Abstract
Bornaviruses (e.g., Borna disease virus, BDV; Borna disease virus 1, BoDV-1) are nonsegmented, negative-sense single-stranded RNA viruses whose most distinctive feature among animal nonsegmented negative-strand RNA (NNS-RNA) viruses is that genome transcription and replication occur in the host cell nucleus rather than predominantly in the cytoplasm[1–3]. Molecular studies of BDV have defined a compact ~8.9–9 kb genome with multiple open reading frames organized into a small number of transcription units and flanked by extracistronic terminal sequences that function as promoters and regulatory boundaries for viral RNA synthesis[4–8]. Gene expression is complex and includes production of both mono- and polycistronic poly(A)+ RNAs, frequent readthrough at termination sites, and use of splicing for expression of key products including the polymerase (L) in some transcript contexts[4–6, 9, 10]. Functional reconstitution and minigenome systems have mapped cis-acting promoter requirements in the 3′ genomic end and shown that N and P encapsidate templates recognized by the L/P polymerase complex to generate both replicative and transcript products[11, 12]. Nuclear organization and trafficking further shape the replication program, with viral “speckles of transcripts” (vSPOTs) and CRM1-dependent export of nucleoprotein contributing to RNP dynamics and compartmentalization of poly(A)+ versus poly(A)− viral RNAs[3, 13–15].
Keywords
- Bornavirus[1]
- Borna disease virus[4]
- nonsegmented negative-strand RNA virus[4]
- nuclear replication[2]
- transcription readthrough[5]
- vSPOTs[3]
- minigenome[11]
- CRM1 export[15]
- phosphoprotein P[15]
- RNA-dependent RNA polymerase L[16]
Introduction
Bornaviruses are NNS-RNA viruses whose promoter and gene-start consensus sequences align with patterns observed across Mononegavirales families, indicating a shared negative-strand transcription logic at the level of cis-acting initiation elements[4, 17]. A central mechanistic distinction is that, while most mononegaviruses mainly replicate in the cytoplasm, bornaviruses replicate in the nucleus, where they establish specialized nuclear sites for viral RNA synthesis[1, 3]. Experimental evidence specifically supports that BDV replication and transcription take place in the nuclei of infected cells and are associated with infectious ribonucleoprotein complexes (BDV-RNPs), emphasizing that bornaviral RNA synthesis is executed in the context of nuclear RNPs[4, 13]. Nuclear localization is further supported by cell fractionation showing that most newly synthesized genomic-polarity BDV RNA is poly(A)− and found largely in the nuclear fraction, consistent with nuclear replication of the genomic RNA[13].
Genome organization
BDV genome sequencing and mapping identified a linear ~8.9 kb genome with major open reading frames predicted across the genome and flanked by noncoding sequences at both termini[4, 5]. In one report, five major ORFs (I–V) were predicted in an 8,903-nt BDV genome sequence, while another description of the BDV genome (8,910 nt) similarly noted antisense information for five major ORFs flanked by 53 nt of noncoding sequence at the 3′ terminus and 91 nt at the 5′ terminus[4, 5]. In addition to the five-ORF framing, antigenome-level mapping has described three transcription units and six ORFs, and review-level summaries likewise describe BDV as encoding six ORFs in three transcription units framed by complementary termini reminiscent of other NNS RNA viruses[6, 7].
The largest coding region (ORF V) encodes a predicted ~170 kDa protein with strong homology to the L-protein family of NNS-RNA virus polymerases, placing the viral RNA-dependent RNA polymerase at the 5′-proximal end of the gene set in the canonical negative-strand layout[4]. Conserved sequence analysis identified highest homology in a putative catalytic domain with invariant and conservatively maintained residues clustered into four highly conserved motifs (A–D), consistent with conserved enzymatic constraints on polymerase function among NNS-RNA viruses[18]. Infectious BDV-RNPs have been reported to contain only one BDV RNA species, the poly(A)−, negative-polarity 9-kb RNA, supporting that the genome-length poly(A)− RNA is the encapsidated template species in infectious RNPs[13].
Bornavirus genomes also carry regulatory boundary regions typical of NNS-RNA viruses, with ORFs flanked by nontranslated boundary sequences that include transcription initiation and stop signals[8]. Terminal sequences can base-pair to form a panhandle-like structure, and in BDV the alignment of genomic termini allowed formation of a terminal panhandle with the first 3 nucleotides unpaired, linking terminal complementarity to promoter architecture without requiring perfect duplexing[5]. Importantly, analyses of BDV genomic termini reported both lack of perfect terminal complementarity and increased terminal sequence heterogeneity during long-term persistence, indicating that cis-acting terminal regions are variable yet functionally tolerated across infection states[11].
