YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity

The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity.

A global map of the Zika virus phosphoproteome reveals host-driven regulation of viral budding

Flaviviruses are enveloped, positive-strand RNA viruses that cause millions of infections in the human population annually. Although Zika virus (ZIKV) had been detected in humans as early as the 1950s, its reemergence in South America in 2015 resulted in a global health crisis. While flaviviruses encode 10 proteins that can be post-translationally modified by host enzymes, little is known regarding post-translational modifications (PTMs) of the flavivirus proteome. We used mass spectrometry to comprehensively identify host-driven PTMs on the ZIKV proteome. This approach allowed us to identify 43 PTMs across 8 ZIKV proteins, including several that are highly conserved within the Flavivirus genus. Notably, we found two phosphosites on the ZIKV envelope protein that are functionally important for viral propagation and appear to regulate viral budding. Additionally, we discovered 115 host kinases that interacted with ZIKV proteins and determined that Bosutinib, an FDA-approved tyrosine kinase inhibitor that targets ZIKV interacting host kinases, impairs ZIKV growth. Thus, we have defined a high-resolution map of host-driven PTMs on ZIKV proteins as well as cellular interacting kinases, uncovered a novel mechanism of host driven-regulation of ZIKV budding, and identified an FDA-approved inhibitor of ZIKV growth.

Lipid droplets are beneficial for rabies virus replication by facilitating viral budding

Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets have been reported to play a role in pathogenesis of several viruses. However, its role on RABV infection remains unclear. Here, we initially found that RABV infection upregulated lipid droplet (LD) production in multiple cells and mouse brains. After the treatment of atorvastatin, a specific inhibitor of LD, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhance the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglycerides synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication and their biogenesis is regulated via NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but its role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier and transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.

Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

Structural Insight into the Interaction of Sendai Virus C Protein with Alix To Stimulate Viral Budding

The Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae , harbors an accessory protein, named C protein, which facilitates the viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles through the binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, siRNA-mediated gene knockdown studies suggested that neither Alix nor C protein are related to the SeV budding. In the present study, we determined the crystal structure of a complex comprising of the C -terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å, to investigate the role of the association in the SeV budding. The structure revealed that a novel consensus sequence, LxxW, which is conserved among the Respirovirus C proteins, is important for the Alix-binding. SeV possessing a mutated C protein with a reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h post-infection compared with non-mutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, on the binding to Alix. Altogether, these results highlight the critical role of C protein in the SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting in young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been extensively studied to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating the viral reproduction, however, its role in budding remains controversial. In the present study, the crystal structure of the C -terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of a cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutated C proteins with different binding affinity to Alix, and showed that the interaction between C and Alix is vital for the viral budding. These findings provide new insights into the development of a new antiviral drugs against hPIV1.

The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

Phosphatidylserine (PS) has been shown to be a critical lipid factor in the assembly and spread of numerous lipid enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA approved drug, fendiline, to reduce PS clustering subsequently reducing virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were employed including imaging, viral budding and viral entry assays. Fendiline reduced the PS content in mammalian cells and PS in the plasma membrane, reducing the ability of VP40 to form new virus particles. Further, particles that do form from fendiline treated cells have altered particle morphology and decreased infectivity capacity. These complementary studies reveal the mechanism by which filovirus matrix proteins cluster PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

Biophysical characterization of the SARS-CoV-2 E protein

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic which continues to wreak havoc across the world, over a year and a half after its effects were first reported in the general media. Current fundamental research efforts largely focus on the SARS-CoV-2 Spike protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length E protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

Ilaprazole and other novel prazole-based compounds that bind Tsg101 inhibit viral budding of HSV-1/2 and HIV from cells

In many enveloped virus families, including HIV and HSV, a crucial, yet unexploited, step in the viral life cycle is releasing particles from the infected cell membranes. This release process is mediated by host ESCRT complex proteins, which are recruited by viral structural proteins and provides the mechanical means for membrane scission and subsequent viral budding. The prazole drug, tenatoprazole, was previously shown to bind to ESCRT complex member Tsg101 and to quantitatively block the release of infectious HIV-1 from cells in culture. In this report we show that tenatoprazole and a related prazole drug, ilaprazole, effectively block infectious Herpes Simplex Virus (HSV)-1/2 release from Vero cells in culture. By electron microscopy, we found that both prazole drugs block the transit of HSV particles through the cell nuclear membrane resulting in their accumulation in the nucleus. Ilaprazole also quantitatively blocks the release of HIV-1 from 293T cells with an EC50 of 0.8-1.2 μM, which is much more potent than tenatoprazole. Our results indicate that prazole-based compounds may represent a class of drugs with potential to be broad-spectrum antiviral agents against multiple enveloped viruses, by interrupting cellular Tsg101 interaction with maturing virus, thus blocking the budding process that releases particles from the cell. Importance These results provide the basis for the development of drugs that target enveloped virus budding that can be used ultimately to control multiple virus infections in humans.

S-acylation controls SARS-Cov-2 membrane lipid organization and enhances infectivity

SARS-CoV-2 virions are surrounded by a lipid bilayer which contains membrane proteins such as Spike, responsible for target-cell binding and virus fusion, the envelope protein E and the accessory protein Orf3a. Here, we show that during SARS-CoV-2 infection, all three proteins become lipid modified, through action of the S- acyltransferase ZDHHC20. Particularly striking is the rapid acylation of Spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics and biochemical approaches, we show that this massive lipidation controls Spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid rich lipid nanodomains, in the early Golgi where viral budding occurs. ZDHHC20-mediated acylation allows the formation of viruses with enhanced fusion capacity and overall infectivity. Our study points towards S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.