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Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope (Env) glycoprotein drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report the cryo-electron microscopy structures of the FV Env ectodomain in the pre- and post-fusion states, which unexpectedly demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the viral fusogens. We describe the structural features that are unique to the FV Env and propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive membrane fusion and viral entry. The structural knowledge on the FV Env now provides a framework for functional investigations, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors.

We have developed a flexible platform for delivery of proteins to target cell interiors using paramyxovirus-like particles. The key enabling feature is an appendage, 15 to 30 amino acid residues in length, that is added to cargo proteins and that induces them to bind to the viral matrix (M) protein during virus-like particle (VLP) assembly. The cargo is then incorporated within the VLPs as they bud, using the same interactions that normally direct viral genome packaging. The appendage can also serve as an epitope tag for cargo detection using a nucleocapsid (NP) protein-specific monoclonal antibody. Using this approach, we generated Renilla luciferase-loaded VLPs, green fluorescent protein-loaded VLPs, superoxide dismutase-loaded VLPs, and Cre recombinase-loaded VLPs. In each case, the VLPs could efficiently deliver their functional cargos to target cells and, in the case of Cre recombinase, to target cell nuclei. The strategy was employed using two different VLP production platforms, one based on parainfluenza virus 5 (PIV5) and the other based on Nipah virus, and in both cases efficient cargo packaging and delivery could be achieved. These findings provide a foundation for development of paramyxovirus-like particles as tools for safe and efficient delivery of therapeutic proteins to cells and tissues. IMPORTANCE Therapeutic proteins including transcription factors and genome editors have enormous clinical potential but are currently limited in part due to the challenges of safely and efficiently delivering these proteins to the interiors of target cells. Here, we have developed a new strategy for protein delivery based on manipulation of paramyxovirus genome packaging interactions.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of ferlaviruses into four genogroups, and criteria for definition of ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups., Competing Interests: The authors have declared that no competing interests exist.

The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the "a" or "d" hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and phosphoproteins (P). In particular, we focus on measles, Nipah, and Hendra viruses and highlight both commonalities and differences with respect to the closely related Sendai virus. The molecular mechanisms that control the disorder-to-order transition undergone by the intrinsically disordered C-terminal domain (N TAIL ) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins are described in detail. By having a significant residual disorder, N TAIL -XD complexes are illustrative examples of "fuzziness", whose possible functional significance is discussed. Finally, the relevance of N-P interactions as promising targets for innovative antiviral approaches is underscored, and the functional advantages of structural disorder for paramyxoviruses are pinpointed.

Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti-IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN-α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll-like receptor (TLR) 7- and TLR9-dependent IFN-α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti-IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Paramyxovirus V proteins bind to MDA5 (melanoma differentiation-associated gene 5) and LGP2 (laboratory of genetics and physiology gene 2) but not RIG-I (retinoic acid-inducible gene I). The results demonstrate MDA5 R806 is essential for inhibition by diverse V proteins. Complementary substitution for the analogous RIG-I L714 confers V protein recognition. The analogous LGP2 R455 is required for recognition by measles V protein, but not other V proteins. These findings indicate that paramyxoviruses use a single amino acid to distinguish MDA5 from RIG-I and have evolved distinct contact sites for LGP2 interference.

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion.

During cell entry, enveloped viruses fuse their viral membrane with a cellular membrane in a process driven by energetically favorable, large-scale conformational rearrangements of their fusion proteins. Structures of the pre- and postfusion states of the fusion proteins including paramyxovirus PIV5 F and influenza virus hemagglutinin suggest that this occurs via two intermediates. Following formation of an initial complex, the proteins structurally elongate, driving a hydrophobic N-terminal "fusion peptide" away from the protein surface into the target membrane. Paradoxically, this first conformation change moves the viral and cellular bilayers further apart. Next, the fusion proteins form a hairpin that drives the two membranes into close opposition. While the pre- and postfusion hairpin forms have been characterized crystallographically, the transiently extended prehairpin intermediate has not been visualized. To provide evidence for this extended intermediate we measured the interbilayer spacing of a paramyxovirus trapped in the process of fusing with solid-supported bilayers. A gold-labeled peptide that binds the prehairpin intermediate was used to stabilize and specifically image F-proteins in the prehairpin intermediate. The interbilayer spacing is precisely that predicted from a computational model of the prehairpin, providing strong evidence for its structure and functional role. Moreover, the F-proteins in the prehairpin conformation preferentially localize to a patch between the target and viral membranes, consistent with the fact that the formation of the prehairpin is triggered by local contacts between F- and neighboring viral receptor-binding proteins (HN) only when HN binds lipids in its target membrane.

