Assembly Of Virus Particles Research Articles

Dengue virus capsid anchor modulates the efficiency of polyprotein processing and assembly of viral particles.

The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavages mediated by both viral and host proteases. Among these, sequential cleavage at the N and C termini of the hydrophobic capsid anchor (Ca) is crucial in deciding the fate of viral infection. Here, using a refined dengue pseudovirus production system, along with cleavage and furin inhibition assays, immunoblotting and secondary structure prediction analysis, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and viral particle assembly. Replacement of the DENV2 Ca with the homologous regions from West nile or Zika viruses or, alternatively, increasing its length, improved cleavage and hence particle assembly. Further, we showed that substitution of the Ca conserved proline residue (P110) to alanine abolishes pseudovirus production, regardless of the Ca sequence length. Besides providing the results of a biochemical analysis of DENV2 structural polyprotein processing, this study also presents a system for efficient production of dengue pseudoviruses.

Chronic hepatitis delta: A state-of-the-art review and new therapies.

Chronic delta hepatitis is the most severe form of viral hepatitis affecting nearly 65 million people worldwide. Individuals with this devastating illness are at higher risk for developing cirrhosis and hepatocellular carcinoma. Delta virus is a defective RNA virus that requires hepatitis B surface antigen for propagation in humans. Infection can occur in the form of a co-infection with hepatitis B, which can be self-limiting, vs superinfection in a patient with established hepatitis B infection, which often leads to chronicity in majority of cases. Current noninvasive tools to assess for advanced liver disease have limited utility in delta hepatitis. Guidelines recommend treatment with pegylated interferon, but this is limited to patients with compensated disease and is efficacious in about 30% of those treated. Due to limited treatment options, novel agents are being investigated and include entry, assembly and export inhibitors of viral particles in addition to stimulators of the host immune response. Future clinical trials should take into consideration the interaction of hepatitis B and hepatitis D as suppression of one virus can lead to the activation of the other. Also, surrogate markers of treatment efficacy have been proposed.

Progeny Varicella-Zoster Virus Capsids Exit the Nucleus but Never Undergo Secondary Envelopment during Autophagic Flux Inhibition by Bafilomycin A1.

Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that inhibitors of autophagy reduce VZV infectivity. We selected the vacuolar proton ATPase inhibitor bafilomycin A1 for analysis because of its well-known antiautophagy property of impeding acidification during the late stage of autophagic flux. We documented that bafilomycin treatment from 48 to 72 h postinfection lowered VZV titers substantially (P ≤ 0.008). Because we were unable to define the site of the block in the infectious cycle by confocal microscopy, we turned to electron microscopy. Capsids were observed in the nucleus, in the perinuclear space, and in the cytoplasm adjacent to Golgi apparatus vesicles. Many of the capsids had an aberrant appearance, as has been observed previously in infections not treated with bafilomycin. In contrast to prior untreated infections, however, secondary envelopment of capsids was not seen in the trans-Golgi network, nor were prototypical enveloped particles with capsids (virions) seen in cytoplasmic vesicles after bafilomycin treatment. Instead, multiple particles with varying diameters without capsids (light particles) were seen in large virus assembly compartments near the disorganized Golgi apparatus. Bafilomycin treatment also led to increased numbers of multivesicular bodies in the cytoplasm, some of which contained remnants of the Golgi apparatus. In summary, we have defined a previously unrecognized property of bafilomycin whereby it disrupted the site of secondary envelopment of VZV capsids by altering the pH of the trans-Golgi network and thereby preventing the correct formation of virus assembly compartments.IMPORTANCE This study of VZV assembly in the presence of bafilomycin A1 emphasizes the importance of the Golgi apparatus/trans-Golgi network as a platform in the alphaherpesvirus life cycle. We have previously shown that VZV induces levels of autophagy far above the basal levels of autophagy in human skin, a major site of VZV assembly. The current study documented that bafilomycin treatment led to impaired assembly of VZV capsids after primary envelopment/de-envelopment but before secondary reenvelopment. This VZV study also complemented prior herpes simplex virus 1 and pseudorabies virus studies investigating two other inhibitors of endoplasmic reticulum (ER)/Golgi apparatus function: brefeldin A and monensin. Studies with porcine herpesvirus demonstrated that primary enveloped particles accumulated in the perinuclear space in the presence of brefeldin A, while studies with herpes simplex virus 1 documented an impaired secondary assembly of enveloped viral particles in the presence of monensin.

Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types.

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells.IMPORTANCE All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection.

Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag.

The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.

