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1.  Measles Virus C Protein Interferes with Beta Interferon Transcription in the Nucleus 
Journal of Virology  2012;86(2):796-805.
Transcriptional induction of beta interferon (IFN-β) through pattern recognition receptors is a key event in the host defense against invading viruses. Infection of cells by paramyxoviruses, like measles virus (MV) (genus Morbillivirus), is sensed predominantly by the ubiquitous cytoplasmic helicase RIG-I, recognizing viral 5′-triphosphate RNAs, and to some degree by MDA5. While MDA5 activation is effectively prevented by the MV V protein, the viral mechanisms for inhibition of MDA5-independent induction of IFN-β remained obscure. Here, we identify the 186-amino-acid MV C protein, which shuttles between the nucleus and the cytoplasm, as a major viral inhibitor of IFN-β transcription in human cells. Activation of the transcription factor IRF3 by upstream kinases and nuclear import of activated IRF3 were not affected in the presence of C protein, suggesting a nuclear target. Notably, C proteins of wild-type MV isolates, which are poor IFN-β inducers, were found to comprise a canonical nuclear localization signal (NLS), whereas the NLSs of all vaccine strains, irrespective of their origins, were mutated. Site-directed mutagenesis of the C proteins from an MV wild-type isolate and from the vaccine virus strain Schwarz confirmed a correlation of nuclear localization and inhibition of IFN-β transcription. A functional NLS and efficient nuclear accumulation are therefore critical for MV C to retain its potential to downregulate IFN-β induction. We suggest that a defect in efficient nuclear import of C protein contributes to attenuation of MV vaccine strains.
doi:10.1128/JVI.05899-11
PMCID: PMC3255862  PMID: 22072748
2.  Complete Genome Sequence of a Wild-Type Measles Virus Isolated during the Spring 2013 Epidemic in Germany 
Genome Announcements  2014;2(2):e00157-14.
Measles virus induces an acute disease with rash and fever. Despite ongoing vaccination and elimination campaigns, the measles virus still sustains long-lasting transmission chains in Europe. Here we report the complete genome sequence of a wild-type measles virus isolated from a patient in Munich (MVi/Muenchen.DEU/19.13[D8]) during a German measles outbreak in 2013.
doi:10.1128/genomeA.00157-14
PMCID: PMC3990739  PMID: 24744323
3.  A Modified Screening System for Loss-of-Function and Dominant Negative Alleles of Essential MCMV Genes 
PLoS ONE  2014;9(4):e94918.
Inactivation of gene products by dominant negative mutants is a valuable tool to assign functions to yet uncharacterized proteins, to map protein-protein interactions or to dissect physiological pathways. Detailed functional and structural knowledge about the target protein would allow the construction of inhibitory mutants by targeted mutagenesis. Yet, such data are limited for the majority of viral proteins, so that the target gene needs to be subjected to random mutagenesis to identify suitable mutants. However, for cytomegaloviruses this requires a two-step screening approach, which is time-consuming and labor-intensive. Here, we report the establishment of a high-throughput suitable screening system for the identification of inhibitory alleles of essential genes of the murine cytomegalovirus (MCMV). In this screen, the site-specific recombination of a specifically modified MCMV genome was transferred from the bacterial background to permissive host cells, thereby combining the genetic engineering and the rescue test in one step. Using a reference set of characterized pM53 mutants it was shown that the novel system is applicable to identify non-complementing as well as inhibitory mutants in a high-throughput suitable setup. The new cis-complementation assay was also applied to a basic genetic characterization of pM99, which was identified as essential for MCMV growth. We believe that the here described novel genetic screening approach can be adapted for the genetic characterization of essential genes of any large DNA viruses.