To concisely summarize genome and gene-expression architecture as described in these sources, the table below contrasts several frequently cited organizational features.
Transcription and gene expression
Bornavirus gene expression in BDV includes multiple polyadenylated subgenomic RNAs complementary to the negative-sense genome, with one study identifying nine species of polyadenylated subgenomic RNAs, including six polycistronic poly(A)+ RNAs and monocistronic mRNAs corresponding to ORFs I, II, and IV[4]. In the same analysis, monocistronic poly(A)+ RNAs for ORFs III and V were not detected, indicating that expression of some coding regions is not dominated by simple monocistronic messages in BDV[4]. Northern analyses also showed that BDV transcribes both mono- and polycistronic RNAs and uses termination/polyadenylation signals reminiscent of other negative-strand RNA viruses, consistent with a stop–start transcriptional program that nevertheless yields frequent transcript continuity beyond termination sites[5].
Termination and polyadenylation involve discrete sites and a recognizable signal. Northern hybridization experiments supported the use of specific termination sites (T2, T3, T5, and T7) and identified a termination/polyadenylation signal consensus sequence, providing molecular landmarks for transcript boundaries and readthrough propensity[5]. BDV is also notable for a high frequency of readthrough transcripts relative to other negative-strand RNA viruses, implying that termination efficiency is systematically tuned and can be mechanistically essential[5]. Indeed, readthrough of T3 is essential for expression of p190 (polymerase protein), directly linking termination suppression to polymerase availability and thus to the capacity for replication and transcription[9].
BDV mRNAs are capped and polyadenylated, demonstrating that mRNA maturation is consistent with production of translation-competent RNAs despite the nuclear replication niche[19]. Additional coding diversity is generated by splicing, as the 2.8-kb and 7.1-kb RNAs contain two introns that are differentially spliced to yield RNAs encoding multiple products including G and polymerase-related proteins, and separate statements indicate that L expression requires splicing and suppression of termination[6, 10]. At the level of initiation and promoter structure, perfect terminal complementarity does not appear to be required for high promoter activity, and increased terminal complementarity did not promote replication versus transcription by the BDV polymerase, indicating that the replication–transcription balance is not simply a function of terminal base-pairing strength[11].
Replication cycle overview
Bornavirus replication is defined by nuclear RNA synthesis and nuclear organization of viral RNPs. Multiple sources provide evidence that BDV replication and transcription occur in the nucleus in association with infectious BDV-RNPs, establishing that the functional template for RNA synthesis is an RNP complex operating in the nuclear environment[4, 13]. Quantitative fractionation and labeling experiments further showed that most newly synthesized BDV genomic-polarity RNA is poly(A)− and nuclear, supporting the conclusion that genome replication takes place in the nucleus of infected cells[13].
A consistent compartmentalization pattern emerges for different viral RNA classes. The BDV 9-kb poly(A)− RNA is largely nuclear, while newly synthesized poly(A)+ RNAs are transported to the cytoplasmic compartment, indicating that mRNA export and genome retention are separated across the nuclear envelope[13, 14]. Transport of BDV mRNAs has been shown to be energy-dependent, implying active export rather than passive diffusion as a key control point for gene expression in nuclear-bornavirus infection[20].
Bornaviruses also assemble nuclear viral factories described as “viral Speckles Of Transcripts” (vSPOTs), providing a structural context for concentrated transcription/replication and potentially coordinated RNA processing in the nucleus[1]. In BoDV-1, vSPOTs formed by P protein-driven liquid–liquid phase separation are present in the nucleus and interact closely with chromatin, including docking on neuronal DNA double-strand breaks, linking replication sites to specific nuclear substructures[3]. Consistent with the centrality of RNPs, BDV-RNP complexes containing only the 9-kb RNA species have been reported to possess polymerase activity required for transcription and to carry the genetic information needed to direct synthesis of BDV macromolecules and production of infectious BDV particles, connecting genome-length RNPs to downstream steps in the viral life cycle[21].
RNP and polymerase machinery
The BDV genome-length RNA is packaged into RNP complexes that contain N and the viral RNA-dependent RNA polymerase complex, defining the core machinery as a nucleocapsid template bound by the polymerase complex[15]. The RdRp complex consists of P and L and is responsible for replication and transcription of the viral genome, establishing L as the catalytic subunit and P as an essential polymerase cofactor in BDV[15]. Structural and functional descriptions further define L as a ~192 kDa RdRp forming the core of the replication complex, performing transcription and replication to produce viral mRNA and new genome copies, and associating with P for efficient RNA synthesis[16].
