Coronaviral ORF6 protein mediates inter-organelle contacts and modulates host cell lipid flux for virus production.

Lipid droplets (LDs) form inter-organelle contacts with the endoplasmic reticulum (ER) that promote their biogenesis, while LD contacts with mitochondria enhance ß-oxidation of contained fatty acids. Viruses have been shown to take advantage of lipid droplets to promote viral production, but it remains unclear whether they also modulate the interactions between LDs and other organelles. Here, we showed that coronavirus ORF6 protein targets LDs and is localized to the mitochondria-LD and ER-LD contact sites, where it regulates LD biogenesis and lipolysis. At the molecular level, we find that ORF6 inserts into the LD lipid monolayer via its two amphipathic helices. ORF6 further interacts with ER membrane proteins BAP31 and USE1 to mediate ER-LDs contact formation. Additionally, ORF6 interacts with the SAM complex in the mitochondrial outer membrane to link mitochondria to LDs. In doing so, ORF6 promotes cellular lipolysis and LD biogenesis to reprogram host cell lipid flux and facilitate viral production.

SARS-CoV-2 USES ENDOSOMAL MEMBRANES TO BUILD REPLICATION ORGANELLES

Background: The health emergency caused by the COVID-19 pandemic has evidenced that the frequency of spillover episodes of viruses infecting bats to other species, including humans, has significantly increased compared to previous decades. Besides SARS-CoV-2, six other human coronaviruses (NL63, 229E, OC43, HKU1, SARS-CoV and MERS-CoV) emerged in the 20th and 21st century, most likely because of cross-species transmission events from bats. While many of these coronaviruses cause mild respiratory infections, MERS-CoV, SARS-CoV and SARS-CoV-2 can cause severe respiratory distress, particularly in immunocompromised individuals. However, unlike SARS-CoV and MERS-CoV, SARS-CoV-2 is highly contagious, very stable, with many person-to-person transmissions, which can occur even before individuals exhibit any symptoms. While vaccines are readily available, the emergence of new SARS-CoV-2 variants along with the increasing incidence of individuals developing long COVID urge to develop antivirals specific to treat COVID-19. To reach this goal, we need to have a working knowledge of the host-SARS-CoV-2 interactions to identify targets for therapeutic intervention. Method(s): Following that rationale, we focused on understanding how SARSCoV- 2 generates replication organelles (ROs). All coronaviruses need to remodel cellular membranes to create these structures to allow the active replication and transcription of their genome. Due to their relevance for virus replication, disabling RO formation represents a promising strategy to fight SARS-CoV-2. However, the biogenesis mechanism, the origin, and type of these replication organelles are still a major focus of debate. To identify the cellular membranes that SARS-CoV-2 uses to generate ROs we used multiple cell lines and primary cells that were evaluated by fluorescence microscopy, genetic engineering, compounds that specifically inhibit cellular processes, and immunoprecipitation assays to validate protein-protein interactions. We also used RT-qPCR to assess viral genome replication. Result(s): SARS-CoV-2 uses the viral protein NSP6 to remodel endosomal membranes juxtaposed to the ER to generate replication organelles. Specifically, the virus depends on Clathrin, COPB1, and Rab5 for efficient SARSCoV- 2 RNA synthesis. Conclusion(s): Uncovering the origins and mechanism(s) by which SARS-CoV-2 assembles ROs opens new avenues to develop strategies to interfere with RO biogenesis and halt virus replication.

Antiviral Activity of Acetylsalicylic Acid against Bunyamwera Virus in Cell Culture.

The Bunyavirales order is a large group of RNA viruses that includes important pathogens for humans, animals and plants. With high-throughput screening of clinically tested compounds we have looked for potential inhibitors of the endonuclease domain of a bunyavirus RNA polymerase. From a list of fifteen top candidates, five compounds were selected and their antiviral properties studied with Bunyamwera virus (BUNV), a prototypic bunyavirus widely used for studies about the biology of this group of viruses and to test antivirals. Four compounds (silibinin A, myricetin, L-phenylalanine and p-aminohippuric acid) showed no antiviral activity in BUNV-infected Vero cells. On the contrary, acetylsalicylic acid (ASA) efficiently inhibited BUNV infection with a half maximal inhibitory concentration (IC50) of 2.02 mM. In cell culture supernatants, ASA reduced viral titer up to three logarithmic units. A significant dose-dependent reduction of the expression levels of Gc and N viral proteins was also measured. Immunofluorescence and confocal microscopy showed that ASA protects the Golgi complex from the characteristic BUNV-induced fragmentation in Vero cells. Electron microscopy showed that ASA inhibits the assembly of Golgi-associated BUNV spherules that are the replication organelles of bunyaviruses. As a consequence, the assembly of new viral particles is also significantly reduced. Considering its availability and low cost, the potential usability of ASA to treat bunyavirus infections deserves further investigation.