doi:10.1371/journal.pone.0094918
PMCID: PMC3986410  PMID: 24733555
4.  Revealing the secrets of neuronal circuits with recombinant rabies virus technology 
An understanding of how the brain processes information requires knowledge of the architecture of its underlying neuronal circuits, as well as insights into the relationship between architecture and physiological function. A range of sophisticated tools is needed to acquire this knowledge, and recombinant rabies virus (RABV) is becoming an increasingly important part of this essential toolbox. RABV has been recognized for years for its properties as a synapse-specific trans-neuronal tracer. A novel genetically modified variant now enables the investigation of specific monosynaptic connections. This technology, in combination with other genetic, physiological, optical, and computational tools, has enormous potential for the visualization of neuronal circuits, and for monitoring and manipulating their activity. Here we will summarize the latest developments in this fast moving field and provide a perspective for the use of this technology for the dissection of neuronal circuit structure and function in the normal and diseased brain.
doi:10.3389/fncir.2013.00002
PMCID: PMC3553424  PMID: 23355811
connectivity; wiring diagram; neuronal tracer; synapses; monosynaptic
6.  The Measles Virus V Protein Binds to p65 (RelA) To Suppress NF-κB Activity▿  
Journal of Virology  2011;85(7):3162-3171.
Nuclear factor κB (NF-κB) transcription factors are involved in controlling numerous cellular processes, including inflammation, innate and adaptive immunity, and cell survival. Here we show that the immunosuppressive measles virus (MV; Morbillivirus genus, Paramyxoviridae) has evolved multiple functions to interfere with canonical NF-κB signaling in epithelial cells. The MV P, V, and C proteins, also involved in preventing host cell interferon responses, were found to individually suppress NF-κB-dependent reporter gene expression in response to activation of the tumor necrosis factor (TNF) receptor, RIG-I-like receptors, or Toll-like receptors. NF-κB activity was most efficiently suppressed in the presence of V, while expression of P or C resulted in moderate inhibition. As indicated by reporter gene assays involving overexpression of the IκB kinase (IKK) complex, which phosphorylates the inhibitor of κB to liberate NF-κB, V protein targets a downstream step in the signaling cascade. Coimmunoprecipitation experiments revealed that V specifically binds to the Rel homology domain of the NF-κB subunit p65 but not of p50. Notably, the short C-terminal domain of the V protein, which is also involved in binding STAT2, IRF7, and MDA5, was sufficient for the interaction and for preventing reporter gene activity. As observed by confocal microscopy, the presence of V abolished nuclear translocation of p65 upon TNF-α stimulation. Thus, MV V appears to prevent NF-κB-dependent gene expression by retaining p65 in the cytoplasm. These findings reveal NF-κB as a key target of MV and stress the importance of the V protein as the major viral immune-modulatory factor.
doi:10.1128/JVI.02342-10
PMCID: PMC3067892  PMID: 21270162
7.  Immunogenicity Studies in Carnivores Using a Rabies Virus Construct with a Site-Directed Deletion in the Phosphoprotein 
Different approaches have been applied to develop highly attenuated rabies virus vaccines for oral vaccination of mesocarnivores. One prototype vaccine construct is SAD dIND1, which contains a deletion in the P-gene severely limiting the inhibition of type-1 interferon induction. Immunogenicity studies in foxes and skunks were undertaken to investigate whether this highly attenuated vaccine would be more immunogenic than the parental SAD B19 vaccine strain. In foxes, it was demonstrated that SAD dIND1 protected the animals against a rabies infection after a single oral dose, although virus neutralizing antibody titres were lower than in foxes orally vaccinated with the SAD B19 virus as observed in previous experiments. In contrast, skunks receiving 107.5 FFU SAD dIND1 did not develop virus neutralizing antibodies and were not protected against a subsequent rabies infection.
doi:10.4061/2011/898171
PMCID: PMC3177460  PMID: 21991446
8.  Genetic Dissection of Interferon-Antagonistic Functions of Rabies Virus Phosphoprotein: Inhibition of Interferon Regulatory Factor 3 Activation Is Important for Pathogenicity▿  
Journal of Virology  2010;85(2):842-852.