Protein interaction properties of P provide mechanistic insight into polymerase function. P self-associates in oligomers through a central oligomerization domain and bridges the RdRp and nucleocapsid, and it also chaperones N to maintain it in an RNA-free form needed for replication, supporting a model in which P coordinates both enzyme recruitment and template readiness[3]. Consistently, experimental disruption of P oligomerization in a minireplicon assay addressed whether oligomerization-defective P mutants could reconstitute functional polymerase complexes, and none of the P mutants supported reporter expression, indicating that P oligomerization is required for polymerase activity under these conditions[22]. In addition, the cofactor activity of P for BDV RdRp is negatively regulated by phosphorylation, providing an explicit post-translational regulatory lever for polymerase function[15].
Bornavirus nucleoprotein isoforms also connect protein function to intracellular localization. Experiments expressing p40 and p38 showed that p40 is primarily nuclear whereas p38 is primarily cytoplasmic, yet both bind the BDV phosphoprotein P, suggesting that isoform-dependent localization coupled with P binding may modulate where RNP assembly and/or function occurs[9]. Consistent with a nuclear targeting role for P, P contains a strong bipartite nuclear localization signal (NLS) at its amino-terminus and additional weaker NLS motifs, supporting nuclear import of polymerase-containing complexes as a mechanistic prerequisite for nuclear RNA synthesis[9].
Finally, the accessory protein X can directly down-modulate RNA synthesis in reconstituted systems. When BDV polymerase complexes were reconstituted in cells expressing X protein, no plus-strand minigenome RNA was detected, suggesting that X inhibits both viral transcription and replication in that assay context[23]. These observations together support a machinery model in which L and P form the catalytic core, P oligomerization and phosphorylation regulate polymerase competence, and accessory factors such as X can suppress polymerase output under defined conditions[15, 23].
Nuclear trafficking
Bornaviruses couple nuclear replication with regulated nucleocytoplasmic trafficking of RNP components and RNA species. Direct evidence indicates that BDV replication and transcription take place in nuclei where infectious BDV RNPs are present, and fractionation indicates that newly synthesized genomic-polarity RNA is strongly enriched in the nuclear fraction, providing a functional impetus for nuclear import and export pathways to coordinate the life cycle[13, 21]. RNA transport assays indicate that newly synthesized BDV poly(A)+ RNAs are efficiently transported to the cytoplasmic compartment while the 9-kb genomic RNA remains mostly nuclear, demonstrating RNA-class-specific export behavior[14]. Moreover, mRNA transport is energy-dependent, with negligible export in the absence of ATP, supporting active, host-factor-dependent export mechanisms for viral mRNAs[20].
For protein trafficking, CRM1-dependent export is implicated for nucleoprotein. The nuclear export of N is via a CRM1-dependent pathway consistent with an NES, and a separate statement similarly reports that nuclear export of BoDV-N occurs via a CRM1-dependent pathway, indicating conservation of this export route within bornaviruses[15, 24]. Broader reviews further state that several BDV proteins (including nucleoprotein, phosphoprotein, X, and L) contribute to nucleocytoplasmic trafficking of BDV RNP, and that directional control is likely determined by the ratios and interactions between NLS and NES elements in the RNP, framing trafficking as an emergent property of competing localization signals rather than a single determinant[25].
Nuclear viral factory formation provides an additional trafficking-relevant organizational level. Bornaviruses assemble vSPOTs in the nucleus, and BoDV-1 vSPOTs are formed by P protein-driven liquid–liquid phase separation and interact closely with chromatin, which can constrain diffusion and potentially direct the positioning of transcription/replication sites relative to host nuclear landmarks[1, 3].
Reverse genetics and promoter elements
Bornavirus cis-acting regulatory signals are concentrated in nontranslated boundary and terminal regions. In BDV, ORFs are flanked by nontranslated boundary sequences containing regulatory signals for transcription initiation and stop, consistent with a Mononegavirales-like architecture of gene-start and gene-end signals guiding polymerase behavior along the genome[8]. A deduced BDV start consensus sequence (UNCNNNUUNN) is identical to that inferred by comparing NNS-RNA viruses across multiple Mononegavirales families, supporting conservation of the initiation motif used by the polymerase machinery[4, 17]. Termination/polyadenylation signals include a conserved AUUUUU six-mer at the 5′ end of each termination/polyadenylation signal followed by GG (or CG in ORF II), while putative signals could not be identified for ORF III in one analysis, indicating both conservation and gaps in motif detectability across gene boundaries[4].
