RetroCHMP3 blocks budding of enveloped viruses without blocking cytokinesis.

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.

Minor electrostatic changes robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

A fatty acid ordered plasma membrane environment is critical for Ebola virus matrix protein assembly and budding.

Plasma membrane (PM) domains and order phases have been shown to play a key role in the assembly, release, and entry of several lipid-enveloped viruses. In the present study, we provide a mechanistic understanding of the Ebola virus (EBOV) matrix protein VP40 interaction with PM lipids and their effect on VP40 oligomerization, a crucial step for viral assembly and budding. VP40 matrix formation is sufficient to induce changes in the PM fluidity. We demonstrate that the distance between the lipid headgroups, the fatty acid tail saturation and the PM order are important factors for the stability of VP40 binding and oligomerization at the PM. Use of FDA-approved drugs to fluidize the PM, destabilizes the viral matrix assembly leading to a reduction in budding efficiency. Overall, these findings support an EBOV assembly mechanism that reaches beyond lipid headgroup specificity by using ordered PM lipid regions independent of cholesterol.

Oropouche Virus Glycoprotein Topology and Cellular Requirements for Glycoprotein Secretion.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.

Mutagenesis of the di-leucine motif in the cytoplasmic tail of newcastle disease virus fusion protein modulates the viral fusion ability and pathogenesis.

BACKGROUND: Newcastle disease virus (NDV) is a highly infectious viral disease, which can affect chickens and many other kinds of birds. The main virulence factor of NDV, the fusion (F) protein, is located on the viral envelope and plays a major role in the virus' ability to penetrate cells and cause host cell fusion during infection. Multiple highly conserved tyrosine and di-leucine (LL) motifs in the cytoplasmic tail (CT) of the virus may contribute to F protein functionality in the viral life cycle. METHODS: To examine the contribution of the LL motif in the biosynthesis, transport, and function of the F protein, we constructed and rescued a NDV mutant strain, rSG10*-F/L537A, with an L537A mutation using a reverse genetic system. Subsequently, we compared the differences in the syncytium formation ability, pathogenicity, and replication levels of wild-type rSG10* and the mutated strain. RESULTS: Compared with rSG10*, rSG10*-F/L537A had attenuated syncytial formation and pathogenicity, caused by a viral budding defect. Further studies showed that the LL-motif mutation did not affect the replication, transcription, or translation of the virus genome but affected the expression of the F protein at the cell surface. CONCLUSIONS: We concluded that the LL motif in the NDV F CT affected the regulation of F protein expression at the cell surface, thus modulating the viral fusion ability and pathogenic phenotype.

Hepatitis B virus movement through the hepatocyte: An update.

Viruses are obligate intracellular pathogens that utilize cellular machinery for many aspects of their propagation and effective egress of virus particles from host cells is one important determinant of virus infectivity. Hijacking host cell processes applies in particular to the hepatitis B virus (HBV), as its DNA genome with about 3 kb in size is one of the smallest viral genomes known. HBV is a leading cause of liver disease and still displays one of the most successful pathogens in human populations worldwide. The extremely successful spread of this virus is explained by its efficient transmission strategies and its versatile particle types, including virions, empty envelopes, naked capsids, and others. HBV exploits distinct host trafficking machineries to assemble and release its particle types including nucleocytoplasmic shuttling transport, secretory, and exocytic pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Understanding how HBV uses and subverts host membrane trafficking systems offers the chance of obtaining new mechanistic insights into the regulation and function of this essential cellular processes. It can also help to identify potential targets for antiviral interventions. Here, I will provide an overview of HBV maturation, assembly, and budding, with a focus on recent advances, and will point out areas where questions remain that can benefit from future studies. Unless otherwise indicated, almost all presented knowledge was gained from cell culture-based, HBV in vitro-replication and in vitro-infection systems.

SARS-CoV-2 viral budding and entry can be modeled using BSL-2 level virus-like particles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses, is restricted to BSL-3 facilities. Such BSL-3 classification makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.

Viruses go modular.

