Host Factor Requirements of the Rotavirus Viroplasm

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

Prevalence and Infant Mortality of Major Congenital Malformations Stratified by Birthweight

Background: Low birthweight and major congenital malformations (MCMs) are key causes of infant mortality. Objectives: The aim of this study was to explore the prevalence of MCMs in infants with low and very low birthweight and analyze the impact of MCMs and birthweight on infant mortality. Methods: We determined prevalence and infant mortality of 28 life-threatening MCMs in very-low-birthweight (75% (10,316) had severe congenital heart disease. The prevalence (per 10,000) of any/cardiac MCM was increased in VLBW (286/176) and LBW (244/143), as compared to NBW infants (38/32). Infant mortality rates were significantly higher in infants with an MCM, as opposed to infants without an MCM, in each birthweight group (VLBW 28.5% vs. 11.5%, LBW 16.7% vs. 0.9%, and NBW 8.6% vs. 0.1%). For most MCMs, observed survival rates in VLBW and LBW infants were lower than expected, as calculated from survival rates of VLBW or LBW infants without an MCM, and NBW infants with an MCM. Conclusions: Infants with an MCM are more often born with LBW or VLBW, as opposed to infants without an MCM. Many MCMs carry significant excess mortality when occurring in VLBW or LBW infants

Rotavirus Spike Protein VP4 Mediates Viroplasm Assembly by Association to Actin Filaments

Rotavirus (RV) viroplasms are cytosolic inclusions where both virus genome replication and primary steps of virus progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, that contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wild-type (wt) VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in coexpression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCE The spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens

Psychiatric In-Patients Are More Likely to Meet Recommended Levels of Health-Enhancing Physical Activity If They Engage in Exercise and Sport Therapy Programs

Background:; People with mental disorders engage in sedentary behaviors more often than their healthy counterparts. In Switzerland, nearly all psychiatric hospitals offer structured exercise and sport therapy as part of their standard therapeutic treatment. However, little is known about the degree to which psychiatric patients make use of these treatment offers. The aim of this study is to examine, in a sample of psychiatric in-patients (a) how many participate in the structured exercise and sport therapy programs offered by the clinic, (b) how many engage in exercise and sport activities on an individual basis, and (c) how many meet recommended levels of health-enhancing physical activity during their stay at the clinic. Furthermore, we examine whether those who engage in exercise and sport activities are more likely to meet internationally accepted physical activity recommendations.; Methods:; 107 psychiatric in-patients (49% women, M; age; = 39.9 years) were recruited at three psychiatric clinics in the German-speaking part of Switzerland. All participants were engaged in treatment and received usual care. Based on accelerometer data, participants were classified as either meeting or not meeting physical activity recommendations (≥150 min of moderate-to-vigorous physical activity per week). Participation in structured and individually performed exercise and sport activities was assessed with the Simple Physical Activity Questionnaire.; Results:; In total, 57% of all patients met physical activity recommendations. 55% participated in structured exercise and sport therapy activities, whereas only 22% of all patients engaged in exercise and sport activities independently. Psychiatric patients were significantly more likely to meet recommended levels of health-enhancing physical activity if they engaged in at least 60 min per week of structured exercise and sport therapy or in at least 30 min of individually performed exercise and sport activity.; Conclusions:; Given that prolonged immobilization and sedentary behavior have harmful effects on patients' physical and mental well-being, promoting exercise and sport activities is an important endeavor in psychiatric care. Clinics currently succeed in involving between 50 and 60% of all patients in sufficient physical activity. While this is encouraging, more systematic efforts are needed to ensure that all patients get enough physical activity

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication

ABSTRACT Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication

Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle.Peer reviewe

The Role of the Host Cytoskeleton in the Formation and Dynamics of Rotavirus Viroplasms

Rotavirus (RV) replicates within viroplasms, membraneless electron-dense globular cytosolic inclusions with liquid–liquid phase properties. In these structures occur the virus transcription, replication, and packaging of the virus genome in newly assembled double-layered particles. The viroplasms are composed of virus proteins (NSP2, NSP5, NSP4, VP1, VP2, VP3, and VP6), single- and double-stranded virus RNAs, and host components such as microtubules, perilipin-1, and chaperonins. The formation, coalescence, maintenance, and perinuclear localization of viroplasms rely on their association with the cytoskeleton. A stabilized microtubule network involving microtubules and kinesin Eg5 and dynein molecular motors is associated with NSP5, NSP2, and VP2, facilitating dynamic processes such as viroplasm coalescence and perinuclear localization. Key post-translation modifications, particularly phosphorylation events of RV proteins NSP5 and NSP2, play pivotal roles in orchestrating these interactions. Actin filaments also contribute, triggering the formation of the viroplasms through the association of soluble cytosolic VP4 with actin and the molecular motor myosin. This review explores the evolving understanding of RV replication, emphasizing the host requirements essential for viroplasm formation and highlighting their dynamic interplay within the host cell

Antiviral potential of 3'-sialyllactose- and 6'-sialyllactose-conjugated dendritic polymers against human and avian influenza viruses

Current treatment options for influenza virus infections in humans are limited and therefore the development of novel antivirals is of high priority. Inhibiting influenza virus attachment to host cells would provide an early and efficient block of the infection and thus, receptor analogs have been considered as options for antiviral treatment. Here, we describe the rapid and efficient synthesis of PAMAM dendrimers conjugated with either 3'-sialyllactose (3SL) or 6'-sialyllactose (6SL) and their potential to inhibit a diverse range of human and avian influenza virus strains. We show in a hemagglutination inhibition (HAI) assay that human IAV strains can be inhibited by (6SL)- and to a lesser extent also by (3SL)-conjugated PAMAM dendrimers. In contrast, avian strains could only be inhibited by (3SL)-conjugated dendrimers. Importantly, the differential sensitivities of human and avian IAV to the two types of sialyllactose-conjugated dendrimers could be confirmed in cell-based neutralization assays. Based on our findings, we suggest to further develop both, (3SL)- and (6SL)-conjugated PAMAM dendrimers, as influenza virus inhibitors

Implicit attitudes towards exercise and physical activity behaviour among in-patients with psychiatric disorders

The current body of evidence suggests that in healthy participants, implicit attitudes towards physical activity explain variance in exercise behaviour beyond explicit cognitive processes. However, such relationships have not been examined in psychiatric patients, although this may contribute to a better understanding of the motivational and volitional resources needed to self-regulate their exercise behaviour. Therefore, the present cross-sectional study aimed to assess implicit attitudes towards exercise among psychiatric in-patients, and to correlate these implicit attitudes with their physical activity levels. Patients (N = 101) showing a psychiatric disorder, but no severe cognitive impairment, were directly recruited from psychiatric clinics. Their physical activity levels were assessed using both accelerometers and self-reports. Additionally, patients reported psychiatric symptoms and performed a single-target implicit association test (ST-IAT) with exercise employed as the target category. Of all patients, 39% showed a preference for exercise, whereas 13% showed an aversion towards exercise. The implicit attitudes of the remaining participants were equally strong for both concepts. Based on correlational analysis (correcting for age, sex, psychiatric symptoms severity, and ST-IAT sequence), no association was found between ST-IAT score, or self-reported and objectively assessed physical activity. Consequently, the link between exercise behaviour and implicit attitudes towards physical activity found in healthy participants could not be observed in psychiatric patients