Mutation of interfacial residues disrupts subunit folding and particle assembly of Physalis mottle tymovirus

Virus-like particles (VLPs) serve as excellent model systems to identify the pathways of virus assembly. To gain insights into the assembly mechanisms of the Physalis mottle tymovirus (PhMV), six interfacial residues, identified based on the crystal structure of the native and recombinant capsids, were targeted for mutagenesis. The Q37E, Y67A, R68Q, D83A, I123A, and S145A mutants of the PhMV recombinant coat protein (rCP) expressed in Escherichia coli were soluble. However, except for the S145A mutant, which assembled into VLPs similar to that of wild type rCP capsids, all the other mutants failed to assemble into VLPs. Furthermore, the purified Q37E, Y67A, R68Q, D83A, and I123A rCP mutants existed essentially as partially folded monomers as revealed by sucrose density gradient analysis, circular dichroism, fluorescence, thermal, and urea denaturation studies. The rCP mutants locked into such conformations probably lack the structural signals/features that would allow them to assemble into capsids. Thus, the mutation of residues involved in inter-subunit interactions in PhMV disrupts both subunit folding and particle assembly

Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication

Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling

In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors

A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγcnull-humanized mouse model. The UL133-UL138NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations

Development and validation of new algorithms to improve contact detection and robustness in finite element simulations

Approximately half of all numerical problems in crashworthiness analysis involve impact dynamics, and accurate contact algorithms are critical to capture the structures’ behavior. Conventional contact algorithms use the principle of preventing ‘slave’ nodes from penetrating ‘master’ segments. Only nodes are checked in these contact algorithms and the connectivity of the nodes (in the slave side) are not considered. Additionally, to achieve efficiency, the conventional contact algorithms use different methods to eliminate element pairs that would unlikely come in contact and simplify the geometry while searching for penetration between the contact pairs. These eliminations and simplifications, sometimes, cause inaccuracy in the results. In this research, a new contact algorithm has been developed and implemented in an explicit nonlinear large displacement finite element code (DYNA3D). A new global search method and a new local search method for contact search have been implemented in the algorithm. The new global search method uses the concept of enclosing spheres around nodes combined with bucket-sorting. Unlike in the current algorithms where bucket-sort checks for presence of nodes in the buckets, bucket-sort in the new global search check for intersections of enclosed spheres with the buckets. In the new local search method, effort is made to represent accurate geometry of the contact surface. The element surfaces are offset by their thickness and, edges and corners are represented using beams of circular cross-section and spheres respectively. Using this configuration, problems associated in finding penetration in a skewed mesh are eliminated. Constant stiffness that is used in computing contact force in current contact algorithms is replaced by exponentially varying stiffness in the new contact algorithm. When compared to the constant stiffness, the varying stiffness applies significantly higher forces when the penetration becomes large. The new contact algorithm has been implemented in DYNA3D and validated. Element level and component level examples have been used to check accuracy of the contact algorithm. Using these examples, gap between the contact surfaces and stress variation along the contact surface are checked. Using the new contact algorithm, the gap distance was found to be accurate and stress variation was found to be minimal. The new contact algorithm has few limitations which need to be addressed before it can be used to solve general three dimensional problems. Provisions should be made to the contact algorithm to include segments from solid elements, and rectangular & varying cross-sectional beam elements in the contact and to delete failed elements from the contact. Care should be taken not to include severely warped elements and initially penetrated elements in the contact definition

Virus-Like Particles: Models for Assembly Studies and Foreign Epitope Carriers

Virus-like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. When expressed in a suitable heterologous system, viral structural proteins involved in capsid or envelope formation often self-assemble into VLPs in the absence of other viral components usually required for virus assembly, such as multiple structural or nonstructural proteins and viral genomes. Protein–protein interactions in VLPs are relatively strong and can result in the formation of stable structures. Several experiments have been reported that may help answer questions regarding the requirements for VLP formation. Knowledge on the assembly process of VLPs is crucial to define the usefulness of such particles for the presentation of their own or foreign epitopes as carriers for transiently expressed proteins as a means of vaccine production. The aim of the present chapter is to outline recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are (1) the requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. To date, reviews in these areas have mainly focused on results obtained with a specific virus genus or family of viruses (1, 2, 3, 4 and 5) and the reader is advised to refer to these reviews for complementary information. Virus-like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. When expressed in a suitable heterologous system, viral structural proteins involved in capsid or envelope formation often self-assemble into VLPs in the absence of other viral components usually required for virus assembly, such as multiple structural or nonstructural proteins and viral genomes. Protein–protein interactions in VLPs are relatively strong and can result in the formation of stable structures. Several experiments have been reported that may help answer questions regarding the requirements for VLP formation. Knowledge on the assembly process of VLPs is crucial to define the usefulness of such particles for the presentation of their own or foreign epitopes as carriers for transiently expressed proteins as a means of vaccine production. The aim of the present chapter is to outline recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are (1) the requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. To date, reviews in these areas have mainly focused on results obtained with a specific virus genus or family of viruses (1, 2, 3, 4 and 5) and the reader is advised to refer to these reviews for complementary information. Virus-like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. When expressed in a suitable heterologous system, viral structural proteins involved in capsid or envelope formation often self-assemble into VLPs in the absence of other viral components usually required for virus assembly, such as multiple structural or nonstructural proteins and viral genomes. Protein–protein interactions in VLPs are relatively strong and can result in the formation of stable structures. Several experiments have been reported that may help answer questions regarding the requirements for VLP formation. Knowledge on the assembly process of VLPs is crucial to define the usefulness of such particles for the presentation of their own or foreign epitopes as carriers for transiently expressed proteins as a means of vaccine production. The aim of the present chapter is to outline recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are (1) the requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. To date, reviews in these areas have mainly focused on results obtained with a specific virus genus or family of viruses (1, 2, 3, 4 and 5) and the reader is advised to refer to these reviews for complementary information

Virus-Like Particles: Models for Assembly Studies and Foreign Epitope Carriers

Virus-Like Particles: Models for Assembly Studies and Foreign Epitope Carrier

Inhibition of EGFR signal promotes viral replication.

<p>Fibroblasts infected with 0.5 MOI of WT virus were treated with DMSO, (A) 5 μM AG1478 or (B) 20 μM LY294002 at 18hpi. Cells and media were collected over a time course and virus yields were determined by TCID₅₀. Values represent the average of three independent experiments with SEM shown. Statistical significance was determined by two-way ANOVA with Bonferroni correction for differences between vehicle controls and drug treatments are indicated by asterisks (* p-value<0.05; ** p-value<0.01).</p