Cyclopeptides design as blockers against HCV p7 channel in silico

ABSTRACTThe protein p7 in the hepatitis C virus (HCV) is a 63-residue transmembrane protein that oligomerizes to form an ion channel with cation selectivity. This protein is essential for the assembly and release of infectious viral particles. Structural analysis indicates that the bottleneck of the p7 hexamer channel is located on Asparagines at position 9 in the conical region with a pore radius of 3.9 Å, which is reduced to nearly 3.0 Å upon protonation. Amantadine and BIT225 binds in the hydrophobic pocket on the lipid-facing side of the p7 channel as allosteric inhibitors to stabilise the channel pore in a closed conformation. Here, we designed a series of cyclopeptides as inner channel blockers in the conical region based on chemical and physical characteristics of the channel bottleneck for the HCV p7 protein. As a result, cyclic-4-Asp is found to bind in the channel lumen between the gating residues Ile6 and Asn9 with a favourable binding affinity, perfectly fitting with the hydrophobic pocket in the conical region. Compared with amantadine and BIT225, we believe that the cylcopeptides have potential to be effective inhibitors against HCV p7 protein.

Cholesterol Binding to the Transmembrane Region of a Group 2 Hemagglutinin (HA) of Influenza Virus Is Essential for Virus Replication, Affecting both Virus Assembly and HA Fusion Activity.

Hemagglutinin (HA) of influenza virus is incorporated into cholesterol-enriched nanodomains of the plasma membrane. Phylogenetic group 2 HAs contain the conserved cholesterol consensus motif (CCM) YKLW in the transmembrane region. We previously reported that mutations in the CCM retarded intracellular transport of HA and decreased its nanodomain association. Here, we analyzed whether cholesterol interacts with the CCM. Incorporation of photocholesterol into HA was significantly reduced if the whole CCM is replaced by alanine, both using immunoprecipitated HA and when HA is embedded in the membrane. We next used reverse genetics to investigate the significance of the CCM for virus replication. No virus was rescued if the whole motif is exchanged (YKLW4A); singly (LA) or doubly (YK2A and LW2A) mutated virus showed decreased titers and a comparative fitness disadvantage. In polarized cells, transport of HA mutants to the apical membrane was not disturbed. Reduced amounts of HA and cholesterol were incorporated into the viral membrane. Mutant viruses exhibit a decrease in hemolysis, which is only partially corrected if the membrane is replenished with cholesterol. More specifically, viruses have a defect in hemifusion, as demonstrated by fluorescence dequenching. Cells expressing HA YKLW4A fuse with erythrocytes, but the number of events is reduced. Even after acidification unfused erythrocytes remain cell bound, a phenomenon not observed with wild-type HA. We conclude that cholesterol binding to a group 2 HA is essential for virus replication. It has pleiotropic effects on virus assembly and membrane fusion, mainly on lipid mixing and possibly a preceding step.IMPORTANCE The glycoprotein HA is a major pathogenicity factor of influenza viruses. Whereas the structure and function of HA's ectodomain is known in great detail, similar data for the membrane-anchoring part of the protein are missing. Here, we demonstrate that the transmembrane region of a group 2 HA interacts with cholesterol, the major lipid of the plasma membrane and the defining element of the viral budding site nanodomains of the plasma membrane. The cholesterol binding motif is essential for virus replication. Its partial removal affects various steps of the viral life cycle, such as assembly of new virus particles and their subsequent cell entry via membrane fusion. A cholesterol binding pocket in group 2 HAs might be a promising target for a small lipophilic drug that inactivates the virus.

Role of the Ubiquitin Proteasome System (UPS) in the HIV-1 Life Cycle.

Given that the ubiquitin proteasome system (UPS) is the major protein degradation process in the regulation of a wide variety of cellular processes in eukaryotic cells, including alteration of cellular location, modulation of protein activity, and regulation of protein interaction, it is reasonable to suggest that the infecting HIV-1 and the invaded hosts exploit the UPS in a contest for survival and proliferation. However, to date, regulation of the HIV-1 life cycle has been mainly explained by the stage-specific expression of HIV-1 viral genes, not by elimination processes of the synthesized proteins after completion of their duties in the infected cells, which is also quintessential for understanding the molecular processes of the virus life cycle and thereby HIV-1 pathogenesis. In fact, several previous publications have indicated that the UPS plays a critical role in the regulation of the proteasomal degradation of viral and cellular counterparts at every step of the HIV-1 life cycle, from the virus entry to release of the assembled virus particles, which is integral for the regulation of survival and proliferation of the infecting HIV-1 and to replication restriction of the invading virus in the host. However, it is unknown whether and how these individual events taking place at different stages of the HIV-1 life cycle are orchestrated as an overall strategy to overcome the restrictions conferred by the host cells. Thus, in this review, we overview the interplay between HIV-1 viral and cellular proteins for restrictions/competitions for proliferation of the virus in the infected cell, which could open a new avenue for the development of therapeutics against HIV-1 via targeting a specific step of the proteasome degradation pathway during the HIV-1 life cycle.

