Rotaviruses (RVs) continue to be a leading cause of infantile gastroenteritis and a significant global disease burden despite four approved vaccines. RV particles are icosahedral non-enveloped with a complex architecture composed of three concentric protein shells encapsidating 11 segments of dsRNA, which code for six structural (VPs) and six non-structural proteins (NSPs). With the atomic structure of VP3, which we recently determined, the atomic details of all the RV capsid proteins are known as the structural determinants required for glycan-mediated cell attachment and conformational changes the capsid undergoes during endosomal entry. However, the structural underpinnings of the fascinating stages in RV replication following cell entry, from endogenous transcription and release of capped nascent transcripts to the final stages of how RVs acquire their infectious form, remain unclear. From our recent exciting discoveries on the RV capping enzyme VP3, multifunctional NSP2, and the membrane-associated glycoprotein NSP4, which play critical roles in key events in RV replication and morphogenesis, we now propose novel hypothesis-driven experiments to provide unprecedented mechanistic details underlying these events in RV-infected cells. In probing these complex molecular events, we will make use of remarkable new technological advances in high-resolution cryo-EM imaging, single-particle and focused sub-particle refinement and reconstruction pipelines, cellular cryo-ET with cryo-FIB-SEM-CLEM and sub-tomogram reconstructions, combined with crystallography and complementary biophysical and cell-based techniques such as reverse genetics, and gene silencing. The focus of Aim 1 is on endogenous transcription. Based on our atomic structure of the capping enzyme VP3, we will investigate the structural basis of how VP3, with its unique multidomain tetrameric organization, integrates four essential enzymatic activities for capping, how transcripts traverse through the multiple domains for capping, and how VP3 caps the nascent transcript exiting from VP1 within the actively transcribing double-layered particles (DLPs). In Aim 2, our focus is to investigate structure-based mechanisms of how the RNA-binding protein NSP2 with multiple enzymatic activities performs dual functions, based on hypotheses from our recent studies, first, in trafficking RV proteins and capped mRNAs from the cytoplasm into the viroplasm, and second, as a scaffolding protein in regulating the formation of replication intermediates and core assembly. In Aim 3, our focus is on the final stages of RV morphogenesis. Exploiting our successful expression and purification of the first functionally active NSP4, we will determine its atomic structure and investigate the structural basis of how NSP4 facilitates the 'budding' of assembling DLPs from the viroplasm, inhibits their endogenous transcription and incorporates VP4 spikes onto DLPs to form transiently enveloped particles (TEPs), and deter...