Toxicity of the disinfectant benzalkonium chloride (C14) towards cyanobacterium Microcystis results from its impact on the photosynthetic apparatus and cell metabolism

Quaternary ammonium compounds (QACs) are commonly used in a variety of consumer and commercial products, typically as a component of disinfectants. During the COVID-19 pandemic, QACs became one of the primary agents utilized to inactivate the SARS-CoV-2 virus on surfaces. However, the ecotoxicological effects of QACs upon aquatic organisms have not been fully assessed. In this study, we examined the effects of a widely used QAC (benzalkonium chloride-C14, BAC-14) on two toxigenic Microcystis strains and one non-toxigenic freshwater Microcystis strain and carried out an analysis focused on primary, adaptive and compensatory stress responses at apical (growth and photosynthesis) and metabolic levels. This analysis revealed that the two toxic Microcystis strains were more tolerant than the non-toxic strain, with 96 hr-EC50 values of 0.70, 0.76, and 0.38 mg/L BAC-14 for toxigenic M. aeruginosa FACHB-905, toxigenic M. aeruginosa FACHB-469, and non-toxigenic M. wesenbergii FACHB-908, respectively. The photosynthetic activities of the Microcystis, assessed via Fv/Fm values, were significantly suppressed under 0.4 mg/L BAC-14. Furthermore, this analysis revealed that BAC-14 altered 14, 12, and 8 metabolic pathways in M. aeruginosa FACHB-905, M. aeruginosa FACHB-469, and M. wesenbergii FACHB-908, respectively. It is noteworthy that BAC-14 enhanced the level of extracellular microcystin production in the toxigenic Microcystis strains, although cell growth was not significantly affected. Collectively, these data show that BAC-14 disrupted the physiological and metabolic status of Microcystis cells and stimulated the production and release of microcystin, which could result in damage to aquatic systems. © 2022

COVID-19 Biogenesis and Intracellular Transport.

SARS-CoV-2 is responsible for the COVID-19 pandemic. The structure of SARS-CoV-2 and most of its proteins of have been deciphered. SARS-CoV-2 enters cells through the endocytic pathway and perforates the endosomes' membranes, and its (+) RNA appears in the cytosol. Then, SARS-CoV-2 starts to use the protein machines of host cells and their membranes for its biogenesis. SARS-CoV-2 generates a replication organelle in the reticulo-vesicular network of the zippered endoplasmic reticulum and double membrane vesicles. Then, viral proteins start to oligomerize and are subjected to budding within the ER exit sites, and its virions are passed through the Golgi complex, where the proteins are subjected to glycosylation and appear in post-Golgi carriers. After their fusion with the plasma membrane, glycosylated virions are secreted into the lumen of airways or (seemingly rarely) into the space between epithelial cells. This review focuses on the biology of SARS-CoV-2's interactions with cells and its transport within cells. Our analysis revealed a significant number of unclear points related to intracellular transport in SARS-CoV-2-infected cells.

Subversion of autophagy machinery and organelle-specific autophagy by SARS-CoV-2 and coronaviruses.

As a new emerging severe coronavirus, the knowledge on the SARS-CoV-2 and COVID-19 remains very limited, whereas many concepts can be learned from the homologous coronaviruses. Macroautophagy/autophagy is finely regulated by SARS-CoV-2 infection and plays important roles in SARS-CoV-2 infection and pathogenesis. This review will explore the subversion and mechanism of the autophagy-related machinery, vacuoles and organelle-specific autophagy during infection of SARS-CoV-2 and coronaviruses to provide meaningful insights into the autophagy-related therapeutic strategies for infectious diseases of SARS-CoV-2 and coronaviruses.

