Distribution and suitability of pulmonary surfactants as a vehicle for topically applied antibodies in healthy and SARS-CoV-2 infected rodent lungs.

The use of natural pulmonary surfactants (PS) as a drug delivery vehicle for biologics is a more recent therapeutic modality. Herein, we tested different contents of PS regarding their physicochemical properties under stress conditions. The PS content of 12.25 mg/ml (Formulation B) showed desired properties such as an isotonic osmolality ∼300 mOsm/kg and an acceptable viscosity of 8.61 cSt, being lower than in commercially available PS solutions. Formulation B passed the specifications of surface lowering capacities of >80 % total lung capacity and physiologically desired formulation properties were independent of the antibody used in the composition. The identified formulation showed the capability of significantly increasing the oxygen saturation in ex vivo isolated perfused rat lungs, compared to a control and up to 30 min post lavage. In the in vivo setting, we showed that intratracheal administration of a human mAB with and without pulmonary surfactant led to higher amounts of delivered antibody within the alveolar tissue compared to intravenous administration. The antibody with the PS formulation remained longer in the alveolar tissues than the antibody without the PS formulation. Further, SARS-CoV-2 infected Golden Syrian hamsters showed that the intranasally applied antibody reached the site of infection in the alveoli and could be detected in the alveolar region 24 h after the last administration. With this work, we demonstrated that pulmonary surfactants can be used as a pulmonary drug delivery mechanism for antibodies and may subsequently improve the antibody efficacy by increasing the residence time at the desired site of action in the alveolar tissue.

SARS-CoV-2 induced changes in the glycosylation pattern in the respiratory tract of Golden Syrian hamsters.

Even after more than two years of intensive research, not all of the pathophysiological processes of Coronavirus Disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, have been fully elucidated. The initial virus-host interaction at the respiratory epithelium plays a crucial role in the course and progression of the infection, and is highly dependent on the glycosylation pattern of the host cell and of the secreted mucins. Glycans are polysaccharides that can be attached to proteins and thereby add to their stability and functionality. Lectins are glycan-binding proteins that recognize specific glycan motifs, and lectin histochemistry is a suitable tool to visualize and examine glycosylation pattern changes in tissues. In this study we used lectins with different glycan-specificities for the visualization of glycosylation pattern changes in the respiratory tract of SARS-CoV-2 infected Golden Syrian hamsters. While some lectins (LEL, STL) enable the visualization of the damage to alveolar type 1 pneumocytes, other lectins, e.g., GSLI, visualized the loss and subsequent hyperplasia of type 2 pneumocytes. UEAI staining was co-localized with KI67, a proliferation marker. Double staining of lectins LEL, STL and WGA with specific immune cell markers (Iba1, CD68) showed co-localization and the dominant infiltration of monocyte-derived macrophages into infected alveolar tissue. The elucidation of the glycosylation pattern of the respiratory tract cells in uninfected and infected Golden Syrian hamsters revealed physiological and pathological aspects of the disease that may open new possibilities for therapeutic development.

Compellingly high SARS-CoV-2 susceptibility of Golden Syrian hamsters suggests multiple zoonotic infections of pet hamsters during the COVID-19 pandemic.

Golden Syrian hamsters (Mesocricetus auratus) are used as a research model for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Millions of Golden Syrian hamsters are also kept as pets in close contact to humans. To determine the minimum infective dose (MID) for assessing the zoonotic transmission risk, and to define the optimal infection dose for experimental studies, we orotracheally inoculated hamsters with SARS-CoV-2 doses from 1 * 105 to 1 * 10-4 tissue culture infectious dose 50 (TCID50). Body weight and virus shedding were monitored daily. 1 * 10-3 TCID50 was defined as the MID, and this was still sufficient to induce virus shedding at levels up to 102.75 TCID50/ml, equaling the estimated MID for humans. Virological and histological data revealed 1 * 102 TCID50 as the optimal dose for experimental infections. This compelling high susceptibility leading to productive infections in Golden Syrian hamsters must be considered as a potential source of SARS-CoV-2 infection for humans that come into close contact with pet hamsters.