The rabies virus (RV) phosphoprotein (P) is a type I interferon (IFN) antagonist preventing both transcriptional induction of IFN and IFN-mediated JAK/STAT signaling. In addition, P is an essential cofactor of the viral polymerase and is required for encapsidation of viral RNA into nucleoprotein during replication. By site-directed mutagenesis, we have identified a domain of P required for efficient inhibition of IFN induction. Phosphoproteins lacking amino acids (aa) 176 to 181, 182 to 186, or 176 to 186 were severely compromised in counteracting phosphorylation of IRF3 and IRF7 by TBK1 or IKKi while retaining the full capacity of preventing nuclear import of activated STATs and of supporting virus transcription and replication. Recombinant RV carrying the mutated phosphoproteins (the SAD ΔInd1, SAD ΔInd2, and SAD ΔInd1/2 viruses) activated IRF3 and beta IFN (IFN-β) transcription in infected cells but still blocked STAT-mediated expression of IFN-stimulated genes. Due to a somewhat higher transcription rate, the SAD ΔInd1 virus activated IRF3 more efficiently than the SAD ΔInd2 virus. After intracerebral injection into mouse brains at high doses, the SAD ΔInd1 virus was completely apathogenic for wild-type (wt) mice, while the SAD ΔInd2 virus was partially attenuated and caused a slower progression of lethal rabies than wt RV. Neurovirulence of IFN-resistant RV thus correlates with the capacity of the virus to prevent activation of IRF3 and IRF7.
doi:10.1128/JVI.01427-10
PMCID: PMC3020028  PMID: 21084487
9.  Retrograde neuronal tracing with a deletion-mutant rabies virus 
Nature methods  2006;4(1):47-49.
We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.
doi:10.1038/NMETH999
PMCID: PMC2755236  PMID: 17179932
10.  Attenuation of Rabies Virus Replication and Virulence by Picornavirus Internal Ribosome Entry Site Elements▿  
Journal of Virology  2008;83(4):1911-1919.
Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and relies on the canonical 5′-end-dependent translation of capped viral mRNAs. Here, we have used internal ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2 IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-β) promoter, and increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2 IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-α receptor (IFNAR−/−). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demonstrated only in newborn IFNAR−/− mice. Translational control of individual genes is a promising strategy to attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study the function of IRES elements in detail.
doi:10.1128/JVI.02055-08
PMCID: PMC2643788  PMID: 19073737
11.  Measles Virus V Protein Is a Decoy Substrate for IκB Kinase α and Prevents Toll-Like Receptor 7/9-Mediated Interferon Induction▿  
Journal of Virology  2008;82(24):12365-12373.
The central role of plasmacytoid dendritic cells (pDC) in activating host immune responses stems from their high capacity to express alpha interferon (IFN-α) after stimulation of Toll-like receptors 7 and 9 (TLR7 and -9). This involves the adapter MyD88 and the kinases interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, and IκB kinase α (IKKα), which activates IFN regulatory factor 7 (IRF7) and is independent of the canonical kinases TBK1 and IKKɛ. We have recently shown that the immunosuppressive measles virus (MV) abolishes TLR7/9/MyD88-dependent IFN induction in human pDC (Schlender et al., J. Virol. 79:5507-5515, 2005), but the molecular mechanisms remained elusive. Here, we have reconstituted the pathway in cell lines and identified IKKα and IRF7 as specific targets of the MV V protein (MV-V). Binding of MV-V to IKKα resulted in phosphorylation of V on the expense of IRF7 phosphorylation by IKKα in vitro and in living cells. This corroborates the role of IKKα as the kinase phosphorylating IRF7. MV-V in addition bound to IRF7 and to phosphomimetic IRF7 and inhibited IRF7 transcriptional activity. Binding to both IKKα and IRF7 required the 68-amino-acid unique C-terminal domain of V. Inhibition of TLR/MyD88-dependent IFN induction by MV-V is unique among paramyxovirus V proteins and should contribute to the unique immunosuppressive phenotype of measles. The mechanisms employed by MV-V inspire strategies to interfere with immunopathological TLR/MyD88 signaling.
doi:10.1128/JVI.01321-08
PMCID: PMC2593327  PMID: 18922877
12.  Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons 
Neuron  2007;53(5):639-647.