The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.

Paramyxovirinae envelope glycoproteins constitute a premier model to dissect how specific and dynamic interactions in multisubunit membrane protein complexes can control deep-seated conformational rearrangements. However, individual residues that determine reciprocal specificity of the viral attachment and fusion (F) proteins have not been identified. We have developed an assay based on a pair of canine distemper virus (CDV) F proteins (strains Onderstepoort (ODP) and Lederle) that share approximately 95% identity but differ in their ability to form functional complexes with the measles virus (MV) attachment protein (H). Characterization of CDV F chimeras and mutagenesis reveals four residues in CDV F-ODP (positions 164, 219, 233, and 317) required for productive interaction with MV H. Mutating these residues to the Lederle type disrupts triggering of F-ODP by MV H without affecting functionality when co-expressed with CDV H. Co-immunoprecipitation shows a stronger physical interaction of F-ODP than F-Lederle with MV H. Mutagenesis of MV F highlights the MV residues homologous to CDV F residues 233 and 317 as determinants for physical glycoprotein interaction and fusion activity under homotypic conditions. In assay reversal, the introduction of sections of the CDV H stalk into MV H shows a five-residue fragment (residues 110-114) to mediate specificity for CDV F-Lederle. All of the MV H stalk chimeras are surface-expressed, show hemadsorption activity, and trigger MV F. Combining the five-residue H chimera with the CDV F-ODP quadruple mutant partially restores activity, indicating that the residues identified in either glycoprotein contribute interdependently to the formation of functional complexes. Their localization in structural models of F and H suggests that placement in particular of F residue 233 in close proximity to the 110-114 region of H is structurally conceivable.

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.

In this study we report the development and optimization of two minigenome rescue systems for Nipah virus, a member of the Paramyxoviridae family. One is mediated by the T7 RNA polymerase supplied either by a constitutively expressing cell line or by transfection of expression plasmids and is thus independent from infection with a helper virus. The other approach is based on RNA polymerase I-driven transcription, a unique approach for paramyxovirus reverse genetics technology. Minigenome rescue was evaluated by reporter gene activities of (i) the two different minigenome transcription systems, (ii) genomic versus antigenomic-oriented minigenomes, (iii) different ratios of the viral protein expression plasmids, and (iv) time course experiments. The high efficiency and reliability of the established systems allowed for downscaling to 96-well plates. This served as a basis for the development of a high-throughput screening system for potential antivirals that target replication and transcription of Nipah virus without the need of high bio-containment. Using this system we were able to identify two compounds that reduced minigenome activity.

Menangle virus (MenV), which was isolated in Australia in 1997 during an outbreak of severe reproductive disease in pigs, is a novel member of the genus Rubulavirus in the family Paramyxoviridae. Although successfully eradicated from the affected piggery, fruit bats are considered to be the natural reservoir of the virus and therefore an ongoing risk of re-introduction to the pig population exists. Accordingly, reagents to facilitate serological surveillance are required to enhance the diagnostic capability for MenV, which is a newly recognized cause of disease in pigs with the potential to severely affect production in naive breeding herds. To address this need, recombinant MenV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. Using the expression vector pFGG3 under control of the GAL7 promoter, high yields of recombinant MenV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology, although not in length, to authentic nucleocapsids from virus-infected cells. Electron microscopy analysis also showed that yeast-expressed N protein which lacked the C-terminal tail (amino acid residues 400-519) formed significantly longer and denser nucleocapsid-like particles. Nucleocapsid-like particles derived from the full-length recombinant protein were stable and readily purified by CsCl gradient ultracentrifugation. When used as coating antigen in an indirect ELISA, the recombinant N protein reacted with sera derived from pigs experimentally infected with MenV and a simple serological assay to detect MenV-specific antibodies in pigs, fruit bats and humans could be designed on this basis.

