Functional characterization of the genomic promoter of borna disease virus (BDV): implications of 3'-terminal sequence heterogeneity for BDV persistence

Borna disease virus (BDV) is an enveloped virus with a genome organization characteristic of Mononegavirales. However, based on its unique features, BDV is considered the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. We have described the establishment of a reverse genetics system for the rescue of BDV RNA analogues, or minigenomes, that is based on the use of polymerase I/polymerase II. Using this BDV minigenome rescue system, we have examined the functional implications of the reported sequence heterogeneity found at the 5' and 3' termini of the BDV genome and also defined the minimal BDV genomic promoter within the 3'-terminal 25 nucleotides. Our results suggest that the accumulation of RNA genome species containing truncations of one to three nucleotides at their 3' termini may contribute to modulate BDV RNA replication and gene expression during long-term persistence.     

Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus

The nonsegmented negative-strand RNA viruses (order Mononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3' end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.     

Detecting remote sequence homology in disordered proteins: discovery of conserved motifs in the N-termini of Mononegavirales phosphoproteins

Paramyxovirinae are a large group of viruses that includes measles virus and parainfluenza viruses. The viral Phosphoprotein (P) plays a central role in viral replication. It is composed of a highly variable, disordered N-terminus and a conserved C-terminus. A second viral protein alternatively expressed, the V protein, also contains the N-terminus of P, fused to a zinc finger. We suspected that, despite their high variability, the N-termini of P/V might all be homologous; however, using standard approaches, we could previously identify sequence conservation only in some Paramyxovirinae. We now compared the N-termini using sensitive sequence similarity search programs, able to detect residual similarities unnoticeable by conventional approaches. We discovered that all Paramyxovirinae share a short sequence motif in their first 40 amino acids, which we called soyuz1. Despite its short length (11-16aa), several arguments allow us to conclude that soyuz1 probably evolved by homologous descent, unlike linear motifs. Conservation across such evolutionary distances suggests that soyuz1 plays a crucial role and experimental data suggest that it binds the viral nucleoprotein to prevent its illegitimate self-assembly. In some Paramyxovirinae, the N-terminus of P/V contains a second motif, soyuz2, which might play a role in blocking interferon signaling. Finally, we discovered that the P of related Mononegavirales contain similarly overlooked motifs in their N-termini, and that their C-termini share a previously unnoticed structural similarity suggesting a common origin. Our results suggest several testable hypotheses regarding the replication of Mononegavirales and suggest that disordered regions with little overall sequence similarity, common in viral and eukaryotic proteins, might contain currently overlooked motifs (intermediate in length between linear motifs and disordered domains) that could be detected simply by comparing orthologous proteins.     

Filoviruses as emerging pathogens

Of all the animal virus families that cause disease in man least is known about members of Filoviridae. This family, which contains Marburg and Ebola viruses, is one member of the order Mononegavirales, RNA viruses with a single negative genomic strand. The natural history of the filoviruses is unknown, but they have on occasion caused small but lethal monkey or human epidemics. Each outbreak has been contained by increasing the precautions taken during contact with sick humans or non-human primates. Nevertheless, because of our ignorance of the genetics and natural history of these viruses, their established high pathogenicity for man and other primates, and their potential for causing aerosol infections, they continue to be of considerable concern for biomedical science.     

Borna disease virus nucleoprotein requires both nuclear localization and export activities for viral nucleocytoplasmic shuttling

Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the order Mononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.     

The 24-angstrom structure of respiratory syncytial virus nucleocapsid protein-RNA decameric rings

Respiratory syncytial virus (RSV), a nonsegmented, negative-sense RNA-containing virus, is a common cause of lower respiratory tract disease. Expression of RSV nucleocapsid protein (N) in insect cells using the baculovirus expression system leads to the formation of N-RNA complexes that are morphologically indistinguishable from viral nucleocapsids. When imaged in an electron microscope, three distinct types of structures were observed: tightly wound short-pitch helices, highly extended helices, and rings. Negative stain images of N-RNA rings were used to calculate a three-dimensional reconstruction at 24 A resolution, revealing features similar to those observed in nucleocapsids from other viruses of the order Mononegavirales. The reconstructed N-RNA rings comprise 10 N monomers and have an external radius of 83 A and an internal radius of 40 A. Comparison of this structure with crystallographic data from rabies virus and vesicular stomatitis virus N-RNA rings reveals striking morphological similarities.     

Negative-sense RNA viruses: An underexplored platform for examining virus–host lipid interactions

Viruses are pathogenic agents that can infect all varieties of organisms, including plants, animals, and humans. These microscopic particles are genetically simple as they encode a limited number of proteins that undertake a wide range of functions. While structurally distinct, viruses often share common characteristics that have evolved to aid in their infectious life cycles. A commonly underappreciated characteristic of many deadly viruses is a lipid envelope that surrounds their protein and genetic contents. Notably, the lipid envelope is formed from the host cell the virus infects. Lipid-enveloped viruses comprise a diverse range of pathogenic viruses, which often lead to high fatality rates and many lack effective therapeutics and/or vaccines. This perspective primarily focuses on the negative-sense RNA viruses from the order Mononegavirales, which obtain their lipid envelope from the host plasma membrane. Specifically, the perspective highlights the common themes of host cell lipid and membrane biology necessary for virus replication, assembly, and budding.     

Negri bodies and other virus membrane-less replication compartments

Viruses reshape the organization of the cell interior to achieve different steps of their cellular cycle. Particularly, viral replication and assembly often take place in viral factories where specific viral and cellular proteins as well as nucleic acids concentrate. Viral factories can be either membrane-delimited or devoid of any cellular membranes. In the latter case, they are referred as membrane-less replication compartments. The most emblematic ones are the Negri bodies, which are inclusion bodies that constitute the hallmark of rabies virus infection. Interestingly, Negri bodies and several other viral replication compartments have been shown to arise from a liquid-liquid phase separation process and, thus, constitute a new class of liquid organelles. This is a paradigm shift in the field of virus replication. Here, we review the different aspects of membrane-less virus replication compartments with a focus on the Mononegavirales order and discuss their interactions with the host cell machineries and the cytoskeleton. We particularly examine the interplay between viral factories and the cellular innate immune response, of which several components also form membrane-less condensates in infected cells.     

Open Reading Frame III of Borna Disease Virus Encodes a Nonglycosylated Matrix Protein

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.     

Characterization of conserved viral leader RNA sequences that stimulate innate immunity through TLRs

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.