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

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.

Differential Localization of Structural and Non-Structural Proteins during the Bluetongue Virus Replication Cycle.

Members of the Reoviridae family assemble virus factories within the cytoplasm of infected cells to replicate and assemble virus particles. Bluetongue virus (BTV) forms virus inclusion bodies (VIBs) that are aggregates of viral RNA, certain viral proteins, and host factors, and have been shown to be sites of the initial assembly of transcriptionally active virus-like particles. This study sought to characterize the formation, composition, and ultrastructure of VIBs, particularly in relation to virus replication. In this study we have utilized various microscopic techniques, including structured illumination microscopy, and virological assays to show for the first time that the outer capsid protein VP5, which is essential for virus maturation, is also associated with VIBs. The addition of VP5 to assembled virus cores exiting VIBs is required to arrest transcriptionally active core particles, facilitating virus maturation. Furthermore, we observed a time-dependent association of the glycosylated non-structural protein 3 (NS3) with VIBs, and report on the importance of the two polybasic motifs within NS3 that facilitate virus trafficking and egress from infected cells at the plasma membrane. Thus, the presence of VP5 and the dynamic nature of NS3 association with VIBs that we report here provide novel insight into these previously less well-characterized processes.

Hsp90 Chaperones Bluetongue Virus Proteins and Prevents Proteasomal Degradation.

The molecular chaperone machinery is important for the maintenance of protein homeostasis within the cells. The principle activities of the chaperone machinery are to facilitate protein folding and organize conformationally dynamic client proteins. Prominent among the members of the chaperone family are heat shock protein 70 (Hsp70) and 90 (Hsp90). Like cellular proteins, viral proteins depend upon molecular chaperones to mediate their stabilization and folding. Bluetongue virus (BTV), which is a model system for the Reoviridae family, is a nonenveloped arbovirus that causes hemorrhagic disease in ruminants. This constitutes a significant burden upon animals of commercial significance, such as sheep and cattle. Here, for the first time, we examined the role of chaperone proteins in the viral lifecycle of BTV. Using a combination of molecular, biochemical, and microscopic techniques, we examined the function of Hsp90 and its relevance to BTV replication. We demonstrate that Hsp70, the chaperone that is commonly usurped by viral proteins, does not influence virus replication, while Hsp90 activity is important for virus replication by stabilizing BTV proteins and preventing their degradation via the ubiquitin-proteasome pathway. To our knowledge this is the first report showing the involvement of Hsp90 as a modulator of BTV infection.IMPORTANCE Protein chaperones are instrumental for maintaining protein homeostasis, enabling correct protein folding and organization; prominent members include heat shock proteins 70 and 90. Virus infections place a large burden on this homeostasis. Identifying and understanding the underlying mechanisms that facilitate Bluetongue virus replication and spread through the usurpation of host factors is of primary importance for the development of intervention strategies. Our data identify and show that heat shock protein 90, but not heat shock protein 70, stabilizes bluetongue virus proteins, safeguarding them from proteasomal degradation.

Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection.

Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signaling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analyzed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication.

Elucidating virus entry using a tetracysteine-tagged virus.

Fluorescent tags constitute an invaluable tool in facilitating a deeper understanding of the mechanistic processes governing virus-host interactions. However, when selecting a fluorescent tag for in vivo imaging of cells, a number of parameters and aspects must be considered. These include whether the tag may affect and interfere with protein conformation or localization, cell toxicity, spectral overlap, photo-stability and background. Cumulatively, these constitute challenges to be overcome. Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a non-enveloped virus that is comprised of two architecturally complex capsids. The outer capsid, composed of two proteins, VP2 and VP5, together facilitate BTV attachment, entry and the delivery of the transcriptionally active core in the cell cytoplasm. Previously, the significance of the endocytic pathway for BTV entry was reported, although a detailed analysis of the role of each protein during virus trafficking remained elusive due to the unavailability of a tagged virus. Described here is the successful modification, and validation, of a segmented genome belonging to a complex and large capsid virus to introduce tags for fluorescence visualization. The data generated from this approach highlighted the sequential dissociation of VP2 and VP5, driven by decreasing pH during the transition from early to late endosomes, and their retention therein as the virus particles progress along the endocytic pathway. Furthermore, the described tagging technology and methodology may prove transferable and allow for the labeling of other non-enveloped complex viruses.