Unlabelled: Previous studies have implicated CDV in the pathogenesis of Paget's disease; however, there has been no direct evidence that CDV can infect human cells. We studied the effects of CDV on osteoclastogenesis in vitro and showed that CDV had a dose-dependent effect on osteoclastogenesis, through a possible mechanism involving activation of NF-kappaB and sequestosome 1/p62., Introduction: Paget's disease is characterized by a dramatic increase in size and number of osteoclasts. The etiology of the disorder is still unclear; however, evidence points to either a viral infection or a genetic susceptibility or a combination of both. Previously, we have shown that canine distemper virus (CDV) RNA is present in Pagetic bone. However, the effects of CDV on human osteoclast formation in vitro have not been studied previously., Materials and Methods: Replicate cultures (n = 5) of purified human osteoclast precursors were infected with increasing doses of CDV and cultured on dentine slices for 14 days. Osteoclasts were stained for TRACP, and the dentine slices were examined for evidence of resorption. Control cells were incubated in the absence of virus. In each case, 10 high-power microscopy fields were analyzed. Immunocytochemical analyses were performed for p65, Gab2, sequestosome 1/p62, and ubiquitin., Results: CDV dose-dependently increased osteoclast number and size (p < 0.0001, ANOVA), and there was a concomitant increase in resorption (p < 0.0001, ANOVA). CDV infection induced nuclear translocation of p65 and led to a dramatic increase in sequestosome 1/p62 and ubiquitin expression., Conclusions: These results provide the first conclusive proof that CDV can infect and replicate in human osteoclast precursors, raising possible zoonotic implications for CDV. The increased osteoclastogenesis is accompanied by NF-kappaB and sequestosome 1/p62 activation. This study provides further evidence for the possible role of paramyxoviruses in the pathogenesis of Paget's disease.

The frequency and complications of respiratory viral infections (RVI) were studied in 50 ambulatory lung transplant patients during a single winter season, using viral antigens, viral cultures and PCR of nasal washes or bronchoalveolar lavages. Patients' survival, episodes of acute rejection and occurrence of bronchiolitis obliterans (BO) or BO syndrome (BOS) were monitored for 1 yr after the study. Overall, 32 (64%) patients had 49 symptomatic episodes. Documented infections included eight due to respiratory syncytial virus (RSV), one due to parainfluenza virus (PIV) and 10 due to influenza (FLU). Four of the FLU infections were serological rises without symptoms. Overall, 17 (34%) patients had documented viral infection; four patients had lower respiratory involvement and two (one RSV, one PIV) were hospitalised for aerosolised ribavirin treatment. After 1 yr there were three (6%) deaths unrelated to RVI. BO or BOS had occurred in one (6%) out of 17 patients with and three (12%) out of 33 without RVI. Respiratory viruses infected one-third of ambulatory lung transplant recipients in a single season. In conclusion, respiratory viral infection was not associated with subsequent graft dysfunction. Larger prospective studies are required to better define the acute and long-term morbidity of these infections.

Paramyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process-an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxoviruses (parainfluenza viruses 1-5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described.

The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy ("stutter" on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their phosphoprotein mRNA are also governed by the "rule of six", i.e. their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Why these two seemingly unrelated processes are so tightly linked in the Paramyxovirinae has been an enigma. This paper will review what is presently known about these two processes that are unique to viruses of this subfamily, and will discuss whether this enigmatic linkage could be due to the phenomenon of RNA virus error catastrophe.

A coronavirus (CoV) has recently been identified as the causative agent of the severe acute respiratory syndrome (SARS) in humans. CoVs enter target cells through fusion of viral and cellular membranes mediated by the viral envelope glycoprotein S. We have determined by x-ray crystallography the structure of a proteolytically stable core fragment from the heptad repeat (HR) regions HR1 and HR2 of the SARS-CoV S protein. We have also determined the structure of an HR1-HR2 S core fragment, containing a shorter HR1 peptide and a C-terminally longer HR2 peptide that extends up to the transmembrane region. In these structures, three HR1 helices form a parallel coiled-coil trimer, whereas three HR2 peptides pack in an oblique and antiparallel fashion into the coiled-coil hydrophobic grooves, adopting mixed extended and alpha-helical conformations as in postfusion paramyxoviruses F proteins structures. Our structure positions a previously proposed internal fusion peptide adjacent to the N-terminus of HR1. Peptides from the HR2 region of SARS-CoV S have been shown to inhibit viral entry and infection in vitro. The structures presented here can thus open the path to the design of small-molecule inhibitors of viral entry and candidate vaccine antigens against this virus.