Hepatitis E Virus Assembly and Release.

Hepatitis E is an underestimated threat to public health, caused by the hepatitis E virus (HEV). HEV is the most common cause of acute viral hepatitis in the world, with no available direct-acting antiviral treatment. According to a recent WHO report, 20 million people become infected with HEV annually, resulting in 44,000 deaths. However, due to the scarcity of efficient in vitro cell culture systems for HEV, our knowledge of the life cycle of HEV is incomplete. Recently, significant progress has been made towards gaining a more comprehensive view of the HEV life cycle, as several in vitro culturing systems have been developed in recent years. Here, we review current knowledge and recent advances with regard to the HEV life cycle, with a particular focus on the assembly and release of viral particles. We also discuss the knowledge gaps in HEV assembly and release. Meanwhile, we highlight experimental platforms that could potentially be utilized to fill these gaps. Lastly, we offer perspectives on the future of research into HEV virology and its interaction with host cells.

RNA Packaging in HIV.

Successful replication of the AIDS retrovirus, HIV, requires that its genomic RNA be packaged in assembling virus particles with high fidelity. However, cellular mRNAs can also be packaged under some conditions. Viral RNA (vRNA) contains a 'packaging signal' (ψ) and is packaged as a dimer, with two vRNA monomers joined by a limited number of base pairs. It has two conformers, only one of which is capable of dimerization and packaging. Recent years have seen important progress on the 3D structure of dimeric ψ. Gag, the protein that assembles into the virus particle, interacts specifically with ψ, but this is obscured under physiological conditions by its high nonspecific affinity for any RNA. New results suggest that vRNA is selected for packaging because ψ nucleates assembly more efficiently than other RNAs.

Detection of lentiviral constructs for release testing of CAR- T cells using digital droplet PCR

Background & Aim Viral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. While viral vectors are efficient at gene delivery and able to stably integrate into the host genome for subsequent replication, they also pose a safety risk if replication competent lentivirus (RCL) remains in the final patient product. FDA guidelines require testing of CAR-T products to ensure absence of RCL before delivery to the patient. Current testing methods include cell based assays that require significant time to produce results or qPCR based assays that provide rapid results but lack the limit of detection for such low copies of target DNA. We describe here the development of digital droplet PCR (ddPCR) for detection of the vesicular stomatitis virus G glycoprotein (VSV-G) envelope sequence, a component required for the assembly of competent viral particles. Validation of this method provides several advantages over qPCR by improving limit of detection, reducing sources of human error by elimination of the standard curve and reducing hands on technical time. Methods, Results & Conclusion We used the BioRad QX200 ddPCR system to test samples of defined concentration over a broad range of conditions. Samples of limiting dilution were tested to prove assay linearity over a broad range of concentrations. Reproducibility was established by running the defined samples in multiple wells or over consecutive days to compare variability over time, within an assay and between assays. Specificity was demonstrated by running replicates of positive and negative samples to determine a false positive rate and limit of detection. Taken together, we have provided experimental and statistical evidence for the use of ddPCR as a reliable release test for the manufacture of CAR-T products. Our results demonstrate the reproducibility, linearity, specificity and sensitivity to detect RCL and assure the safety of patient products in a rapid manner. Future efforts will be focused on adapting this technology to detect other valuable targets throughout cell product manufacturing.

Near-Neighbor Interactions in the NS3-4A Protease of HCV Impact Replicative Fitness of Drug-Resistant Viral Variants

