Curcumin Inhibits Replication of Human Parainfluenza Virus Type 3 by Affecting Viral Inclusion Body Formation

Human Parainfluenza Virus Type 3 (HPIV3) is one of the main pathogens that cause acute lower respiratory tract infections in infants and young children. However, there are currently no effective antiviral drugs and vaccines. Herein, we found that a natural compound, curcumin, inhibits HPIV3 infection and has antiviral effects on entry and replication of the virus life cycle. Immunofluorescence and western blotting experiments revealed that curcumin disrupts F-actin and inhibits viral inclusion body (IB) formation, thus inhibiting virus replication. Curcumin can also downregulate cellular PI4KB and interrupt its colocalization in viral IBs. This study verified the antiviral ability of curcumin on HPIV3 infection and preliminarily elucidated its influence on viral replication, providing a theoretical basis for antiviral drug development of HPIV3 and other parainfluenza viruses.

New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections

Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane-less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to different protein functions. P keeps N soluble after expression to allow a concerted binding of N to the viral RNA. This results in the encapsidation of the viral genome by N, while P acts additionally as a cofactor for the viral polymerase, enabling viral transcription and replication. Here, we will review the formation and function of those viral inclusion bodies upon infection with NSVs with respect to their nature as biomolecular condensates.

Oral ulcerations in a patient with autosomal dominant hyper-IgE syndrome (AD-HIES)

A 23-year-old woman with autosomal dominant hyper-IgE syndrome complicated by recurrent pneumonia and sinusitis presented with 1 week of multiple painful oral ulcers unresponsive to empiric antiviral and antifungal treatment. Her ulcers progressively worsened and she required hospitalisation for intravenous hydration and pain control. PCR swab of an ulcer was positive for varicella-zoster virus. Her symptoms never fully resolved despite antiviral therapy, and within 2 weeks, she relapsed with new and worsening ulcers. Biopsy revealed chronic active inflammation with no evidence of viral inclusion bodies or fungal hyphae. She was diagnosed with recurrent aphthous stomatitis and referred to a local dentist for CO2 laser treatments with rapid resolution of her symptoms. This case highlights the broad differential for recurrent oral ulcers in people with a primary immunodeficiency. It also raises awareness of the benefits of laser therapy for aphthous stomatitis treatment and the importance of partnering with our colleagues in dentistry.

A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication

ABSTRACT Many viruses use specific viral proteins to bind calcium ions (Ca2+) for stability or to modify host cell pathways; however, to date, no Ca2+ binding protein has been reported in bluetongue virus (BTV), the causative agent of bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV nonstructural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by alanine. Using circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in the presence of Ca2+ revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain that wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus, and the NS2 phosphorylation level and assembly of viral inclusion bodies (VIBs) were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly. IMPORTANCE After entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.

Measles Virus Forms Inclusion Bodies with Properties of Liquid Organelles

ABSTRACT Nonsegmented negative-strand RNA viruses, including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane bound, and they serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here, we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying that they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggests that Mononegavirales have broadly evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells. IMPORTANCE Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur, and globally ∼100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WD repeat-containing protein 5 (WDR5) enhances MeV growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here, we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies, that these structures display properties characteristic of phase-separated liquid organelles, and that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.

Genetic Polymorphisms and Molecular Mechanisms Mediating Oncolytic Potency of Reovirus Strains

ABSTRACTThe Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapeutic agent. In this study, a comprehensive phenotypic and genetic comparison of T3D virus stocks from various laboratories and commercial sources revealed that T3D laboratory strains differ substantially in their oncolytic activitiesin vitroandin vivo. Superior replication of the most-oncolytic T3D lab strain was attributed to several mechanistic advantages: virus-cell binding, viral RNA transcriptase activity, viral inclusion morphology, and differential activation of RIG-I versus NFκB-dependent signalling pathways. Viral S4, M1 and L3 gene segments were each independently associated with a distinct mechanistic advantage. Furthermore, the specific missense polymorphisms that governed replication potency were identified, and utilized to generate a hybrid of T3D laboratory strains with further-augmented replication in tumor cells. Together, the results depict an elaborate balance between reovirus replication and host-cell signaling to achieve optimal oncolytic reovirus efficacy.