We report direct, real-time electrical detection of single virus particles with high selectivity by using nanowire field effect transistors. Measurements made with nanowire arrays modified with antibodies for influenza A showed discrete conductance changes characteristic of binding and unbinding in the presence of influenza A but not paramyxovirus or adenovirus. Simultaneous electrical and optical measurements using fluorescently labeled influenza A were used to demonstrate conclusively that the conductance changes correspond to binding/unbinding of single viruses at the surface of nanowire devices. pH-dependent studies further show that the detection mechanism is caused by a field effect, and that the nanowire devices can be used to determine rapidly isoelectric points and variations in receptor-virus binding kinetics for different conditions. Lastly, studies of nanowire devices modified with antibodies specific for either influenza or adenovirus show that multiple viruses can be selectively detected in parallel. The possibility of large-scale integration of these nanowire devices suggests potential for simultaneous detection of a large number of distinct viral threats at the single virus level.

CPI(+) and CPI(-) are two canine isolates of simian virus 5 (SV5). CPI(+) was originally isolated from the cerebrospinal fluid of a dog with temporary posterior paralysis and CPI(-) was recovered at 12 days p.i. from the brain tissue of a dog experimentally infected with CPI(+). We have previously shown that the V protein of SV5 blocks interferon (IFN) signalling by targeting STAT1 for degradation. Here we report that whilst CPI(+) targets STAT1 for degradation, CPI(-) fails to and as a consequence, CPI(+) blocks IFN signalling but CPI(-) does not. Three amino acid differences in the P/V N-terminal common domain of the V protein are responsible for the observed difference in the abilities of CPI(+) and CPI(-) to block IFN signalling. In cells persistently infected with CPI(-) the virus may become repressed in response to IFN, under which circumstances virus glycoproteins are lost from the surface of infected cells and virus nucleocapsid proteins accumulate in cytoplasmic inclusion bodies. We suggest that in vivo cells infected with IFN-resistant viruses (in which there would be continuous virus protein synthesis) may be more susceptible to killing by cytotoxic T cells than cells infected with IFN-sensitive viruses (in which virus protein synthesis was repressed), and a model of virus persistence is put forward in which there is alternating selection of IFN-resistant and IFN-sensitive viruses depending upon the state of the adaptive immune response.

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.

We studied the effects of eight protease inhibitors on the multicycle replications of various orthomyxoviruses and paramyxoviruses. Among the compounds, nafamostat mesilate, camostat mesilate, gabexate mesilate, and aprotinin, which are widely used in the treatment of pancreatitis, inhibited influenza virus A and B replication at concentrations that were significantly lower than their cytotoxic thresholds in vitro. None of the protease inhibitors had activity against respiratory syncytial virus, measles virus, or parainfluenza virus type 3 at the highest concentrations tested. Camostat mesilate was found to be the most selective inhibitor. Its 50% effective concentration for influenza virus A replication was 2.2 micrograms/ml, and the selectivity index, which was based on the ratio of the 50% inhibitory concentration for host cell proliferation to the 50% effective concentration for influenza virus A replication, was 680. When the in ovo antiviral activity of the compounds was tested by using chicken embryos, camostat mesilate at a dose of 10 micrograms/g markedly reduced the hemagglutinin titers of influenza viruses A and B.

The role of residues in the conserved hydrophobic N-terminal fusion peptide of the paramyxovirus fusion (F) protein in causing cell-cell fusion was examined. Mutations were introduced into the cDNA encoding the simian virus 5 (SV5) F protein, the altered F proteins were expressed by using an eukaryotic vector, and their ability to mediate syncytium formation was determined. The mutant F proteins contained both single- and multiple-amino-acid substitutions, and they exhibited a variety of intracellular transport properties and fusion phenotypes. The data indicate that many substitutions in the conserved amino acids of the simian virus 5 F fusion peptide can be tolerated without loss of biological activity. Mutant F proteins which were not transported to the cell surface did not cause cell-cell fusion, but all of the mutants which were transported to the cell surface were fusion competent, exhibiting fusion properties similar to or better than those of the wild-type F protein. Mutant F proteins containing glycine-to-alanine substitutions had altered intracellular transport characteristics, yet they exhibited a great increase in fusion activity. The potential structural implications of this substitution and the possible importance of these glycine residues in maintaining appropriate levels of fusion activity are discussed.