A variety of amino acid substitutions in the NS3-4A protease of the hepatitis C virus lead to protease inhibitor (PI) resistance. Many of these significantly impair the replication fitness of the resistant variants in a genotype- and subtype-dependent manner, a critical factor in determining the probability with which resistant variants will persist. However, the underlying molecular mechanisms are unknown. Here, we present a novel residue-interaction network approach to determine how near-neighbor interactions of PI resistance mutations in NS3-4A can impact protease functional sites dependent on their genomic background. We constructed subtype-specific consensus residue networks for subtypes 1a and 1b from protease structure ensembles combined with biological properties of protein residues and evolutionary amino acid conservation. By applying local and global network topology analysis and visual exploration, we characterize PI resistance-associated sites and outline differences in near-neighbor interactions. We find local residue-interaction patterns and features at protease functional sites that are subtype specific. The noncovalent bonding patterns indicate higher fitness costs conferred by PI resistance mutations in a subtype 1b genomic background and explain the prevalence of Q80K and R155K in subtype 1a. Based on local residue interactions, we predict a subtype-specific role for the protease residue NS3–Q80 in molecular mechanisms related to the assembly of infectious virus particles that is supported by experimental data on the capacity of Q80K variants to replicate and produce infectious virus in subtype 1a and 1b cell culture.

The Role of ApoE in HCV Infection and Comorbidity.

Hepatitis C virus (HCV) is an RNA virus that can efficiently establish chronic infection in humans. The overlap between the HCV replication cycle and lipid metabolism is considered to be one of the primary means by which HCV efficiently develops chronic infections. In the blood, HCV is complex with lipoproteins to form heterogeneous lipo-viro-particles (LVPs). Furthermore, apolipoprotein E (ApoE), which binds to receptors during lipoprotein transport and regulates lipid metabolism, is localized on the surface of LVPs. ApoE not only participate in the attachment and entry of HCV on the cell surface but also the assembly and release of HCV viral particles from cells. Moreover, in the blood, ApoE can also alter the infectivity of HCV and be used by HCV to escape recognition by the host immune system. In addition, because ApoE can also affect the antioxidant and immunomodulatory/anti-inflammatory properties of the host organism, the long-term binding and utilization of host ApoE during chronic HCV infection not only leads to liver lipid metabolic disorders but may also lead to increased morbidity and mortality associated with systemic comorbidities.

Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus.

Rabies virus (RABV), the causative agent of rabies, is highly neurovirulent for warm-blooded animals with a mortality rate of up to 100%. The RABV matrix protein (M) is required for virus particle assembly and budding. However, little is known about antigenic differences in the M protein. In this study, five monoclonal antibodies (mAbs), designated 3B9, 4A1, 2B11, 2C1, and 4B11, against the RABV M protein were generated using a recombinant M protein. All five mAbs reacted with the CVS-11 strain but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting. The epitope targeted by these mAbs was further identified by peptide scanning using GST-fused peptides. The 25PPYDDD30 peptide was defined as the minimal linear epitope. Alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III, while the other strains resembling ERA and SRVA9 within the clade had another variable epitope, 25PLDDDD30. A Y27D mutation within the epitope was found among the rest of the RABV strains distributed in different clades. However, a single D28G mutation eliminated the reactivity of these five mAbs. In addition, the mAbs were able to recognize wildtype RABV strain in indirect immunofluorescence and western blotting and detect RABV-infected brain tissue using immunohistochemistry. The newly established mAbs and identified epitope may facilitate future investigations in the structure and function of the M protein and the development of diagnostic methods for the detection of different RABV strains worldwide. Most importantly, the epitope recognized by the mAbs against M protein might serve as a novel target for the development of a vaccine targeting RABV virulent strains.

Model-based analysis of influenza A virus replication in genetically engineered cell lines elucidates the impact of host cell factors on key kinetic parameters of virus growth.

The best measure to limit spread of contagious diseases caused by influenza A viruses (IAVs) is annual vaccination. The growing global demand for low-cost vaccines requires the establishment of high-yield production processes. One possible option to address this challenge is the engineering of novel vaccine producer cell lines by manipulating gene expression of host cell factors relevant for virus replication. To support detailed characterization of engineered cell lines, we fitted an ordinary differential equation (ODE)-based model of intracellular IAV replication previously established by our group to experimental data obtained from infection studies in human A549 cells. Model predictions indicate that steps of viral RNA synthesis, their regulation and particle assembly and virus budding are promising targets for cell line engineering. The importance of these steps was confirmed in four of five single gene overexpression cell lines (SGOs) that showed small, but reproducible changes in early dynamics of RNA synthesis and virus release. Model-based analysis suggests, however, that overexpression of the selected host cell factors negatively influences specific RNA synthesis rates. Still, virus yield was rescued by an increase in the virus release rate. Based on parameter estimations obtained for SGOs, we predicted that there is a potential benefit associated with overexpressing multiple host cell genes in one cell line, which was validated experimentally. Overall, this model-based study on IAV replication in engineered cell lines provides a step forward in the dynamic and quantitative characterization of IAV-host cell interactions. Furthermore, it suggests targets for gene editing and indicates that overexpression of multiple host cell factors may be beneficial for the design of novel producer cell lines.

Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles.

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses.

Structural determinants of the interaction between influenza A virus matrix protein M1 and lipid membranes

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1–protein interactions and multimerization have not been clarified, yet.In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95–105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.

Unravelling a co-nsP-iracy: the role of chikungunya virus non-structural protein 3 in replication and pathogenesis

Chikungunya virus (CHIKV) is a member of the Alphavirus genus, transmitted to humans by mosquitoes of the Aedesgenera. Infection with CHIKV causes chikungunya fever, which in many cases can lead to chronic joint disease, leaving patients with reduced ambulation. Despite its rising potential as a threat to global health, no effective vaccine or antiviral agent for protection or treatment are available. The CHIKV non-structural protein 3 (nsP3) is essential to the virus lifecycle and is believed to be a component of the genome replication complex. However, to date, the exact role of this protein has yet been determined. Although a conserved polyproline motif in the C-terminal hypervariable domain of nsP3 has been reported to interact with cellular SH3 domains, the function of this motif remains enigmatic. To address this question we generated a panel of mutations in this motif and tested the phenotype in the context of both a subgenomic replicon and full-length infectious virus, in both mammalian and mosquito-derived cell lines. Most of the mutations were well tolerated in the sub-genomic replicon, however, a subset either attenuated or completely abolished production of infectious CHIKV. These results suggest that as well as its role in genome replication, nsP3 also functions during assembly and release of infectious virus particles and that the C-terminal polyproline motif is a critical determinant of this function.

Analysis of the multifunctionality of Marburg virus VP40.

The Marburg virus (MARV) matrix protein, VP40, is a multifunctional protein that is essential for the assembly and release of viral particles, inhibition of the interferon response and viral transcription/replication. VP40 is assumed to be present as soluble monomers and membrane-bound higher-order oligomers. To investigate the functional relevance of oligomerization and lipid binding of VP40 we constructed mutants with impaired VP40-VP40 or VP40-lipid interactions and tested their capacity to bind the plasma membrane, to form virus-like particles (VLPs) and to inhibit viral RNA synthesis. All of the analysed VP40 mutants formed perinuclear aggregates and were defective in their delivery to the plasma membrane and in VLP production. The VP40 mutants that were competent for oligomerization but lacked VP40-lipid interactions formed fibril-like structures, influenced MARV inclusion body formation and inhibited viral transcription/replication more strongly than the VP40 wild-type. Altogether, mutations that interfere with VP40's transition from monomer to higher-order oligomers and/or lipid interactions destroy the protein's multifunctionality.

Mechanism of Tetramer Dissociation, Unfolding, and Oligomer Assembly of Pneumovirus M2-1 Transcription Antiterminators.

Among Mononegavirales, the Pneumovirus family stands out by its RNA polymerase processivity that relies on a transcription antiterminator, the M2-1 protein, which also plays a key role in viral particle assembly. Biophysical and structural evidence shows that this RNA-binding tetramer is strongly modulated by a CCCH Zn2+ binding motif. We show that while the global dissociation/unfolding free energy is 10 kcal mol–1, more stable for the respiratory syncytial virus M2-1, the human metapneumovirus (HMPV) counterpart shows a 7 kcal mol–1 higher intersubunit affinity. Removal of Zn2+ from both homologues leads to an apo-monomer of identical secondary structure that further undergoes a slow irreversible oligomerization. Mutation of the histidine residue of the Zn2+ motif to cysteine or alanine leads directly to large oligomers, strongly suggesting that metal coordination has an exquisite precision for modulating the quaternary arrangement. Zn2+ removal is very slow and requires subdenaturing concentrations of guanidine chloride, suggesting a likely local folding energy barrier. Exploring a broad combination of denaturant and ethylenediaminetetraacetic acid conditions, we showed that the metapneumovirus protein has to overcome a higher energy barrier to trigger Zn2+ removal-driven dissociation, in concordance with a slower dissociation kinetics. In silico modeling of open and close conformations for both M2-1 tetramers together with interaction energy calculations reveals that the gradual opening of protomers decreases the number of intersubunit contacts. Half of the interaction energy holding each protomer in the tetramer comes from the CCCH motif, while HMPV-M2-1 harbors additional contacts between the CCCH motif of one subunit and the core domain of a protomer located in trans, allowing the rationalization of the experimental data obtained. Overall, the evidence points at a key role of the CCCH motif in switching between structural and consequently functional alternatives of the M2-1 protein.