Probenecid-Induced IgA Nephropathy Is Reversible

Introduction: IgA nephropathy is usually idiopathic in nature but can have a genetic predisposition & it can also be secondary to autoimmune diseases, vasculitis, infections, liver disease, or anti-VEGFdrugs like bevacizumab, & covid vaccine. We present a case of probenecid-induced IgA nephropathy. Case Description: A 65-year-old male with chronic gout developed progressive chronic kidney disease over a 3-year period. He had been on probenecid 1 g twice daily for his gout for 15 yrs. His sodium iothalamate clearance deteriorated to 58 mL/min, with a serum creatinine of 1.5 mg/dL. All other serologic tests were negative. His 72-hour lead level was also normal. He did not have SLE, granulomatous disease, or systemic rheumatic disorders. Suprisingly he did not have proteinuria or hematuria. Renal biopsy revealed IgA nephropathy, with segmental mesangial hypercellularity, mild arterial sclerosis, & tubular atrophy. Cytoplasmic lipofuscin pigment was noted on PAS stain capillary loops with focal thickening of the glomerular basement membrane, engorged capillary loops, & thickened tubular basement membrane. Immunofluorescence studies showed diffuse segmental paramesangial granules of IgA [3+] & fibrinogen as well as paramesangial granules of IgG [2+], Kappa & lambda. Electron microscopy showed swollen podocytes with increased cytoplasmic organelles and vacuolization, focal foot process effacement, & electron-dense deposits in the paramesangium & mesangium. His MEST score was zero. 6 months after discontinuation of probenecid, the patient's iothalamate GFR significantly improved to 79 mL/min, followed by 82 mL/min 6 months later. He never had proteinuria, hematuria, or casts throughout his disease course. Five years later his GFR was 88 ml/min with a serum creatinine of 1.1 mg/dl. Discussion(s): Probenecid has pleiotropic effects on the human immune system. It inhibits Pannexin-1 channels which are known to modulate T-cell function. Probenecid also regulates TRPV -2 channels as an agonist. These channels are also present on human immune B and T cell lymphocytes. Probenecid inhibits VEGF in retinal endothelial cells, & bevacizumab, an anti-VEGF monoclonal antibody, has been shown to cause IgA nephropathy. We conclude that probenecid can be a cause of IgA nephropathy which is reversible upon drug discontinuation.

Chapter 26 - Phase separation and infectious diseases

Infectious diseases continue to represent a major threat to the humankind. This is reiterated by the current COVID-19 pandemic that affected almost 550 million people worldwide and caused more than 6.35 million deaths. It is clear that in addition to the existing preventive measures and treatments for various pathogens, better understanding is needed of the relationship between pathogen infection and the human antiinfection immune response and of the specific mechanisms underlying these complex processes. There is a constant warfare between the hosts and infectious pathogens, where humans have evolved a very effective and broadly amended antiinfection immune system, but, in their turn, pathogens have evolved a multitude of immune escape mechanisms to efficiently oppose it. It is recognized now that liquid–liquid phase separation (LLPS) occupies a special place among the important molecular mechanisms of the antiinfection immune response. Some illustrative examples of the roles of LLPS in the antiinfection immune response are considered in this chapter.

The double-membrane vesicle (DMV): a virus-induced organelle dedicated to the replication of SARS-CoV-2 and other positive-sense single-stranded RNA viruses.

Positive single-strand RNA (+ RNA) viruses can remodel host cell membranes to induce a replication organelle (RO) isolating the replication of their genome from innate immunity mechanisms. Some of these viruses, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMVs) for this purpose. Viral non-structural proteins are essential for DMV biogenesis, but they cannot form without an original membrane from a host cell organelle and a significant supply of lipids. The endoplasmic reticulum (ER) and the initial mechanisms of autophagic processes have been shown to be essential for the biogenesis of SARS-CoV-2 DMVs. However, by analogy with other DMV-inducing viruses, it seems likely that the Golgi apparatus, mitochondria and lipid droplets are also involved. As for hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMVs have been identified. These pores presumably allow the supply of metabolites essential for viral replication within the DMV, together with the export of the newly synthesized viral RNA to form the genome of future virions. It remains unknown whether, as for HCV, DMVs with open pores can coexist with the fully sealed DMVs required for the storage of large amounts of viral RNA. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis and morphology between these two phylogenetically distant viruses. An understanding of the mechanisms of DMV formation and their role in the infectious cycle of SARS-CoV-2 may be essential for the development of new antiviral approaches against this pathogen or other coronaviruses that may emerge in the future.

