The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.

SUMMARYAphidicolin, a tetracyclic diterpenoid which inhibits the DNA polymerase-ct activities of many eukaryotic cells, inhibited herpes simplex virus growth and DNA synthesis in infected cultures and the activity of the virus DNA polymerase in vitro. A wide range of stable aphidicolin sensitivities was represented amongst a collection of virus strains with no prior exposure to this drug, but viruses with polymerase mutations selected for resistance to phosphonoacetic acid (PAA) or to acycloguanosine typically showed increased sensitivity to aphidicolin. Of 16 unrelated PAA-resistant variants, 7 were hypersensitive to aphidicolin. A number of mutants with temperature-sensitive (ts) lesions in the polymerase gene also showed increased aphidicolin sensitivity (e.g. HSV-I[mP17]tsH) or aphidicolin hypersensitivity (e.g. HSV-I[KOS]tsD9, tsC4).Resistance or hypersensitivity of virus growth and DNA synthesis in vivo were correlated with resistance or hypersensitivity of virus DNA polymerase reactions in vitro. Resistance phenotypes were closely linked to the polymerase gene during recombination with outside markers. Moreover, the selection of aphidicolin-resistant mutants from hypersensitive variants with independent PAA resistance or ts mutations in the polymerase gene could result in co-selection for PAA-sensitive and ts + phenotypes. Confirmation that multiple independent mutations could determine aphidicolin hypersensitivity was obtained by studies of recombination between independent hypersensitive variants. Aphidicolin-resistant recombinant progeny were formed with recombination frequencies (0.4 to 2.6~) compatible with intragenic events. With parental hypersensitive variants which were products of limited PAA selection, or with the ts polymerase mutations, aphidicolin-resistant recombinants were PAA-sensitive and/or ts +. The segregation of other markers (ts, plaque morphology) amongst recombinant progeny permitted the orientation of multiple determinants of PAA resistance and aphidicolin hypersensitivity with respect to other markers in the polymerase gene and in other genes. The nature of residues determined at any one of a constellation of separate sites within the polymerase locus can determine resistance or sensitivity to antiviral drugs and aphidicolin hypersensitivity associated with changes at the polymerase locus facilitates high resolution genetic analysis of this locus. INTRODUCTION Herpesviruses characteristically specify the synthesis of a novel DNA polymerase activity which is essential for the replication of virus DNA in productively infected cells. For herpes simplex viruses of type l and type 2 (HSV-1, HSV-2) there is now good evidence that t Present address:

bFlock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3= end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.T he nodaviruses are a family of positive-strand RNA viruses that naturally infect insects and fish. They have a bipartite genome that contains approximately 4,500 bases and encodes three proteins. The most thoroughly studied nodavirus is the insect virus Flock House virus (FHV), which has served as an important model system to investigate the structural and molecular basis of RNA replication, virus assembly, and inhibition of antiviral host responses (1). The larger of the two genomic RNAs, RNA1 (3.1 kb), encodes the RNAdependent RNA polymerase (RdRp; 112 kDa), which is located on the outer membrane of mitochondria in infected cells (2, 3). Viral RNA synthesis induces so-called spherules (4), i.e., membrane invaginations, which are thought to sequester the replication complexes and double-stranded RNA (dsRNA) intermediates to protect them from RNA silencing, a major antiviral pathway activated in insects upon infection with RNA viruses (5). Further protection from RNA silencing is afforded by FHV protein B2 (11.6 kDa), a dsRNA binding protein that is translated from RNA3 (387 nucleotides), a subgenomic RNA derived from the 3= end of RNA1 (6-8). The capsid protein, protein alpha (43 kDa), is translated from RNA2 (1.4 kb), the second genomic RNA segment.Although a seemingly simple virus, FHV uses a sophisticated regulatory system to control its gene expression. This regulation occurs at several levels and is currently incompletely understo...

In both deletion transgenic strains, the subunits near the spokehead were restored, but their firm attachment to the spokestalk required the DPY-30 domain. We postulate that the DPY-30 -helix dimer is a conserved two-prong linker, required for normal motility, organizing duplicated subunits in the radial spoke stalk and formation of a symmetrical spokehead. Further, the dispensable calmodulin-binding region appears to fine-tune the spokehead for regulation of "steering" motility in the green algae. Thus, in general, D/D domains may function to localize molecular modules for both the assembly and modulation of macromolecular complexes.

NDK5 promotes assembly of motile cilia and flagella with its structure and protein phosphorylation–related reactions instead of the canonical NDK activity. The novel mechanisms and dominant-negative effect of mutated functional NDK5 reveal the remarkable versatility of a molecular platform that is used in diverse cellular processes.

Virus-like particles (VLPs) are self-assembling platforms composed of viral structural proteins. They are used for a variety of purposes, ranging from the study of virus assembly to vaccine development. VLPs can be produced in plants, bacteria, yeast, and insect and mammalian cells. The baculovirus expression system is one of the most commonly used systems for production of VLPs in eukaryotic cells. This chapter provides a brief overview of the main strategies used to generate recombinant baculoviruses and the applications of insect virus-derived VLPs in basic and applied research. It then describes detailed protocols for generation of recombinant baculoviruses, screening for their expression of VLPs in insect cells, and VLP purification.

Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ϳ50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single-and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

Nodaviruses are icosahedral viruses with a bipartite, positive-sense RNA genome. The two RNAs are packaged into a single virion by a poorly understood mechanism. We chose two distantly related nodaviruses, Flock House virus and Nodamura virus, to explore formation of viral reassortants as a means to further understand genome recognition and encapsidation. In mixed infections, the viruses were incompatible at the level of RNA replication and their coat proteins segregated into separate populations of progeny particles. RNA packaging, on the other hand, was indiscriminate as all four viral RNAs were detectable in each progeny population. Consistent with the trans-encapsidation phenotype, fluorescence in situ hybridization of viral RNA revealed that the genomes of the two viruses co-localized throughout the cytoplasm. Our results imply that nodaviral RNAs lack rigorously defined packaging signals and that coencapsidation of the viral RNAs does not require a pair of cognate RNA1 and RNA2.