Cellular Casein Kinase 2 and Protein Phosphatase 2A Modulate Replication Site Assembly of Bluetongue Virus.

A number of cytoplasmic replicating viruses produce cytoplasmic inclusion bodies or protein aggregates; however, a hallmark of viruses of the Reoviridae family is that they utilize these sites for purposes of replication and capsid assembly, functioning as viral assembly factories. Here we have used bluetongue virus (BTV) as a model system for this broad family of important viruses to understand the mechanisms regulating inclusion body assembly. Newly synthesized viral proteins interact with sequestered viral RNA molecules prior to capsid assembly and double-stranded RNA synthesis within viral inclusion bodies (VIBs). VIBs are predominantly comprised of a BTV-encoded non-structural protein 2 (NS2). Previous in vitro studies indicated that casein kinase 2 (CK2) mediated the phosphorylation of NS2, which regulated the propensity of NS2 to form larger aggregates. Using targeted pharmacological reagents, specific mutation in the viral genome by reverse genetics and confocal microscopy, here we demonstrate that CK2 activity is important for BTV replication. Furthermore, we show that a novel host cell factor, protein phosphatase 2A, is involved in NS2 dephosphorylation and that, together with CK2, it regulates VIB morphology and virus replication. Thus, these two host enzymes influence the dynamic nature of VIB assembly/disassembly, and these concerted activities may be relevant to the assembly and the release of these cores from VIBs.

Entry of Bluetongue Virus Capsid Requires the Late Endosome-specific Lipid Lysobisphosphatidic Acid.

The entry of viruses into host cells is one of the key processes of infection. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show, utilizing an in vitro liposome penetration assay and cell biology, that bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosome-specific lipid lysobisphosphatidic acid for productive membrane penetration and viral entry. Further, we provide preliminary evidence that lipid lysobisphosphatidic acid facilitates pore expansion during membrane penetration, suggesting a mechanism for lipid factor requirement of BTV. This finding indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry.

Early events in the generation of autophagosomes are required for the formation of membrane structures involved in hepatitis C virus genome replication.

Hepatitis C virus (HCV) infection has been shown to induce autophagy but the mechanisms underpinning this process remain to be elucidated. Induction of autophagy requires the class III phosphatidylinositol 3-kinase, Vps34, which produces phosphatidylinositol 3-phosphate (PI3P) within the endoplasmic reticulum (ER) membrane. This recruits proteins with PI3P binding domains such as the double-FYVE-containing protein 1 (DFCP1). DFCP1 generates cup-shaped protrusions from the ER membrane, termed omegasomes, which provide a platform for the production of autophagosomes. Here we present data demonstrating that both Vps34 and DFCP1 are required for HCV genome replication, in the context of both a subgenomic replicon and virus infection, but did not affect virus entry or initial translation. Using live cell fluorescence microscopy we demonstrated that early during HCV infection the nascent viral genome replication complexes (identified by using non-structural protein NS5A as a marker) transiently colocalize with DFCP1-positive punctae (omegasomes), before the two structures move apart from each other. This observation is reminiscent of the transient association of LC3 and DFCP1 during omegasome formation, and therefore we propose that omegasomes are utilized by HCV to generate the double-membrane vesicles which are the hallmark of HCV replication complexes.

Requirement for chloride channel function during the hepatitis C virus life cycle.

Hepatocytes express an array of plasma membrane and intracellular ion channels, yet their role during the hepatitis C virus (HCV) life cycle remains largely undefined. Here, we show that HCV increases intracellular hepatic chloride (Cl(-)) influx that can be inhibited by selective Cl(-) channel blockers. Through pharmacological and small interfering RNA (siRNA)-mediated silencing, we demonstrate that Cl(-) channel inhibition is detrimental to HCV replication. This represents the first observation of the involvement of Cl(-) channels during the HCV life cycle.