SUMMARY
There has never been a wholesale way of identifying neurons that are monosynaptically connected either to some other cell group or, especially, to a single cell. The best available tools, transsynaptic tracers, are unable to distinguish weak direct connections from strong indirect ones. Furthermore, no tracer has proven potent enough to label any connected neurons whatsoever when starting from a single cell. Here we present a transsynaptic tracer that crosses only one synaptic step, unambiguously identifying cells directly presynaptic to the starting population. Based on rabies virus, it is genetically targetable, allows high-level expression of any gene of interest in the synaptically coupled neurons, and robustly labels connections made to single cells. This technology should enable a far more detailed understanding of neural connectivity than has previously been possible.
doi:10.1016/j.neuron.2007.01.033
PMCID: PMC2629495  PMID: 17329205
13.  Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport▿ †  
Journal of Virology  2007;82(1):237-245.
Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.
doi:10.1128/JVI.01342-07
PMCID: PMC2224359  PMID: 17928343
14.  Inhibition of Interferon Signaling by Rabies Virus Phosphoprotein P: Activation-Dependent Binding of STAT1 and STAT2 
Journal of Virology  2006;80(6):2675-2683.
Rabies virus (RV) phosphoprotein P is an interferon (IFN) antagonist counteracting transcriptional activation of type I IFN (K. Brzózka, S. Finke, and K. K. Conzelmann, J. Virol 79:7673-7681, 2005). We here show that RV P in addition is responsible for preventing IFN-α/β- and IFN-γ-stimulated JAK-STAT signaling in RV-infected cells by the retention of activated STATs in the cytoplasm. Expression of IFN-stimulated response element- and gamma-activated sequence-controlled genes was severely impaired in cells infected with RV SAD L16 or in cells expressing RV P protein from transfected plasmids. In contrast, a recombinant RV expressing small amounts of P had lost the ability to interfere with JAK-STAT signaling. IFN-mediated tyrosine phosphorylation of STAT1 and STAT2 was not impaired in RV P-expressing cells; rather, a defect in STAT recycling was suggested by distinct accumulation of tyrosine-phosphorylated STATs in cell extracts. In the presence of P, activated STAT1 and STAT2 were unable to accumulate in the nucleus. Notably, STAT1 and STAT2 were coprecipitated with RV P only from extracts of cells previously stimulated with IFN-α or IFN-γ, whereas in nonstimulated cells no association of P with STATs was observed. This conditional, IFN activation-dependent binding of tyrosine-phosphorylated STATs by RV P is unique for a viral IFN antagonist. The 10 C-terminal residues of P are required for counteracting JAK-STAT signaling but not for inhibition of transcriptional activation of IFN-β, thus demonstrating two independent functions of RV P in counteracting the host's IFN response.
doi:10.1128/JVI.80.6.2675-2683.2006
PMCID: PMC1395475  PMID: 16501077
15.  Identification of the Rabies Virus Alpha/Beta Interferon Antagonist: Phosphoprotein P Interferes with Phosphorylation of Interferon Regulatory Factor 3 
Journal of Virology  2005;79(12):7673-7681.
