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African swine fever virus threatens pig production worldwide due to the lack of vaccines, for which generation of both deletion and insertion mutants is considered. For development of the latter, operational ASFV promoters of different temporal regulation and strengths are desirable. We therefore compared the capacities of putative promoter sequences from p72, CD2v, p30, viral DNA polymerase and U104L genes to mediate expression of luciferase from transfected plasmids after activation in trans, or p30-, DNA polymerase- and U104L promoters in cis, using respective ASFV recombinants. We identified sequences with promoter activities upstream the viral ORFs, and showed that they differ in both their expression intensity regulating properties and in their temporal regulation. In summary, p30 and DNA polymerase promoters are recommended for high level early regulated transgene expression. For late expression, the p72, CD2v and U104L promoter are suitable. The latter however, only if low level transgene expression is aimed.

Although most non-segmented negative-strand RNA viruses (NNSVs) replicate in the cytoplasm, NNSV proteins often exert host manipulatory functions in the nucleus. Matrix (M) proteins of henipaviruses and other paramyxoviruses shuttle through the nucleus, where host factors may bind for M modification or host-cell manipulation. Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) is an interactor of Hendra and Nipah virus M. Both accumulate in the nucleus in an ANP32B-dependent manner. Here we demonstrate that the nuclear localization signal (NLS) of ANP32B is dispensable for HeV M binding. Specific purification of M-ANP32B but not of M-ANP32A complexes revealed that neither the negatively charged acidic nor the leucine-rich regions of ANP32 proteins per se mediate interactions with henipavirus M proteins. Whereas pneumovirus M did not interact with ANP32B, Newcastle disease virus (NDV, genus Avulavirus), Sendai virus (SeV, genus Respirovirus), Measles virus (MeV, genus Morbillivirus) and Canine distemper virus (CDV, genus Morbillivirus) M were able to form complexes with ANP32B. However, in contrast to NDV M and SeV M, which accumulated in the nucleus ANP32B dependently, both morbillivirus Ms did not accumulate in the nucleus, neither at ANP32B overexpression nor after nuclear protein export inhibition. These results indicate that intracellular compartmentalization of cytoplasmic morbillivirus M and nuclear ANP32B prevented an intracellular interaction. Overall, we provide evidence for a general ability of paramyxovirus M proteins to interact with ANP32B. This suggests a conserved, yet to be clarified mechanism might play a role in host manipulation and immune regulation in infected hosts.

Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCE Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors, and intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L), we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1 gene expression by both P and L. Finally, disruption of the motif in L resulted in a cell-type-specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step in virus replication. IMPORTANCE Regulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown, and it is also not known whether the large enzymatic polymerase subunit L is involved. By fluorescence microscopy analysis of fluorescence-tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and reorganization, primary transcription regulation by DLC1, and regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L, our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well.

The distal portion of chromosome 1p is frequently deleted in several human cancers, suggesting the presence of one or more putative tumor suppressor genes on this chromosomal arm. In human neuroblastoma, a consistently deleted region at 1p36.1-p36.2 has been defined by comparison of molecular loss of heterozygosity (LOH) analyses. Recently we described the identification of a yeast artificial chromosome, YAC 927G4, that spans a translocation/duplication breakpoint within the minimally defined LOH region at 1p36.1-p36.2 in the neuroblastoma cell line NGP. Here we describe the identification of two overlapping P1 artificial chromosomes comprising 220 kb at the distal end of YAC 927G4, which we have used as hybridization probes under modified conditions to screen a composite, normalized cDNA library (IMAGE cDNA library). Hybridization screening resulted in the rapid and comprehensive identification of partial cDNAs of which a portion comprised two novel candidate genes, termed DNB1/ARPh and DNB5, which encode putative proteins of 1011 and 447 amino acids, respectively. The DNB1/ARPh gene, which was found to be ubiquitously expressed in human adult and fetal tissues, is highly related to the DRPLA gene, in which expansion of a CAG triplet appears to be causal in the dentatorubral and pallidolysian atrophy disease phenotype. The DNB5 sequence, in contrast, which is predominantly expressed in brain tissues and fetal kidney, failed to show any similarity to sequences in the public domain. A preliminary assessment of transcription and sequence of both genes in several neuroblastoma cell lines does not, thus far, support a causal role in neuroblastoma. However, further analyses are required to confirm these results.

Membrane envelopment and budding of negative strand RNA viruses (NSVs) is mainly driven by viral matrix proteins (M). In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV) M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV) M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.

Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS., Author Summary Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is unknown. Here we use live cell imaging to track RABV entry at nerve terminals and study its retrograde transport along the axon. First, we demonstrate that RABV interacts with the p75 neurotrophin receptor (p75NTR) at peripheral neuron tips to enter the axon. Then, characterizing RABV retrograde transport along the axon, we showed that the virus moves in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, an endogenous p75NTR ligand. Finally, we determine that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. Hence, RABV not only exploits the neurotrophin transport machinery, but also has a positive influence on transport kinetics, thus facilitating its own arrival at the CNS.

In human neuroblastomas, the distal portion of 1p is frequently deleted, as if one or more tumor suppressor genes from this region were involved in neuroblastoma tumorigenesis. Earlier studies had identified a smallest region of overlapping deletion (SRO) spanning approximately 23 cM between the most distally retained D1S80 and by the proximally retained D1S244. In pursuit of generating a refined delineation of the minimally deleted region, we have analyzed 49 neuroblastomas of different stages for loss of heterozygosity (LOH) from 1pter to 1p35 by employing 26 simple sequence length polymorphisms. Fifteen of the 49 tumors (31%) had LOH; homozygous deletion was not detected. Seven tumors had LOH at all informative loci analyzed, and eight tumors showed a terminal or an interstitial allelic loss of 1p. One small terminal and one interstitial deletion defined a new 1.7 cM SRO, approximately 1 Mbp in physical length, deleted in all tumors between the retained D1S2731 (distal) and D1S2666 (proximal). To determine the genomic complexity of the deleted region shared among tumors, we assembled a physical map of the I Mbp SRO consisting predominantly of bacteriophage P1-derived artificial chromosome (PAC) clones. A total of 55 sequence-tagged site (STS) markers (23 published STSs and short tandem repeats and 32 newly identified STSs from the insert ends of PACs and cosmids) were assembled in a contig, resulting in a sequence-ready physical map with approximately one STS per 20 Kbp. Twelve genes (41BB, CD30, DFFA, DJ1, DR3, FRAP, HKR3, MASP2, MTHFR, RIZ, TNR2, TP73) previously mapped to 1p36 are localized outside this SRO. On the basis of this study, they would be excluded as candidate genes for neuroblastoma tumorigenesis. Ten expressed sequence tags were integrated in the contig, of which five are located outside the SRO. The other five from within the SRO may provide an entrance point for the cloning of candidate genes for neuroblastoma.