Bluetongue virus capsid assembly and maturation.

Maturation is an intrinsic phase of the viral life cycle and is often intertwined with egress. In this review we focus on orbivirus maturation by using Bluetongue virus (BTV) as a representative. BTV, a member of the genus Orbivirus within the family Reoviridae, has over the last three decades been subjected to intense molecular study and is thus one of the best understood viruses. BTV is a non-enveloped virus comprised of two concentric protein shells that encapsidate 10 double-stranded RNA genome segments. Upon cell entry, the outer capsid is shed, releasing the core which does not disassemble into the cytoplasm. The polymerase complex within the core then synthesizes transcripts from each genome segment and extrudes these into the cytoplasm where they act as templates for protein synthesis. Newly synthesized ssRNA then associates with the replicase complex prior to encapsidation by inner and outer protein layers of core within virus-triggered inclusion bodies. Maturation of core occurs outside these inclusion bodies (IBs) via the addition of the outer capsid proteins, which appears to be coupled to a non-lytic, exocytic pathway during early infection. Similar to the enveloped viruses, BTV hijacks the exocytosis and endosomal sorting complex required for trafficking (ESCRT) pathway via a non-structural glycoprotein. This exquisitely detailed understanding is assembled from a broad array of assays, spanning numerous and diverse in vitro and in vivo studies. Presented here are the detailed insights of BTV maturation and egress.

Hepatitis C virus-induced autophagy is independent of the unfolded protein response.

Hepatitis C virus (HCV) has been shown to induce autophagy and the unfolded protein response (UPR), but the mechanistic link between the induction of these two cellular processes remains unclear. We demonstrate here that HCV infection induces autophagy, as judged by accumulation of lipidated LC3-II, and that this induction occurs rapidly after infection, preceding the stimulation of the UPR, which occurs only at later stages, after the viral envelope glycoproteins have been expressed to high levels. Furthermore, both genotype 1b and 2a subgenomic replicons expressing nonstructural (NS3-5B) proteins and JFH-1 virus lacking the envelope glycoproteins potently induced autophagy in the absence of detectable UPR. This ability was also shared by a subgenomic replicon derived from the related GB virus B (GBV-B). We also show that small interfering RNA (siRNA)-mediated silencing of the key UPR inducer, Ire1, has no effect on HCV genome replication or the induction of autophagy, further demonstrating that the UPR is not required for these processes. Lastly, we demonstrate that the HCV replicase does not colocalize with autophagosomes, suggesting that the induction of autophagy is not required to generate the membrane platform for HCV RNA replication.

Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus.

Bunyamwera virus (BUNV) is the prototypic member of both the Orthobunyavirus genus and the Bunyaviridae family of negative stranded RNA viruses. In common with all negative stranded RNA viruses, the BUNV genomic and anti-genomic strands are not naked RNAs, but instead are encapsidated along their entire lengths with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex. This association is critical for the negative strand RNA virus life cycle because only RNPs are active for productive RNA synthesis and RNA packaging. We are interested in understanding the molecular details of how N and RNA components associate within the bunyavirus RNP, and what governs the apparently selective encapsidation of viral replication products. Toward this goal, we recently devised a protocol that allowed generation of native BUNV N protein that maintained solubility under physiological conditions and allowed formation of crystals that yielded high-resolution x-ray diffraction data. Here we extend this work to show that this soluble N protein is able to oligomerize and bind RNA to form a highly uniform RNP complex, which exhibits characteristics in common with the viral RNP. By extracting and sequencing RNAs bound to these model RNPs, we determined the stoichiometry of N-RNA association to be approximately 12 nucleotides per N monomer. In addition, we defined the minimal sequence requirement for BUNV RNA replication. By comparing this minimal sequence to those bound to our model RNP, we conclude that N protein does not obligatorily require a sequence or structure for RNA encapsidation.