PRE-EXPOSURE TO ALCOHOL IMPAIRS ANTI-COVID DRUGS TREATMENTASSOCIATED CELLULAR STRESS RESPONSES IN LIVER CELLS

As the delta and omicron SARS-CoV-2 variants spread across the world, more tools to fight off serious infection have been developed. COVID antiviral drugs that can be taken orally at home could cut serious illness and reduce the risk of hospitalization and death. However, significant population of people consume alcohol before the infection and use of the antiviral drugs, which could potentiate side effects of the drugs on the liver. We investigated the role of alcohol in anti-Covid drug-induced stress responses in live cells. METHODS: HepG2 cells or primary mouse hepatocytes (PMH) were pre-treated with alcohol (50 mMlow dose or 100 mMhigh dose) for 6-24 hours and then treated with the newly developed oral anti-Covid drugs: nirmatrelvir, ritonavir, molnupiravir, and remdesivir at 10- 30 lg/ml for 6-24 hours. Unfolded protein response (UPR)/ER stress molecular markers (e.g. IRE1 GRP78, PERK, Xbp1 and CHOP), Golgi stress response (GSR) markers of GCP60, HSP47 and TFE3, and STAT3 were measured after the treatments. Cell death was assessed through double staining the liver cells with Syntox Green and Hoesche's Blue. RESULTS: ER stress response as indicated by IRE1, Xbp1 and CHOP was insignificant or mild in either HepG2 or PMH treated individually with alcohol at the low dose, nirmatrelvir, ritonavir, molnupiravir, or remdesivir. Alcohol or remdesivir induced moderate GSR based on mRNA increase of GCP60, HSP47 and TFE3, which was accompanied with apparent Golgi fragmentation in either HepG2 or PMH. Cell death rates in HepG2 treated with alcohol, nirmatrelvir, ritonavir, molnupiravir, or remdesivir individually were less than 5%. Pre-exposure to alcohol combined with subsequent treatment with nirmatrelvir, ritonavir molnupiravir, or remdesivir significantly increased both ER stress and GSR markers and expression of phosphorylated STAT3 (p-STAT3). Most significantly, cell death rates in HepG2 or PMH were increased by 2- to 5-fold by pre-alcohol exposure plus ritonavir, nirmatrelvir, molnupiravir, or remdesivir. The organelle stress markers, p-STAT3 and cell death were all further increased in alcoholand anti-Covid drug-treated HepG2 or primary mouse hepatocytes that were pre-infected with the lentiviruses that were pseudotyped with the SARS-CoV-2 spike protein. CONCLUSION: Our results indicate that pre-exposure to alcohol potentiates the liver cells to anti-Covid-19 drugs induced stress responses and cell death.

SARS-2 related HUPRA syndrome presenting with neonatal lactic acidosis

Introduction: Mitochondria are organelles that fulfill the energy requirements for cells, which is essential for their survival and function. Mitochondria function is dependent on both mitochondrial (mtDNA) and nuclear genes (Tucker, 2010). SARS2 is a nuclear gene that encodes the mitochondria seryl-tRNA synthetase precursor. It catalyzes the attachment of serine to tRNA and in the biosynthesis of selenocysteinyl-tRNA in the mitochondria. Pathogenic variation in the gene is associated with HUPRA syndrome, which is characterized by hyperuricemia, pulmonary hypertension, renal failure, and metabolic alkalosis (Rivera, 2013). It is important to recognize this autosomal recessive condition as it presents in infancy, can lead to death, and has recurrence implications for carrier couples. Case Description: We present a term neonate male who experienced tachypnea at birth requiring respiratory support;echocardiogram concerning for pulmonary hypertension and right ventricular hypertrophy requiring ionized nitric oxide. During his hospitalization, he developed lactic acidosis (consistently 10–12 mmol/L, reaching 26 mmol/L), seizures, and his newborn screen results flagged as abnormal for severe combined immunodeficiency (SCID) due to low Tcell count. He was transferred to a tertiary medical center due to continued elevated lactate levels. During admission to the tertiary medical center, he was found to have hyperkalemia, elevated BUN/Cr, and elevated lactate levels. Additionally, pre-prandial and postprandial lactate and pyruvate levels were obtained. It was found that hyperlactatemia was persistent and not related to feedings. The patient developed a presumed pulmonary hypertensive crisis at 8 weeks of age, and in the setting of chronic intrinsic renal dysfunction and chronic lactic acidosis, the family elected to transfer him to the home hospital for compassionate extubation where he died. Notable genetics evaluation findings included urine organic acid results showing markedly and persistently elevated levels of fumaric acid and lactic acid concerning for fumarase deficiency or a mitochondrial oxidative phosphorylation disorder and plasma amino acids showing elevated alanine and proline indicative of lactic acidosis. An array CGH showed 2% areas of homozygosity, consistent with known shared parental ancestry. The results of combined mitochondrial genome and Mitochondrial Nuclear Gene Panel was ordered. The results revealed two SARS2 variants: (c.988C>T,p.R330W and c.173T>A, p.L58Q). Both variants were classified as variants of uncertain significance (VUS) based on ACMG-AMP criteria (Richards, 2015) and parental testing to determine phase is ongoing. Discussion: Pathogenic variants in SARS2 lead to dysfunction of seryltRNA synthetase and is associated with HUPRA syndrome. Our patient harbors two variants in SARS2 classified as VUSes but based on clinical presentation the phenotype is consistent with HUPRA syndrome. The condition was first described in 2011 (Belostotsky, 2011) with 6 reported patients from 3 families (Belostotsky, 2011 and Rivera, 2013). Further study into pathogenic mechanism is important as no treatment exists, and the disease leads to death of the infants affected. Although the disease is very rare, it must be considered in infants with who present with symptoms of failure to thrive, hyperuricemia, pulmonary hypertension, renal failure, and metabolic alkalosis.