The nucleotide and deduced amino acid sequences of two genes of phocid distemper virus (PDV) were determined by cDNA cloning and sequencing. The long open reading frame of the gene encoding the nucleocapsid (N) protein is presented. As with other morbilliviruses, the phosphoprotein (P) gene of PDV was found to be located after the 5' end of the N gene and before the 3' end of the matrix protein gene. The P gene was shown to have the capacity to encode three distinct proteins, P, V and C, in analogy to other morbilliviruses. The results presented provide evidence for editing of the PDV P mRNA transcript by insertion of G residues. When the nucleotide and deduced amino acid sequences of the N, P, V and C genes were aligned with corresponding sequences of other established members of the morbillivirus genus, compelling homology was found between PDV and canine distemper virus (CDV), whereas there was markedly less similarity between PDV and measles virus or rinderpest virus. On the basis of the alignments presented, the estimated amino acid sequence similarity between the N and P genes of PDV and CDV was 84% and 76%, respectively. These differences at the genomic level indicate that the viruses are two separate entities.

Intracellular proteolytic processing of fusion glycoprotein precursors (F0) of paramyxoviruses, i.e. a virulent strain of Newcastle disease virus (NDV), parainfluenza virus type 3 (PIV3) and simian virus 5 (SV5), was examined in NALM6 and BSC40 cells and compared with that in LLCMK2 cells to investigate the distribution of the virus-activating protease(s) among the cells and its substrate specificity. BSC40 cells lack a processing endoprotease of the neuropeptide precursor, pro-opiomelanocortin (POMC), which possesses multiple cleavage sites at pairs of basic residues, Lys-Arg and Arg-Arg, a motif similar to that found in the cleavage site of the F0 proteins. In NALM6 cells, only small amounts of the F0 protein of virulent NDV was cleaved whereas those of PIV3 and SV5 were efficiently cleaved. In BSC40 cells the F0 proteins of these three viruses were cleaved normally as well as in LLCMK2 cells. The processing inhibitors monensin, chloroquine and A23187 suppressed the F0 cleavage in the three cell types. These results indicate that both NALM6 and BSC40 cells possess virus-activating proteases similar to that of LLCMK2 cells, but suggest that the enzyme of NALM6 may be slightly different in its substrate specificity from those of BSC40 and LLCMK2. The results also suggest that the virus-activating proteases are different in their distribution and substrate specificity from the processing enzyme of POMC.

The cellular glucose-regulated protein GRP78-BiP is a member of the HSP70 stress family of gene products, and the protein is a resident component of the endoplasmic reticulum, where it is thought to play a role in the folding and oligomerization of secretory and membrane-bound proteins. GRP78-BiP also binds to malfolded proteins, and this may be one mechanism for preventing their intracellular transport. An induction in synthesis of the GRP78-BiP protein occurs in cells infected with paramyxoviruses (R. W. Peluso, R. A. Lamb, and P. W. Choppin, Proc. Natl. Acad. Sci. USA 75:6120-6124, 1978). We have studied the expression and activity of the GRP78-BiP gene and synthesis of the GRP78-BiP protein during infection with the paramyxovirus simian virus 5 (SV5). We wished to identify the viral component capable of causing activation of GRP78-BiP since GRP78-BiP interacts specifically and transiently with the SV5 hemagglutinin-neuraminidase (HN) glycoprotein during HN folding (D. T. W. Ng, R. E. Randall, and R. A. Lamb, J. Cell Biol. 109:3273-3289, 1989). Expression of cDNAs of the SV5 wild-type HN glycoprotein and a mutant form of HN that is malfolded but not the SV5 F glycoprotein or SV5 cytoplasmic proteins P, V, and M caused increased amounts of GRP78-BiP mRNA to accumulate, as detected by nuclease S1 protection assays. As unfolded or malfolded forms of HN cannot be detected to accumulate during SV5 infection, the data suggest that the flux of HN through the ER in SV5-infected cells can cause activation of GRP78-BiP transcription.