Rabies virus (RV) of the Rhabdoviridae family grows in alpha/beta interferon (IFN)-competent cells, suggesting the existence of viral mechanisms preventing IFN gene expression. We here identify the viral phosphoprotein P as the responsible IFN antagonist. The critical involvement of P was first suggested by the observation that an RV expressing an enhanced green fluorescent protein (eGFP)-P fusion protein (SAD eGFP-P) (S. Finke, K. Brzózka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004) was eliminated in IFN-competent HEp-2 cell cultures, in contrast to wild-type (wt) RV or an RV replicon lacking the genes for matrix protein and glycoprotein. SAD eGFP-P induced transcription of the IFN-β gene and expression of the IFN-responsive MxA and STAT-1 genes. Similarly, an RV expressing low levels of P, which was generated by moving the P gene to a promoter-distal gene position (SAD ΔPLP), lost the ability to prevent IFN induction. The analysis of RV mutants lacking expression of truncated P proteins P2, P3, or P4, which are expressed from internal AUG codons of the wt RV P open reading frame, further showed that full-length P is competent in suppressing IFN-β gene expression. In contrast to wt RV, the IFN-inducing SAD ΔPLP caused S386 phosphorylation, dimerization, and transcriptional activity of IFN regulatory factor 3 (IRF-3). Phosphorylation of IRF-3 by TANK-binding kinase-1 expressed from transfected plasmids was abolished in wt RV-infected cells or by cotransfection of P-encoding plasmids. Thus, RV P is necessary and sufficient to prevent a critical IFN response in virus-infected cells by targeting activation of IRF-3 by an upstream kinase.
doi:10.1128/JVI.79.12.7673-7681.2005
PMCID: PMC1143667  PMID: 15919920
16.  Inhibition of Toll-Like Receptor 7- and 9-Mediated Alpha/Beta Interferon Production in Human Plasmacytoid Dendritic Cells by Respiratory Syncytial Virus and Measles Virus 
Journal of Virology  2005;79(9):5507-5515.
Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.
doi:10.1128/JVI.79.9.5507-5515.2005
PMCID: PMC1082779  PMID: 15827165
18.  Tracking Fluorescence-Labeled Rabies Virus: Enhanced Green Fluorescent Protein-Tagged Phosphoprotein P Supports Virus Gene Expression and Formation of Infectious Particles 
Journal of Virology  2004;78(22):12333-12343.
Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD ΔP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.
doi:10.1128/JVI.78.22.12333-12343.2004
PMCID: PMC525074  PMID: 15507620
19.  Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly 
Journal of Virology  2003;77(22):12074-12082.
Recently, we have shown that the rabies virus (RV) matrix (M) protein regulates the balance of virus RNA synthesis by shifting synthesis activity from transcription to replication (S. Finke, R. Mueller-Waldeck, and K. K. Conzelmann, J. Gen. Virol. 84:1613-1621, 2003). Here we describe the identification of an M residue critical for regulation of RV RNA synthesis. By analyzing the phenotype of heterotypic RV M proteins with respect to RNA synthesis of RV SAD L16, we identified the M proteins of the RV ERA and PV strains as deficient. Comparison of M sequences suggested that a single residue, arginine 58, was critical. A recombinant virus having this amino acid exchanged with a glycine, SAD M(R58G), has lost the abilities to downregulate RV transcription and to stimulate replication. This resulted in an increase in the transcription rate of more than 15-fold, as previously observed for M deletion mutants. Most importantly, the efficiencies of virus assembly and budding were equal for wild-type M and M(R58G), as determined in assays studying the transient complementation of an M- and G-deficient RV construct, NPgrL. In addition, virus particle density, protein composition, and specific infectivity of SAD L16 and SAD M(R58G) viruses were identical. Thus, we have identified mutations that affect the function of M only in regulation of RNA synthesis, but not in assembly and budding, providing evidence that these functions are genetically separable.
doi:10.1128/JVI.77.22.12074-12082.2003
PMCID: PMC254266  PMID: 14581544
20.  Nonstructural Proteins NS1 and NS2 of Bovine Respiratory Syncytial Virus Block Activation of Interferon Regulatory Factor 3 
Journal of Virology  2003;77(16):8661-8668.