The WW domain is a modular protein structure that recognizes the proline-rich Pro-Pro-x-Tyr (PPxY) motif contained in specific target proteins. The compact modular nature of the WW domain makes it ideal for mediating interactions between proteins in complex networks and signaling pathways of the cell (e.g. the Hippo pathway). As a result, WW domains play key roles in a plethora of both normal and disease processes. Intriguingly, RNA and DNA viruses have evolved strategies to hijack cellular WW domain-containing proteins and thereby exploit the modular functions of these host proteins for various steps of the virus life cycle, including entry, replication, and egress. In this review, we summarize key findings in this rapidly expanding field, in which new virus-host interactions continue to be identified. Further unraveling of the molecular aspects of these crucial virus-host interactions will continue to enhance our fundamental understanding of the biology and pathogenesis of these viruses. We anticipate that additional insights into these interactions will help support strategies to develop a new class of small-molecule inhibitors of viral PPxY-host WW-domain interactions that could be used as antiviral therapeutics.

Lipid-protein interactions in virus assembly and budding from the host cell plasma membrane.

Lipid enveloped viruses contain a lipid bilayer coat that protects their genome to help facilitate entry into the new host cell. This lipid bilayer comes from the host cell which they infect. After viral replication, the mature virion hijacks the host cell plasma membrane where it is then released to infect new cells. This process is facilitated by the interaction between phospholipids that make up the plasma membrane and specialized viral matrix proteins. This step in the viral lifecycle may represent a viable therapeutic strategy for small molecules that aim to block enveloped virus spread. In this review, we summarize the current knowledge on the role of plasma membrane lipid-protein interactions on viral assembly and budding.

The Alphavirus E2 Membrane-Proximal Domain Impacts Capsid Interaction and Glycoprotein Lattice Formation.

Alphaviruses are small enveloped RNA viruses that bud from the host cell plasma membrane. Alphavirus particles have a highly organized structure, with a nucleocapsid core containing the RNA genome surrounded by the capsid protein, and a viral envelope containing 80 spikes, each a trimer of heterodimers of the E1 and E2 glycoproteins. The capsid protein and envelope proteins are both arranged in organized lattices that are linked via the interaction of the E2 cytoplasmic tail/endodomain with the capsid protein. We previously characterized the role of two highly conserved histidine residues, H348 and H352, located in an external, juxtamembrane region of the E2 protein termed the D-loop. Alanine substitutions of H348 and H352 inhibit virus growth by impairing late steps in the assembly/budding of virus particles at the plasma membrane. To investigate this budding defect, we selected for revertants of the E2-H348/352A double mutant. We identified eleven second-site revertants with improved virus growth and mutations in the capsid, E2 and E1 proteins. Multiple isolates contained the mutation E2-T402K in the E2 endodomain or E1-T317I in the E1 ectodomain. Both of these mutations were shown to partially restore H348/352A growth and virus assembly/budding, while neither rescued the decreased thermostability of H348/352A. Within the alphavirus particle, these mutations are positioned to affect the E2-capsid interaction or the E1-mediated intertrimer interactions at the 5-fold axis of symmetry. Together, our results support a model in which the E2 D-loop promotes the formation of the glycoprotein lattice and its interactions with the internal capsid protein lattice.IMPORTANCE Alphaviruses include important human pathogens such as Chikungunya and the encephalitic alphaviruses. There are currently no licensed alphavirus vaccines or effective antiviral therapies, and more molecular information on virus particle structure and function is needed. Here, we highlight the important role of the E2 juxtamembrane D-loop in mediating virus budding and particle production. Our results demonstrated that this E2 region affects both the formation of the external glycoprotein lattice and its interactions with the internal capsid protein shell.

Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE: Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

Host cytoskeleton in respiratory syncytial virus assembly and budding.

Respiratory syncytial virus (RSV) is one of the major pathogens responsible for lower respiratory tract infections (LRTI) in young children, the elderly, and the immunosuppressed. Currently, there are no antiviral drugs or vaccines available that effectively target RSV infections, proving a significant challenge in regards to prevention and treatment. An in-depth understanding of the host-virus interactions that underlie assembly and budding would inform new targets for antiviral development.Current research suggests that the polymerised form of actin, the filamentous or F-actin, plays a role in RSV assembly and budding. Treatment with cytochalasin D, which disrupts F-actin, has been shown to inhibit virus release. In addition, the actin cytoskeleton has been shown to interact with the RSV matrix (M) protein, which plays a central role in RSV assembly. For this reason, the interaction between these two components is hypothesised to facilitate the movement of viral components in the cytoplasm and to the budding site. Despite increases in our knowledge of RSV assembly and budding, M-actin interactions are not well understood. In this review, we discuss the current literature on the role of actin cytoskeleton during assembly and budding of RSV with the aim to integrate disparate studies to build a hypothetical model of the various molecular interactions between actin and RSV M protein that facilitate RSV assembly and budding.

