Rotavirus core shell protein sites that regulate intra-particle polymerase activity.

IMPORTANCE: Rotaviruses are important causes of severe gastroenteritis in young children. A characteristic feature of rotaviruses is that they copy ribonucleic acid (RNA) inside of the viral particle. In fact, the viral polymerase (VP1) only functions when it is connected to the viral inner core shell protein (VP2). Here, we employed a biochemical assay to identify which sites of VP2 are critical for regulating VP1 activity. Specifically, we engineered VP2 proteins to contain amino acid changes at structurally defined sites and assayed them for their capacity to support VP1 function in a test tube. Through this work, we were able to identify several VP2 residues that appeared to regulate the activity of the polymerase, positively and negatively. These results are important because they help explain how rotavirus synthesizes its RNA while inside of particles and they identify targets for the future rational design of drugs to prevent rotavirus disease.

Flexibility of the Rotavirus NSP2 C-Terminal Region Supports Factory Formation via Liquid-Liquid Phase Separation.

Many viruses sequester the materials needed for their replication into discrete subcellular factories. For rotaviruses (RVs), these factories are called viroplasms, and they are formed in the host cell cytosol via the process of liquid-liquid phase separation (LLPS). The nonstructural protein 2 (NSP2) and its binding partner, nonstructural protein 5 (NSP5), are critical for viroplasm biogenesis. Yet it is not fully understood how NSP2 and NSP5 cooperate to form factories. The C-terminal region (CTR) of NSP2 (residues 291 to 317) is flexible, allowing it to participate in domain-swapping interactions that promote interoctamer interactions and, presumably, viroplasm formation. Molecular dynamics simulations showed that a lysine-to-glutamic acid change at position 294 (K294E) reduces NSP2 CTR flexibility in silico. To test the impact of reduced NSP2 CTR flexibility during infection, we engineered a mutant RV bearing this change (rRV-NSP2K294E). Single-cycle growth assays revealed a >1.2-log reduction in endpoint titers for rRV-NSP2K294E versus the wild-type control (rRV-WT). Using immunofluorescence assays, we found that rRV-NSP2K294E formed smaller, more numerous viroplasms than rRV-WT. Live-cell imaging experiments confirmed these results and revealed that rRV-NSP2K294E factories had delayed fusion kinetics. Moreover, NSP2K294E and several other CTR mutants formed fewer viroplasm-like structures in NSP5 coexpressing cells than did control NSP2WT. Finally, NSP2K294E exhibited defects in its capacity to induce LLPS droplet formation in vitro when incubated alongside NSP5. These results underscore the importance of NSP2 CTR flexibility in supporting the biogenesis of RV factories. IMPORTANCE Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.

In vitro particle-associated uridyltransferase activity of the rotavirus VP1 polymerase.

Rotaviruses are 11-segmented, double-stranded RNA (dsRNA) viruses with a unique intra-particle RNA synthesis mechanism. During genome replication, the RNA-dependent RNA polymerase (VP1) performs minus-strand RNA (-ssRNA) synthesis on positive-strand RNA (+ssRNA) templates to create dsRNA segments. Recombinant VP1 catalyzes -ssRNA synthesis using substrate NTPs in vitro, but only when the VP2 core shell protein or virus-like particles made of VP2 and VP6 (2/6-VLPs) are included in the reaction. The dsRNA product can be labeled using [α32P]-UTP and separated from the input +ssRNA template by polyacrylamide gel electrophoresis. Here, we report the generation of [α32P]-labeled rotavirus +ssRNA templates in reactions that lacked non-radiolabeled NTPs but contained catalytically-active VP1, 2/6-VLPs, and [α32P]-UTP. Non-radiolabeled UTP competed with [α32P]-UTP to decrease product levels, whereas CTP and GTP had little effect. Interesting, ATP stimulated [α32P]-labeled product production. These results suggest that rotavirus VP1 transferred [α32P]-UMP onto viral + ssRNA in vitro via a particle-associated uridyltransferase activity.

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

A Perfect Ten-Decoy Maps Uncover Polymerase Complexes within Reoviridae Virion.

In this issue of Structure, Kaelber et al. (2020) use cryo-EM and synthetic decoy maps to reveal the patterning of 10 polymerase complexes within FAKV, a Reoviridae family member containing 9 genome segments. Their findings support a model for FAKV assembly that has implications for the entire Reoviridae family.