TARGETING LIPID METABOLISM in COVID-19

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. We demonstrate major effects of SARS-CoV- 2 to modulate cellular lipid metabolism in human cells favoring increased de novo lipid synthesis and lipid remodeling, leading to increased lipid droplet (LD) accumulation in human cells. We provided evidence that LDs participate at two levels of host pathogen interaction in SARS-CoV-2 infection: first, they are important players for virus replication;and second, they are central cell organelles in the amplification of inflammatory mediator production. We demonstrated that SARS-CoV-2 modulates pathways of lipid uptake and lipogenesis leading to increased LD accumulation in human host cells. We further showed that LDs are in close proximity with SARS-CoV-2 suggestive that LDs are recruited as part of replication compartment. Moreover, we demonstrated that inhibition of DGAT-1 blocked LD biogenesis, and reduced virus replication, cell-death and pro-inflammatory mediator production. Collectively, our findings support major roles for LDs in SARS-CoV-2 replication cycle and immune response. Moreover, the finding that the host lipid metabolism and LDs are required for SARS-CoV-2 replication suggests a potential strategy to interfere with SARS-CoV-2 replication and pathogenesis by targeting lipid metabolic pathway enzymes.

Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions.

In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein-an essential viral protein required for viral replication-and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.

Channels and Transporters of the Pulmonary Lamellar Body in Health and Disease.

The lamellar body (LB) of the alveolar type II (ATII) cell is a lysosome-related organelle (LRO) that contains surfactant, a complex mix of mainly lipids and specific surfactant proteins. The major function of surfactant in the lung is the reduction of surface tension and stabilization of alveoli during respiration. Its lack or deficiency may cause various forms of respiratory distress syndrome (RDS). Surfactant is also part of the innate immune system in the lung, defending the organism against air-borne pathogens. The limiting (organelle) membrane that encloses the LB contains various transporters that are in part responsible for translocating lipids and other organic material into the LB. On the other hand, this membrane contains ion transporters and channels that maintain a specific internal ion composition including the acidic pH of about 5. Furthermore, P2X4 receptors, ligand gated ion channels of the danger signal ATP, are expressed in the limiting LB membrane. They play a role in boosting surfactant secretion and fluid clearance. In this review, we discuss the functions of these transporting pathways of the LB, including possible roles in disease and as therapeutic targets, including viral infections such as SARS-CoV-2.

Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites.

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation.

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.

Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication.

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.

Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates.

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.

The Fatty Acid Lipid Metabolism Nexus in COVID-19.

Enteric symptomology seen in early-stage severe acute respiratory syndrome (SARS)-2003 and COVID-19 is evidence of virus replication occurring in the intestine, liver and pancreas. Aberrant lipid metabolism in morbidly obese individuals adversely affects the COVID-19 immune response and increases disease severity. Such observations are in line with the importance of lipid metabolism in COVID-19, and point to the gut as a site for intervention as well as a therapeutic target in treating the disease. Formation of complex lipid membranes and palmitoylation of coronavirus proteins are essential during viral replication and assembly. Inhibition of fatty acid synthase (FASN) and restoration of lipid catabolism by activation of AMP-activated protein kinase (AMPK) impede replication of coronaviruses closely related to SARS-coronavirus-2 (CoV-2). In vitro findings and clinical data reveal that the FASN inhibitor, orlistat, and the AMPK activator, metformin, may inhibit coronavirus replication and reduce systemic inflammation to restore immune homeostasis. Such observations, along with the known mechanisms of action for these types of drugs, suggest that targeting fatty acid lipid metabolism could directly inhibit virus replication while positively impacting the patient's response to COVID-19.