rearranged intracellular membranes. High-resolution three-dimensional images obtained by electron tomography in SARS-CoV-infected cells showed a unique reticulovesicular network of modified endoplasmic reticulum that integrated convoluted membranes (CMs), interconnected double-membrane vesicles (DMVs), and vesicle packets apparently rising from DMV merger.
Alteration of coronavirus frame-shifting efficiency modified the ratio of replicase proteins, affecting viral RNA synthesis and virus production. In this sense, regulation of the ratio between the two vira lpolymerasess nsp8 and nsp12, encoded by ORF1a and ORF1b, respectively, may be involved in controlling the levels of the different sgmRNAs during viral RNA synthesis.
Corona proteins ratios are also posttranscriptionally regulated. Most sgmRNAs are structurally polycistronic but functionally monicistronic, with only the 5′-most ORF being translated into a viral protein. The clearest example of coronavirus translational regulation is the expression of the polyprotein pp1ab, which is generated by a programmed-1 ribosomal frame-shifting mechanism. This process leads to minor levels of most of the RNA-modifying enzymes, encoded by ORF1b, in comparison with those of other replicase enzymes, such as proteases, encoded by ORF1a.