Reverse-genetics and minigenome approaches have directly tested these regulatory elements. An RNA polymerase I/polymerase II system was established for intracellular reconstitution of BDV RNA replication and transcription, enabling controlled dissection of cis- and trans-acting requirements[11]. In this system, a BDV RNA analog (minigenome) is synthesized by cellular RNA polymerase I and contains BDV 5′ and 3′ untranslated cis-acting sequences required for BDV polymerase-mediated RNA synthesis, linking terminal regions to promoter function in cells[11]. Encapsidation of polymerase I-derived minigenome RNA by plasmid-supplied BDV N and P generates a template recognized by the reconstituted BDV polymerase to direct synthesis of full-length antiminigenome RNA (replication) and a subgenomic mRNA encoding a reporter gene, experimentally demonstrating that N and P encapsidation is sufficient to render the RNA competent for polymerase recognition and dual-output RNA synthesis[11].
Promoter mapping further refines the cis-acting model. Downstream sequences (nucleotides 25 to 33) were required for optimal promoter activity, and deletion of nucleotides 34 to 35 abrogated reporter activity in this context, identifying short, position-specific promoter-proximal requirements in the 3′ genomic promoter region[12]. Together, these data support a promoter architecture in which compact terminal sequences and nearby downstream elements govern initiation and productive replication/transcription in the reconstituted polymerase system[11, 12].
Persistence mechanisms
Bornavirus molecular persistence is linked to genome-end dynamics, nuclear replication, and specialized nuclear organization. A “realign-and-elongation” process renews the 3′ termini of v- and cRNA molecules from internal templates during each round of replication and can only occur if the termini are complete, providing an explicit mechanistic step that acts as a quality-control checkpoint for genome integrity[26]. This process provides integrated quality control that suppresses replication of RNA molecules with terminal deletions, connecting end-repair logic to selection against defective replication intermediates[26]. In parallel, analyses of genomic termini during long-term persistent infection reported a higher degree of terminal heterogeneity in RNAs from persistently infected cells compared to acute time points, indicating that persistence is accompanied by shifts in the distribution of genome-end variants[11].
Persistence is also associated with stable nuclear context for RNPs. Bornaviruses have been described as uniquely among known animal NNS RNA viruses replicating and transcribing in the nucleus, which provides the cellular compartmental setting for long-term noncytolytic RNA synthesis and nuclear genome-end processing[2]. In addition, BoDV-1 constructs its vRNPs using host chromatin as a scaffold, a property that can mechanistically support persistence in the nucleus by stabilizing vRNP positioning relative to chromatin[27].
Host-response modulation has also been experimentally linked to infection states. BDV-infected cells secreted less SEAP than mock cells after Pam3CSK4 activation, suggesting that BDV-infected cells actively suppress NF-κB signaling, which provides a molecular-level correlate for altered innate signaling during persistent infection[28].
Open questions
Several molecular questions remain open or are positioned for targeted experimentation based on the mechanistic findings summarized above.
- What sequence and structural determinants control BDV termination efficiency and the high frequency of readthrough transcripts, particularly at T3 where readthrough is essential for polymerase expression[5, 9]?
- How do alternative splicing events in the 2.8-kb and 7.1-kb RNAs interface with transcriptional boundary usage to yield correct stoichiometries of G and polymerase-related products, including cases where L expression requires splicing and suppression of termination[6, 10]?
- What is the quantitative relationship between terminal heterogeneity observed during persistence and promoter activity, given that perfect terminal complementarity is not required for high promoter activity and termini diversify during long-term infection[11]?
- How is the realign-and-elongation quality-control step coordinated with nuclear RNP organization, given that 3′ termini renewal derives from internal templates and suppresses replication of terminally deleted RNAs[26]?
- Which host factors and nuclear landmarks govern the formation and positioning of P-driven phase-separated vSPOTs that interact with chromatin and dock on neuronal DNA double-strand breaks[3]?
- How do CRM1-dependent export of nucleoprotein and energy-dependent mRNA export integrate to regulate the compartmentalization of poly(A)+ RNAs versus poly(A)− genomic RNA across the nuclear envelope[13, 15, 20]?
- By what molecular mechanism does X protein suppress minigenome RNA accumulation, and how does this suppression intersect with P phosphorylation-dependent negative regulation of polymerase cofactor activity[15, 23]?