The complete nucleotide sequence of gene 3 of pneumonia virus of mice has been determined, and the 5' end of the mRNA mapped using a modification of the polymerase chain reaction technique. The gene contains a single open reading frame, beginning with a 5'-proximal AUG initiation codon, encoding a polypeptide with a predicted Mr of 43141. Expression of the gene 3 protein in Escherichia coli and in vitro showed that it reacted with virus-specific antiserum and comigrated with the major nucleocapsid (N) polypeptide. The predicted amino acid sequence has extensive identity with that of the N protein of human respiratory syncytial virus. Comparisons with the amino acid sequences of N proteins of other paramyxoviruses, vesicular stomatitis virus and Ebola virus suggest that these proteins may have retained much of the same structure. These regions of conserved structure would most likely have the common functions of RNA binding and protein/protein interactions in the virus nucleocapsid.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.

The DNA fraction from a line of bovine embryonic kidney cells originally exposed as primary cultures several months earlier to a temperature-sensitive (ts) mutant of respiratory syncytial (RS) virus could be used to transfect human HEp-2 cells with the production of infectious RS virus. The DNA donor cells, designated BEK/RS ts, retained their healthy fibroblastic appearance during continuous cultivation at a temperature (39 degrees) restrictive for growth of the original infecting mutant and showed no evidence for RS virus replication or viral antigen synthesis when directly examined for these activities by conventional methods. The infectious property of the DNA from BEK/RS ts cells was abolished by exposure of the nucleic acid preparation to DNase (but not RNase) or by pretreatment of recipient HEp-2 cells with actinomycin D or mitomycin C. The latter drug treatments substantially enhanced the replication of infecting wild-type RS virus in HEp-2 cells. Viral isolates derived from the progeny of a DNA transfection included clones possessing several genetic markers of the RS ts mutant originally used to infect BEK/RS ts cells and other virus clones that appeared to be either hybrid or wild-type for phenotypic properties such as their temperature sensitivity. An infectious proviral DNA was also detected in a line of virogenic HEp-2 cells (HEp-2/RS) persistently infected with respiratory syncytial virus after exposure to the wild-type strain 2 years earlier.

The kinetics of synthesis and the nature of the oligosaccharides of the glycoproteins of pneumonia virus of mice (PVM) were studied. Tryptic peptide mapping showed that the two major glycosylated polypeptides G1 and G2 were different forms of the same protein. G2 was derived from G1 which in turn appeared to be derived from an unidentified precursor. The G1/G2 protein of PVM is probably a haemagglutinin since a monoclonal antibody directed against it has a high haemagglutination inhibition titre. On the basis of experiments with inhibitors and glycosidases it was deduced that G1 and G2 have both N-linked and O-linked oligosaccharides. The putative fusion protein-equivalent of PVM was shown to possess N-linked oligosaccharides. In the presence of tunicamycin a high mobility form (F1t) appeared to be derived from a precursor (F0t) with the same mobility as the fully glycosylated protein. If by analogy with other paramyxoviruses this represents a cleavage event, the difference in mobility of the precursor and product suggests that the putative F2 product is smaller than the corresponding F2 protein of other paramyxoviruses. However, no F2 candidate protein was detected and evidence for an F1,2 dimer was inconclusive. The glycoproteins of PVM resemble those of respiratory syncytial virus in terms of their pattern of glycosylation, but differ in their processing.

Some evidences have been found that virulency in paramyxoviruses depends on the sensitivity of the cleavage recognition site of the F glycoprotein to serine type proteases. In this report, the interaction energies between the active site of trypsin and the cleavage recognition sites in paramyxoviruses are calculated. Results show that van der Waals energy and electrostatic energy contribute to the sensitivity. The virulencies of some myxo- and retro-viruses are then predicted on the basis of the two calculated interaction energy values.

The assembly of virion surface glycoproteins by enveloped viruses appears to be both specific and non-specific. It is non-specific in the sense that, during a dual infection by various unrelated viruses, there is very common (if not universal) and efficient mixing of envelope glycoproteins from both genotypes in the progeny virions. However, it seems to be specific in the sense that non-viral, cell surface proteins are, in the main, recognized as alien and are excluded from incorporation into virions during budding. These findings suggest that all enveloped viruses may share an essentially common molecular mechanism for the specific assembly and budding of virus glycoproteins while at the same time incorporating a mechanism for the exclusion of glycoproteins of cellular origin. One of the simplest explanations for this phenomenon may be that virus envelope surface structures all evolved from a single ancestral virus.