High-resolution Crystal Structure of Dimeric VP40 From Sudan ebolavirus.

Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly.

Rubella virus assembly requirements and evolutionary relationships with novel rubiviruses.

Rubella virus (RuV) is an enveloped virus that usually causes mild disease in children, but can produce miscarriage or severe congenital birth defects. While in nature RuV only infects humans, the discovery of the related Ruhugu (RuhV) and Rustrela (RusV) viruses highlights the spillover potential of mammalian rubiviruses to humans. RuV buds into the Golgi, but its assembly and exit are not well understood. We identified a potential late domain motif 278PPAY281 at the C-terminus of the RuV E2 envelope protein. Such late domain motifs can promote virus budding by recruiting the cellular ESCRT machinery. An E2 Y281A mutation reduced infectious virus production by >3 logs and inhibited virus particle production. However, RuV was insensitive to inhibition by dominant-negative VPS4, and thus appeared ESCRT-independent. The E2 Y281A mutation did not significantly inhibit the production of the viral structural proteins capsid (Cp), E2, and E1, or dimerization, glycosylation, Golgi transport, and colocalization of E2 and E1. However, E2 Y281A significantly reduced glycoprotein-Cp colocalization and interaction, and inhibited Cp localization to the Golgi. Revertants of the E2 Y281A mutant contained an E2 281V substitution or the second site mutations [E2 N277I + Cp D215A]. These mutations promoted virus growth, particle production, E2/Cp colocalization and Cp-Golgi localization. Both the E2 substitutions 281V and 277I were found at the corresponding positions in the RuhV E2 protein. Taken together, our data identify a key interaction of the RuV E2 endodomain with the Cp during RuV biogenesis, and support the close evolutionary relationship between human and animal rubiviruses. IMPORTANCE: Rubella virus (RuV) is an enveloped virus that only infects humans, where transplacental infection can cause miscarriage or congenital birth defects. We identified a potential late domain, 278PPAY281, at the C terminus of the E2 envelope protein. However, rather than this domain recruiting the cellular ESCRT machinery as predicted, our data indicate that E2 Y281 promotes a critical interaction of the E2 endodomain with the capsid protein, leading to capsid's localization to the Golgi where virus budding occurs. Revertant analysis demonstrated that two substitutions on the E2 protein could partially rescue virus growth and Cp-Golgi localization. Both residues were found at the corresponding positions in Ruhugu virus E2, supporting the close evolutionary relationship between RuV and Ruhugu virus, a recently discovered rubivirus from bats.

A single-point mutation in the rubella virus E1 glycoprotein promotes rescue of recombinant vesicular stomatitis virus.