We have previously shown that the nonstructural (NS) proteins NS1 and NS2 of bovine respiratory syncytial virus (BRSV) mediate resistance to the alpha/beta interferon (IFN)-mediated antiviral response. Here, we show that they, in addition, are able to prevent the induction of beta IFN (IFN-β) after virus infection or double-stranded RNA stimulation. In BRSV-infected MDBK cells upregulation of IFN-stimulated genes (ISGs) such as MxA did not occur, although IFN signaling via JAK/STAT was found intact. In contrast, infection with recombinant BRSVs lacking either or both NS genes resulted in efficient upregulation of ISGs. Biological IFN activity and IFN-β were detected only in supernatants of cells infected with the NS deletion mutants but not with wild-type (wt) BRSV. Subsequent analyses of IFN-β promoter activity showed that infection of cells with the double deletion mutant BRSV ΔNS1/2, but not with BRSV wt, resulted in a significant increase in IFN-β gene promoter activity. Induction of the IFN-β promoter depends on the activation of three distinct transcription factors, NF-κB, ATF-2/c-Jun, and IFN regulatory factor 3 (IRF-3). Whereas NF-κB and ATF-2/c-Jun activities were readily detectable and comparable in both wt BRSV- and BRSV ΔNS1/2-infected cells, phosphorylation and transcriptional activity of IRF-3, however, were observed only after BRSV ΔNS1/2 infection. NS protein-mediated inhibition of IRF-3 activation and IFN induction should have considerable impact on the pathogenesis and immunogenicity of BRSV.
doi:10.1128/JVI.77.16.8661-8668.2003
PMCID: PMC167228  PMID: 12885884
21.  Role of Alpha/Beta Interferons in the Attenuation and Immunogenicity of Recombinant Bovine Respiratory Syncytial Viruses Lacking NS Proteins 
Journal of Virology  2003;77(15):8426-8439.
Alpha/beta interferons (IFN-α/β) are not only a powerful first line of defense against pathogens but also have potent immunomodulatory activities. Many viruses have developed mechanisms of subverting the IFN system to enhance their virulence. Previous studies have demonstrated that the nonstructural (NS) genes of bovine respiratory syncytial virus (BRSV) counteract the antiviral effects of IFN-α/β. Here we demonstrate that, in contrast to wild-type BRSVs, recombinant BRSVs (rBRSVs) lacking the NS proteins, and those lacking NS2 in particular, are strong inducers of IFN-α/β in bovine nasal fibroblasts and bronchoalveolar macrophages. Furthermore, whereas the NS deletion mutants replicated to wild-type rBRSV levels in cells lacking a functional IFN-α/β system, their replication was severely attenuated in IFN-competent cells and in young calves. These results suggest that the NS proteins block the induction of IFN-α/β gene expression and thereby increase the virulence of BRSV. Despite their poor replication in the respiratory tract of young calves, prior infection with virus lacking either the NS1 or the NS2 protein induced serum antibodies and protection against challenge with virulent BRSV. The greater level of protection induced by the NS2, than by the NS1, deletion mutant, was associated with higher BRSV-specific antibody titers and greater priming of BRSV-specific, IFN-γ-producing CD4+ T cells. Since there were no detectable differences in the ability of these mutants to replicate in the bovine respiratory tract, the greater immunogenicity of the NS2 deletion mutant may be associated with the greater ability of this virus to induce IFN-α/β.
doi:10.1128/JVI.77.15.8426-8439.2003
PMCID: PMC165239  PMID: 12857912
22.  Respiratory Syncytial Virus (RSV) Fusion Protein Subunit F2, Not Attachment Protein G, Determines the Specificity of RSV Infection 
Journal of Virology  2003;77(8):4609-4616.
