Sue Crawford, Baylor College of Medicine

Jeanette M. Criglar, Mary K. Estes, Sue E. Crawford Baylor College of Medicine, Houston, TX, USA

Cell Biology of Viral Infection

Rotavirus (RV) replication and progeny virion assembly occur in the cytoplasm of infected cells in 3-dimensional replication factories (viroplasms). Viroplasm assembly is orchestrated by a complex interaction of viral proteins NSP2 and NSP5 with cellular organelles, including the induction of host cell lipid droplet (LD) synthesis. Precisely where and how viroplasms assemble in the cytoplasm, and how they associate with LDs, remains unknown and is crucial for understanding RV replication. By co-immunoprecipitation and mass spectrometry NSP2 interacts with the smooth endoplasmic reticulum (ER) tubule-forming protein reticulon 4 (RTN4), the smooth ER tubule fusogenic protein atlastin (ATL) and the LD initiation site proteins seipin and LDAF1. Using super resolution microscopy, we show for the first time that NSP2 colocalizes with RTN4 on smooth ER tubules early in nascent viroplasm formation. Surprisingly, we observed fusogenic ATL accumulating inside viroplasms, suggesting that ATL may be required for the growth of viroplasms similar to LDs. We also found that NSP2 accumulates and viroplasms assemble near seipin, and the LD-associated protein PLIN1 also accumulates on viroplasms, suggesting that viroplasms form at the same time as the associated LD in a co-assembly process. These data indicate that viroplasms assemble with nascent LDs at the smooth ER. Viroplasm growth may exploit the fusogenic properties of ATL and/or LDs accounting for the ability of small viroplasms to fuse into larger, likely more productive, replication factories. Understanding the mechanisms behind RV-LD interactions offers potential therapeutic targets for disrupting the viral life cycle and mitigating RV pathogenesis and disease.