Abstract
Rotavirus (RV) is the primary etiological agent responsible for severe gastroenteritis and dehydration worldwide in infants and young animals. RV virions are icosahedral, non-enveloped particles with three concentric layers of protein (TLP, triple-layered particle). The inner layer is termed core or single-layered particle (SLP) and is composed of the core-shell protein VP2 that holds underneath each five-fold axis one copy of both the RNA-dependent RNA polymerase (RdRp) VP1 and the guanylylmethyltransferase VP3. Each core encapsidates one copy of each of the eleven double-stranded (ds) RNA genome segments. The second layer is formed by VP6 trimers, making up the double-layered particles (DLP). On top of the DLP, VP7 protein trimers are arranged in icosahedral symmetry alongside VP4 spike protein trimers. Expressing only eleven proteins, they exhibit a high degree of multifunctionality. RV infection triggers the formation of globular cytosolic and electron-dense inclusions referred to as viroplasms.These structures correspond to the sites of genome replication, transcription, and initial steps of virus progeny assembly. The viroplasm building block proteins consist of NSP5, NSP2, and VP2. Additionally, the viroplasms contain VP6, VP1, and VP3 and single and double-stranded viral RNA. Host components such as tubulin, perilipin, and the host proteasome are also found in viroplasms. During infection, viroplasms coalesce from small punctate cytosolic structures at early times to large perinuclear inclusions at late stages of infection. In addition, the microtubule (MT) cytoskeleton plays a direct role in the formation and maintenance of viroplasms. A stabilized MT network is essential for the formation of viroplasms. Also, molecular motors such as kinesin and dynein, which rely on MTs for their function, are required for viroplasm morphogenesis. Moreover, RV infection leads to a reorganization of all three cytoskeletal networks, including also actin and intermediate filaments. This thesis presents evidence that the cytosolic version of protein VP4 interacts with actin, triggering viroplasm formation. The viral spike protein VP4 has never been shown to play a direct role in viroplasm formation. Nevertheless, it actively participates in various rotavirus (RV) life cycle steps, including virus-cell attachment, internalization, regulation of endocytosis, virion morphogenesis, and virus release. Thus, VP4 interacts with the actin-cytoskeleton components, particularly during virus entry and exit processes. This study shows for the first time that VP4 also acts as a catalytic factor in the development of viroplasms. In this study, we employed reverse genetics to construct a recombinant RV, known as rRV/VP4-BAP, which incorporates a biotin acceptor peptide (BAP) into the K145-G150 loop of the VP4 lectin domain, allowing for real-time monitoring. This recombinant virus was replication-competent but exhibited a reduced fitness. Interestingly, infection with rRV/VP4-BAP, in contrast to rRV/wt infection, did not result in a reorganized actin cytoskeleton, and the viroplasms formed were resistant to drugs that disassemble actin and inhibit myosin. Additionally, wild-type (wt) VP4, but not VP4-BAP, seemed to associate with actin filaments. Similarly, the co-expression of VP4 with NSP5 and NSP2 substantially increased the number of viroplasm-like structures (VLS). An intriguing observation was made when a small peptide, designed to mimic loop K145-G150, successfully restored the phenotype of rRV/VP4-BAP. This enhanced the capability to form viroplasms, ultimately enhancing virus progeny production. The presented results establish a direct link between VP4 and the actin cytoskeleton to facilitate viroplasm assembly. In a second project aimed at further investigation of the relationship between RV proteins and host components within viroplasms, we performed a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Following this, tandem mass spectrometry identified all eight subunits comprising the T-complex protein-1 ring complex (TRiC), a cellular chaperonin responsible for folding up to 10% of cytosolic proteins. TRiC showed to be recruited to viroplasms and specifically localizes around newly formed double-layered particles (DLPs). Notably, chemical inhibition of TriC and siRNA-mediated transcriptional silencing of its subunits significantly reduced virus progeny formation. Intriguingly, in TRiC-inhibited RV-infected cells, mostly empty DLPs were present. Sequence-specific direct RNA nanopore sequencing demonstrated that TRiC plays a crucial role in RV replication by controlling the synthesis of dsRNA genome segments, especially (-)ssRNA. Furthermore, TriC associates and regulates the folding of VP2, a cofactor of VP1 triggering virus dsRNA synthesis. This study provides in-cell culture evidence for the regulatory mechanism governing dsRNA genome segment replication within RV viroplasms. In conclusion, this thesis shows the importance of host cell factors on the dynamics of viroplasms. We demonstrate the multifunctionality of RV proteins, exemplified here by VP2 and VP4, elucidating new functional roles within the biological context. Even though many questions remain open, this thesis elucidates specific aspects of virus host interplay and their effect on viroplasm formation and RV genome replication.
Vetter, Janine