Rubella virus (RuV) is an enveloped plus-sense RNA virus and a member of the Rubivirus genus. RuV infection in pregnant women can lead to miscarriage or an array of severe birth defects known as congenital rubella syndrome. Novel rubiviruses were recently discovered in various mammals, highlighting the spillover potential of other rubiviruses to humans. Many features of the rubivirus infection cycle remain unexplored. To promote the study of rubivirus biology, here, we generated replication-competent recombinant VSV-RuV (rVSV-RuV) encoding the RuV transmembrane glycoproteins E2 and E1. Sequencing of rVSV-RuV showed that the RuV glycoproteins acquired a single-point mutation W448R in the E1 transmembrane domain. The E1 W448R mutation did not detectably alter the intracellular expression, processing, glycosylation, colocalization, or dimerization of the E2 and E1 glycoproteins. Nonetheless, the mutation enhanced the incorporation of RuV E2/E1 into VSV particles, which bud from the plasma membrane rather than the RuV budding site in the Golgi. Neutralization by E1 antibodies, calcium dependence, and cell tropism were comparable between WT-RuV and either rVSV-RuV or RuV containing the E1 W448R mutation. However, the E1 W448R mutation strongly shifted the threshold for the acid pH-triggered virus fusion reaction, from pH 6.2 for the WT RuV to pH 5.5 for the mutant. These results suggest that the increased resistance of the mutant RuV E1 to acidic pH promotes the ability of viral envelope proteins to generate infectious rVSV and provide insights into the regulation of RuV fusion during virus entry and exit.IMPORTANCERubella virus (RuV) infection in pregnant women can cause miscarriage or severe fetal birth defects. While a highly effective vaccine has been developed, RuV cases are still a significant problem in areas with inadequate vaccine coverage. In addition, related viruses have recently been discovered in mammals, such as bats and mice, leading to concerns about potential virus spillover to humans. To facilitate studies of RuV biology, here, we generated and characterized a replication-competent vesicular stomatitis virus encoding the RuV glycoproteins (rVSV-RuV). Sequence analysis of rVSV-RuV identified a single-point mutation in the transmembrane region of the E1 glycoprotein. While the overall properties of rVSV-RuV are similar to those of WT-RuV, the mutation caused a marked shift in the pH dependence of virus membrane fusion. Together, our studies of rVSV-RuV and the identified W448R mutation expand our understanding of rubivirus biology and provide new tools for its study.

Phage display identification of high-affinity ligands for human TSG101-UEV: A structural and thermodynamic study of PTAP recognition.

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disrupting TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of novel host-oriented antivirals with a broad spectrum of action. Nonetheless, finding inhibitors with good properties as therapeutic agents remains a challenge since the key determinants of binding affinity and specificity are still poorly understood. Here we present a detailed thermodynamic, structural, and dynamic characterization viral PTAP Late domain recognition by TSG101-UEV, combining isothermal titration calorimetry, X-ray diffraction structural studies, molecular dynamics simulations, and computational analysis of intramolecular communication pathways. Our analysis highlights key contributions from conserved hydrophobic contacts and water-mediated hydrogen bonds at the PTAP binding interface. We have identified additional electrostatic hotspots adjacent to the core motif that modulate affinity. Using competitive phage display screening we have improved affinity by 1-2 orders of magnitude, producing novel peptides with low micromolar affinities that combine critical elements found in the best natural binders. Molecular dynamics simulations revealed that optimized peptides engage new pockets on the UEV domain surface. This study provides a comprehensive view of the molecular forces directing TSG101-UEV recognition of PTAP motifs, revealing that binding is governed by conserved structural elements yet tuneable through targeted optimization. These insights open new venues to design inhibitors targeting TSG101-dependent pathways with potential application as novel broad-spectrum antivirals.

Minor changes in electrostatics robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine (PS) and PI(4,5)P2 in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles (VLPs). In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

Antibody bivalency improves antiviral efficacy by inhibiting virion release independently of Fc gamma receptors.

Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined. Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (FcγR) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely FcγR dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity. These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development.

When in Need of an ESCRT: The Nature of Virus Assembly Sites Suggests Mechanistic Parallels between Nuclear Virus Egress and Retroviral Budding.

The proper assembly and dissemination of progeny virions is a fundamental step in virus replication. As a whole, viruses have evolved a myriad of strategies to exploit cellular compartments and mechanisms to ensure a successful round of infection. For enveloped viruses such as retroviruses and herpesviruses, acquisition and incorporation of cellular membrane is an essential process during the formation of infectious viral particles. To do this, these viruses have evolved to hijack the host Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II, and -III) to coordinate the sculpting of cellular membrane at virus assembly and dissemination sites, in seemingly different, yet fundamentally similar ways. For instance, at the plasma membrane, ESCRT-I recruitment is essential for HIV-1 assembly and budding, while it is dispensable for the release of HSV-1. Further, HSV-1 was shown to recruit ESCRT-III for nuclear particle assembly and egress, a process not used by retroviruses during replication. Although the cooption of ESCRTs occurs in two separate subcellular compartments and at two distinct steps for these viral lifecycles, the role fulfilled by ESCRTs at these sites appears to be conserved. This review discusses recent findings that shed some light on the potential parallels between retroviral budding and nuclear egress and proposes a model where HSV-1 nuclear egress may occur through an ESCRT-dependent mechanism.