Human respiratory syncytial virus (HRSV) and bovine RSV (BRSV) infect human beings and cattle in a species-specific manner. We have here analyzed the contribution of RSV envelope proteins to species-specific entry into cells. In contrast to permanent cell lines, primary cells of human or bovine origin, including differentiated respiratory epithelia, peripheral blood lymphocytes, and macrophages, showed a pronounced species-specific permissivity for HRSV and BRSV infection, respectively. Recombinant BRSV deletion mutants lacking either the small hydrophobic (SH) protein gene or both SH and the attachment glycoprotein (G) gene retained their specificity for bovine cells, whereas corresponding mutants carrying the HRSV F gene specifically infected human cells. To further narrow the responsible region of F, two reciprocal chimeric F constructs were assembled from BRSV and HRSV F1 and F2 subunits. The specificity of recombinant RSV carrying only the chimeric F proteins strictly correlated with the origin of the membrane-distal F2 domain. A contribution of G to the specificity of entry could be excluded after reintroduction of BRSV or HRSV G. Virus with F1 and G from BRSV and with only F2 from HRSV specifically infected human cells, whereas virus expressing F1 and G from HRSV and F2 from BRSV specifically infected bovine cells. The introduction of G enhanced the infectivities of both chimeric viruses to equal degrees. Thus, the role of the nominal attachment protein G is confined to facilitating infection in a non-species-specific manner, most probably by binding to cell surface glycosaminoglycans. The identification of the F2 subunit as the determinant of RSV host cell specificity facilitates identification of virus receptors and should allow for development of reagents specifically interfering with RSV entry.
doi:10.1128/JVI.77.8.4609-4616.2003
PMCID: PMC152164  PMID: 12663767
23.  Cleavage at the Furin Consensus Sequence RAR/KR109 and Presence of the Intervening Peptide of the Respiratory Syncytial Virus Fusion Protein Are Dispensable for Virus Replication in Cell Culture 
Journal of Virology  2002;76(18):9218-9224.
Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR136) and FCS-2 (RAR/KR109). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR109, RANN109 or SANN109, respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR109 or the RANN109 motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN109 mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.
doi:10.1128/JVI.76.18.9218-9224.2002
PMCID: PMC136468  PMID: 12186905
24.  Respiratory Syncytial Virus (RSV) Nonstructural (NS) Proteins as Host Range Determinants: a Chimeric Bovine RSV with NS Genes from Human RSV Is Attenuated in Interferon-Competent Bovine Cells 
Journal of Virology  2002;76(9):4287-4293.
Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-α/β) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-α, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.
doi:10.1128/JVI.76.9.4287-4293.2002
PMCID: PMC155099  PMID: 11932394
25.  Respiratory Syncytial Virus Fusion Protein Mediates Inhibition of Mitogen-Induced T-Cell Proliferation by Contact 
Journal of Virology  2002;76(3):1163-1170.
Human respiratory syncytial virus (HRSV) and bovine respiratory syncytial virus (BRSV) are major pathogens in infants and calves, respectively. Experimental BRSV infection of calves and lambs is associated with lymphopenia and a reduction in responsiveness of peripheral blood lymphocytes (PBLs) to mitogens ex vivo. In this report, we show that in vitro mitogen-induced proliferation of PBLs is inhibited after contact with RSV-infected and UV-inactivated cells or with cells expressing RSV envelope proteins on the cell surface. The protein responsible was identified as the RSV fusion protein (F), as cells infected with a recombinant RSV expressing F as the single envelope protein or cells transfected with a plasmid encoding F were able to induce this effect. Thus, direct contact with RSV F is necessary and sufficient to inhibit proliferation of PBLs. Interestingly, F derived from HRSV was more efficient in inhibiting human PBL proliferation, while F from BRSV was more efficient in inhibiting bovine PBLs. Since various T-cell activation markers were upregulated after presenter cell contact, T lymphocytes are viable and may still be activated by mitogen. However, a significant fraction of PBLs were delayed or defective in G0/G1 to S-phase transit.
doi:10.1128/JVI.76.3.1163-1170.2002
PMCID: PMC135829  PMID: 11773392

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