Herpes simplex virus type-1 cVAC formation in neuronal cells is mediated by dynein motor function and glycoprotein retrieval from the plasma membrane.

Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection.IMPORTANCEMany viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.

In situ fate of Chikungunya virus replication organelles.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.

The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments.

BACKGROUND: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. METHODS: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. RESULTS: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. CONCLUSIONS: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

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.

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. 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.

Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories.

Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication, and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis.

Shedding light on reovirus assembly-Multimodal imaging of viral factories.

Avian (ortho)reovirus (ARV), which belongs to Reoviridae family, is a major domestic fowl pathogen and is the causative agent of viral tenosynovitis and chronic respiratory disease in chicken. ARV replicates within cytoplasmic inclusions, so-called viral factories, that form by phase separation and thus belong to a wider class of biological condensates. Here, we evaluate different optical imaging methods that have been developed or adapted to follow formation, fluidity and composition of viral factories and compare them with the complementary structural information obtained by well-established transmission electron microscopy and electron tomography. The molecular and cellular biology aspects for setting up and following virus infection in cells by imaging are described first. We then demonstrate that a wide-field version of fluorescence recovery after photobleaching is an effective tool to measure fluidity of mobile viral factories. A new technique, holotomographic phase microscopy, is then used for imaging of viral factory formation in live cells in three dimensions. Confocal Raman microscopy of infected cells provides "chemical" contrast for label-free segmentation of images and addresses important questions about biomolecular concentrations within viral factories and other biological condensates. Optical imaging is complemented by electron microscopy and tomography which supply higher resolution structural detail, including visualization of individual virions within the three-dimensional cellular context.

The Formation and Function of Birnaviridae Virus Factories.

The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid-liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes.

Molten Globule Driven and Self-downmodulated Phase Separation of a Viral Factory Scaffold.

Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".

Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.

Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. IMPORTANCE Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.

A Fijivirus Major Viroplasm Protein Shows RNA-Stimulated ATPase Activity by Adopting Pentameric and Hexameric Assemblies of Dimers.

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.

The endocytic recycling compartment serves as a viral factory for hepatitis E virus.

Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.

The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers.

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.

Tuning the Dynamics of Viral-Factories-Inspired Compartments Formed by Peptide-RNA Liquid-Liquid Phase Separation.

Viral factories are intracellular microcompartments formed by mammalian viruses in their host cells, and contain necessary machinery for viral genome replication, capsid assembly, and maturation, thus serving as "factories" for formation of new viral particles. Recent evidence suggests that these compartments are formed by liquid-liquid phase separation (LLPS) of viral proteins and nucleic acids and present dynamic properties. In this work, inspired by the remarkable functionalities of viral factories, dynamic compartments that are formed by complexation between a minimalistic, disordered peptide and RNA are designed. By systematic studies using sequence variants it is shown that the material properties of the compartments can be modulated by changes to the peptide sequence, at the single amino acid level. Moreover, by taking this approach to the next step, liquid compartments with light-induced tunable dynamics are developed. The results demonstrate that the material properties of liquid droplets can be temporally regulated by increasing peptide polarity and charge, and that these changes can be further utilized for controlled partitioning and release of payloads from the compartments.

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.

Herpesvirus Replication Compartments: Dynamic Biomolecular Condensates?

Recent progress has provided clear evidence that many RNA-viruses form cytoplasmic biomolecular condensates mediated by liquid-liquid phase separation to facilitate their replication. In contrast, seemingly contradictory data exist for herpesviruses, which replicate their DNA genomes in nuclear membrane-less replication compartments (RCs). Here, we review the current literature and comment on nuclear condensate formation by herpesviruses, specifically with regard to RC formation. Based on data obtained with human cytomegalovirus (human herpesvirus 5), we propose that liquid and homogenous early RCs convert into more heterogeneous RCs with complex properties over the course of infection. We highlight how the advent of DNA replication leads to the maturation of these biomolecular condensates, likely by adding an additional DNA scaffold.

VMP1 and TMEM41B are essential for DMV formation during ß-coronavirus infection.

ß-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3-nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.

Rotavirus exploits SREBP pathway for hyper lipid biogenesis during replication.

Species A rotavirus (RVA) is one of the pathogens causing severe acute gastroenteritis in young children and animals worldwide. RVA replicates and assembles its immature particle within electron dense compartments known as viroplasm. Despite the importance of lipid droplet (LD) formation in the RVA viroplasm, the upstream molecules modulating LD formation have remained elusive. Here, we demonstrate that RVA infection reprogrammes sterol regulatory element binding proteins (SREBPs)-dependent lipogenic pathways in virus-infected cells. Interestingly, silencing of SREBPs significantly reduced RVA protein synthesis, genome replication and progeny virus production. Moreover, knockout of SREBP-1c gene conferred resistance to RVA-induced diarrhoea, reduction of RVA replication, and mitigation of small intestinal pathology in mice. This study identifies SREBPs-mediated lipogenic reprogramming in RVA-infected host cells for facilitating virus replication and SREBPs as a potential target for developing therapeutics against RVA infection.