Turkey rhinotracheitis (TRT) virus is a paramyxovirus associated with recent outbreaks of acute respiratory disease in turkeys. On morphological criteria it resembles the pneumoviruses more than the other members of the paramyxovirus family. In this communication we report the identification of five virus-induced polypeptides in TRT virus-infected BS-C-1 cells. The Mr of these polypeptides were estimated as 38K, 35K, 30K, 19K and 15K from their electrophoretic mobility in SDS-polyacrylamide 6 to 15% gradient gels. The virus specificity of four (the 38K, 35K, 30K and 19K Mr polypeptides) of these five was confirmed by their predominance as products of in vitro translation of mRNA from TRT virus-infected BS-C-1 cells. An additional virus-specific polypeptide with an estimated Mr of 22K was revealed by in vitro translation and may be either a non-structural polypeptide or a minor structural protein. All six polypeptides were immunoprecipitated by a murine antiserum. Three other polypeptides (129K, 57K and 45K) were also immunoprecipitated. The 57K, 45K and 15K Mr polypeptides were glycosylated, and the 57K glycopolypeptide may be a disulphide-bonded dimer of the 45K and 15K glycopolypeptides. Another major glycopolypeptide of 83K Mr was observed but its virus-specificity could not be confirmed. Taken together our results tentatively identify eight or nine TRT virus-specified polypeptides and are consistent with the classification of TRT virus as a new member of the genus Pneumovirus.

cDNAs encoding the mRNAs for the fusion protein (F) and the hemagglutinin/neuraminidase protein (HN) of the paramyxovirus simian virus 5 have been inserted into a eukaryotic expression vector under the control of the simian virus 40 late promoter. The F and HN proteins synthesized in recombinant infected cells are indistinguishable in terms of electrophoretic mobility and glycosylation from the proteins synthesized in simian virus 5-infected cells. In addition, the expressed F and HN proteins have been shown to be anchored in the plasma membrane in a biologically active form by indirect live cell immunofluorescence, the F-mediated formation of syncytia, and the ability of HN to cause the hemadsorption of erythrocytes to the infected cell surface.

A murine polyclonal antiserum and monoclonal antibodies have been employed to identify pneumonia virus of mice (PVM) polypeptides in infected cells and to study post-translational modifications. Immunoprecipitation experiments using a murine polyclonal antiserum and a monoclonal antibody directed against a 39K protein have established an antigenic relationship between two PVM proteins and the N and P proteins of human respiratory syncytial virus. Although 20 virus-specific polypeptides have been identified in lysates of infected cells, evidence is presented that some of these are related and that the number of unique polypeptides probably does not exceed 11 or 12. The phosphoproteins of PVM have a pattern of mobilities more like that of the recently described pneumovirus causing rhinotracheitis in turkeys than that of human or bovine respiratory syncytial virus.

Infection of several types of cultured cells with the paramyxoviruses simian virus 5 and Sendai virus stimulates synthesis of four polypeptides (I-IV) with molecular weights of approximately 99,000, 97,000, 86,000, and 78,000, respectively. That these are host polypeptides encoded in cellular mRNAs has been shown by the inhibition of their synthesis by actinomycin D and by the similarity of the peptide maps of them and of polypeptides with the same electrophoretic mobility from uninfected cells. Peptide mapping as well as identical migration in polyacrylamide gels has also indicated that polypeptides I, II, and IV are the same as plasma membrane polypeptides whose synthesis is enhanced in cells transformed by Rous sarcoma virus and in normal cells by glucose deprivation or treatment with 2-deoxyglucose. Polypeptides I and II appear to be the same polypeptides, with the observed differences in migration reflecting the glycosylation of polypeptide I, a relationship previously shown to exist between polypeptides in glucose-deprived and glucose-fed cells. Infection with paramyxoviruses does not significantly increase the transport of glucose by cells, and the maintenance of a high concentration of glucose in the medium does not prevent the enhanced synthesis of these polypeptides. This is in contrast to the situation in transformed cells in which stimulation of synthesis of these polypeptides is secondary to depletion of glucose in the medium due to increased glucose uptake by the cells. Thus, although paramyxovirus infection and transformation by Rous sarcoma virus result in stimulation of the synthesis of the same membrane polypeptides, the mechanism of stimulation differs.