Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5′ triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5′ triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5′ monophosphate-specific exonuclease indicates that the RIG-I-activating 5′ triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5′ end as a strategy of viruses to evade the innate immune response.
Analysis of the composition and regulation of the Borna disease virus (BDV) polymerase complex has so far been limited by the lack of a functional assay. To establish such an assay on the basis of an artificial minigenome, we constructed expression vectors encoding either nucleoprotein (N), phosphoprotein (P), X protein, or polymerase (L) of BDV under the control of the chicken -actin promoter. A Flag-tagged version of L colocalized with virus-encoded N and P in characteristic nuclear dots of BDV-infected cells and increased viral N-protein levels in persistently infected Vero cells. Vector-driven expression of L, N, and P in BSR-T7 cells together with a negative-sense BDV minigenome carrying a chloramphenicol acetyltransferase (CAT) reporter gene resulted in efficient synthesis of CAT protein. Induction of CAT protein synthesis strongly depended on a 10-to 30-fold molar excess of the N-encoding plasmid over the P-encoding plasmid. Cotransfection of even small amounts of plasmid encoding the viral X protein reduced CAT synthesis to background levels. Thus, the N-to-P stoichiometry seems to play a central role in the regulation of the BDV polymerase complex. Our data further suggest a negative regulatory function for the X protein of BDV.Borna disease virus (BDV) is a neurotropic, enveloped virus with a nonsegmented negative-strand RNA genome (4). In naturally and experimentally infected animals, BDV establishes a noncytolytic, persistent infection of the central nervous system that frequently results in a severe immune-mediated neurological disorder (10). Successful experimental infection of a broad range of warm-blooded animals has been reported (25). Serological evidence suggests that BDV or a BDV-like virus infects humans (2).The compact BDV genome of approximately 8,900 nucleotides (nt) is transcribed and replicated in the nucleus and encodes at least six viral proteins organized in three transcription units (3,4,9). BDV employs a variety of strategies to regulate viral protein levels including overlapping open reading frames (ORFs), transcription units, and transcriptional signals, read-through of transcription termination signals, and alternative splicing of polycistronic mRNA (11,21,22). On the basis of these unique properties, BDV has been classified as the prototypic member of a new virus family, Bornaviridae, in the order Mononegavirales.Negative-strand RNA viruses initiate infection by introducing their genetic material in the form of ribonucleoprotein complexes into the host cells. These complexes, which consist of the single-stranded RNA genome associated with the RNAbinding protein nucleoprotein (N), the polymerase cofactor phosphoprotein (P), and the RNA-dependent RNA polymerase L, are transcriptionally active and direct the synthesis of viral mRNAs (8,17). Upon production of sufficient amounts of newly synthesized viral proteins, replication is initiated and new nucleocapsids can be formed. A consequence of this multiplication strategy is that, in contrast to the situation of positive-strand RNA...
De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the Mononegavirales with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the Mononegavirales with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5 termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.The group of negative-strand RNA viruses includes many important human and animal pathogens. Genetic manipulation and molecular analysis of these viruses are complicated by the fact that the naked viral RNA is not infectious. To generate the ribonucleoprotein complex (RNP), the minimal infectious unit of negative-strand RNA viruses, simultaneous expression of viral protein components forming the polymerase (Pol) complex and full-length viral antigenomic RNA (cRNA) from cDNA is required (3,14). During the past decade, such reverse genetics systems have been established for a variety of negative-strand RNA viruses, relying on different DNA-dependent RNA polymerases for the expression of viral cRNA. Negativestrand RNA viruses with a nuclear replication strategy, such as influenza A virus (15) and Thogoto virus (29), were recovered using cellular DNA-dependent RNA Pol I. Recovery of viruses with a cytoplasmic replication strategy, including most of the nonsegmented negative-strand RNA viruses (Mononegavirales) such as measles virus (MV) and rabies virus, relied on the DNA-dependent RNA polymerase from bacteriophage T7 (T7pol) supplied by either stably transfected helper cells or recombinant helper virus (3).Borna disease virus (BDV) contains a nonsegmented RNA genome of negative polarity. BDV replication is strictly noncytolytic and results in the persistent infection of neurons in the central nervous system (6). The genome codes for six genes and is replicated and transcribed in the nucleus (2, 4). Gene expression involves alternative splicing of two intron sequences (5) and results in a complex pattern of viral transcription and replication products. Based on these unique properties, BDV has been c...
Bornaviruses are nonsegmented negative-strand RNA viruses that establish a persistent infection in the nucleus and occasionally integrate a DNA genome copy into the host chromosomal DNA. However, how these viruses achieve intranuclear infection remains unclear. We show that Borna disease virus (BDV), a mammalian bornavirus, closely associates with the cellular chromosome to ensure intranuclear infection. BDV generates viral factories within the nucleus using host chromatin as a scaffold. In addition, the viral ribonucleoprotein (RNP) interacts directly with the host chromosome throughout the cell cycle, using core histones as a docking platform. HMGB1, a host chromatin-remodeling DNA architectural protein, is required to stabilize RNP on chromosomes and for efficient BDV RNA transcription in the nucleus. During metaphase, the association of RNP with mitotic chromosomes allows the viral RNA to segregate into daughter cells and ensure persistent infection. Thus, bornaviruses likely evolved a chromosome-dependent life cycle to achieve stable intranuclear infection.
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