Karl-Klaus Conzelmann

Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons

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.

Infectious rabies viruses from cloned cDNA.

The generation of infectious rabies virus (RV), a non‐segmented negative‐stranded RNA virus of the Rhabdoviridae family, entirely from cloned cDNA is described. Simultaneous intracellular expression of genetically marked full‐length RV antigenome‐like T7 RNA polymerase transcripts and RV N, P and L proteins from transfected plasmids resulted in formation of transcriptionally active nucleocapsids and subsequent assembly and budding of infectious rabies virions. In addition to authentic RV, two novel infectious RVs characterized by predicted transcription patterns were recovered from modified cDNA. Deletion of the entire non‐translated pseudogene region, which is conserved in all naturally occurring RVs, did not impair propagation of the resulting virus in cell culture. This indicates that non‐essential genetic material might be present in the genomes of non‐segmented RNA viruses. The introduction of a functional extra cistron border into the genome of another virus resulted in the transcription of an additional polyadenylated mRNA containing pseudogene sequences. The possibility of manipulating the RV genome by recombinant DNA techniques using the described procedure‐‐potentially applicable also for other negative‐stranded viruses‐‐greatly facilitates the investigation of RV genetics, virus‐host interactions and rabies pathogenesis and provides a tool for the design of new generations of live vaccines.

Molecular Characterization of Porcine Reproductive and Respiratory Syndrome Virus, a Member of the Arterivirus Group

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV)-specific cDNA clones spanning the 3′ terminal 5 kb of the genomic RNA were isolated, sequenced, and used as probes for identification of PRRSV-specific RNAs. The PRRSV genome is a positive-stranded polyadenylated RNA of about 15 kb. In infected cells, a 3′ coterminal nested set of six major subgenomic mRNAs could be demonstrated. Within the 3′ terminal 3.5 kb of the PRRSV genome, six overlapping reading frames (ORFs) were identified, each most likely expressed by one of the subgenomic mRNAs. Amino acid sequence comparisons revealed that the most 3′ terminal ORF (ORF7) encodes the PRRSV nucleocapsid protein with a calculated molecular weight of 14 kDa. It displays 44.8% amino acid identity with the capsid protein of lactate dehydrogenase-elevating virus (LDV) and 23.6% with that of equine arteritis virus (EAV). The product of ORF6, the second 3′ terminal ORF, represents a putative membrane protein and exhibits 53.2 and 27.2% amino acid identity with the corresponding LDV and EAV polypeptides, respectively. Similar to EAV, ORFs 2 through 5 might encode glycosylated viral proteins. The polypeptide deduced from the most 5′ ORF (ORF1b) contains two conserved domains common to EAV and coronavirus polymerases. Genome organization, strategy of gene expression, and the sequence of deduced proteins show that PRRSV belongs to the Arterivirus group of viruses.

Retrograde neuronal tracing with a deletion-mutant rabies virus

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.

Identification of the Rabies Virus Alpha/Beta Interferon Antagonist: Phosphoprotein P Interferes with Phosphorylation of Interferon Regulatory Factor 3

ABSTRACT 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.

Bovine Respiratory Syncytial Virus Nonstructural Proteins NS1 and NS2 Cooperatively Antagonize Alpha/Beta Interferon-Induced Antiviral Response

ABSTRACT The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-α) receptor. Treatment of cells with recombinant universal IFN-α A/D or IFN-β revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-α/β mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.

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

ABSTRACT 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.

Budding of Rabies Virus Particles in the Absence of the Spike Glycoprotein

Budding of enveloped viruses from cellular membranes is believed to de pend on the presence of transmembrane spike proteins interacting with cytoplasmic virus components. To address the mechanism of rhabdovirus budding, we generated rabies virus mutants deficient for the glycoprotein G or the G cytoplasmic tail. We found that spikeless rhabdovirus particles were released from cells infected with the G-deficient mutant, demonstrating that a viral surface protein is not required to drive the budding process. However, particle production is enhanced approximately 6-fold and 30-fold in the presence of tailless G or G, respectively. This reveals that G also possesses an intrinsic and independent exocytosis activity. We propose a model according to which efficient budding of rhabdoviruses is achieved by a concerted action of both core and spike proteins.

Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes.

Protocols to recover negative-stand RNA viruses entirely from cDNA have been established in recent years, opening up this virus group to the detailed analysis of molecular genetics and virus biology. The unique gene-expression strategy of nonsegmented negative-strand RNA viruses, which involves replication of ribonucleoprotein complexes and sequential synthesis of free mRNAs, has also allowed the use of these viruses to express heterologous sequences. There are advantages in terms of easy manipulation of constructs, high capacity for foreign sequences, genetically stable expression, and the possibility of adjusting expression levels. Fascinating prospects for biomedical applications and transient gene therapy are offered by chimeric virus vectors carrying novel envelope protein genes and targeted to defined host cells.

Replication-Dependent Potent IFN-α Induction in Human Plasmacytoid Dendritic Cells by a Single-Stranded RNA Virus

Plasmacytoid dendritic cells sense viral ssRNA or its degradation products via TLR7/8 and CpG motifs within viral DNA via TLR9. Although these two endosomal pathways operate independently of viral replication, little is known about the detection of actively replicating viruses in plasmacytoid dendritic cell (PDC). Replication and transcription of the viral genome of ssRNA viruses as well as many DNA viruses lead to the formation of cytosolic dsRNA absent in noninfected cells. In this study, we used human respiratory syncytial virus (HRSV) encoding a fusion (F) protein for direct cytosolic entry. Both HRSV infection and cytosolic delivery of a 65-nt dsRNA led to potent IFN-α induction in PDC, but not in myeloid dendritic cells. Inactivation of HRSV by UV irradiation abrogated IFN-α induction in PDC. The comparison of two respiratory syncytial virus (RSV) constructs carrying either the HRSV or the bovine RSV F protein revealed that F-mediated cytosolic entry of RSV was absolutely required for IFN-α induction in PDC. HRSV-induced IFN-α production was independent of endosomal acidification and of protein kinase R (PKR) kinase activity, as demonstrated with chloroquine and the PKR inhibitor 2-aminopurine, respectively. In contrast, the induction of IFN-α by the TLR7/8 ligand R848, by the TLR9 ligand CpG-A ODN 2216, and by inactivated influenza virus (TLR7/8 dependent) was completely blocked by 2-aminopurine. IFN-α induction by mouse pathogenic Sendai virus was not affected in PKR- and MyD88-deficient mice, confirming that a ssRNA virus, which is able to directly enter host cells via fusion at the plasma membrane, can be detected by PDC independently of PKR, TLR7/8, and TLR9.