Several residues within the N-terminal arm of vesicular stomatitis virus nucleoprotein play a critical role in protecting viral RNA from nuclease digestion

Chen, Longyun; Yan, Qin; Lu, Guangquan; Hu, Zhulong; Zhang, Guangyuan; Zhang, Shengwei; Ding, Beichen; Jiang, Yanliang; Zhong, Yi; Gong, Peng; Chen, Mingzhou

doi:10.1016/j.virol.2015.01.021

Cited by 11 publications

(7 citation statements)

References 40 publications

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“…The SFV replicon and helper plasmids were kindly donated by Dr. Markus U. Ehrengruber, Department of Biology, Kantonsschule Hohe Promenade, Zurich, Switzerland. The mutated vesicular stomatitis virus rVSV-EGFP-NR7A were prepared as described previously 33,34 .…”

Section: Methodsmentioning

confidence: 99%

Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution

Wang

Zhu

Ding

et al. 2017

Preprint

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2We describe a new light-sheet microscopy method for fast, large-scale and imaging methods were developed to overcome this problem [9][10][11][12][13][14][15] . Recent 50 advances along this line was able to push the imaging speed to ~3 days per 51 . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/240770 doi: bioRxiv preprint first posted online Dec. 29, 2017; 3 mouse brain at synaptic resolution 14 , or ~2.4 hours per mouse brain but at a 52 reduced resolution 15 . Still, it is highly desirable to be able to obtain high 53 resolution images at very high speed for high volume tasks such as whole 54 brain mapping of many animals or of large primates. 56The past decade has also seen various tissue clearing techniques that 57 greatly improve the transparency of brain samples 16 The basic configuration of our system consists of an illumination objective 91 through which a scanning light-beam enters a thick slice sample at a 45-92 degree angle to the sample surface, and an imaging objective positioned 93 perpendicular to the illumination plane (Fig. 1a,b (Supplementary Fig. 1). The geometry and working distance of 98 the objectives constrain the imageable sample thickness to ~300 µm (Fig. 1b). 99The light path is configured such that the effective imaging area on the object 100 plane (corresponding to half of the camera sensor, ~1000 X 2000 pixels) 101covers an oblique section of ~420 µm X 1000 µm using a 20X imaging 102 objective with system magnification reduced to ~13X (Fig. 1c). 104To achieve fast volumetric imaging over a large range, we configured the (Fig. 1c, d). This approach has also been used in the we eliminate virtually all motion blur and achieve high image quality 118 indistinguishable from stationary imaging, with dendritic spines of cortical 119 neurons clearly visible ( Fig. 1e-g). In principle, motion blur can also be 120 reduced using strobe light illumination in a regular light-sheet configuration. 121This however requires much higher light power to achieve similar illumination. 122The synchronized beam scan is thus also optimized in light efficiency. Supplementary Fig. 2a). 134We also developed a system to automatically detect the edges of all mounted 135 brain slices, so that the imaging system can directly move to the target based color VISoR imaging and cell counting in different brain areas (Fig. 3). In the 174 paraventricular nucleus (PVN) of hypothalamus where CRH neurons were 175 observed to form a dense cluster (Fig. 3a,b), more than half of these neurons (Fig. 3b,c, Supplementary Video 6). This is consistent with the role of CRH 180 neurons of the PVN in stress response 28 . Interestingly, in the medial 181 amygdala (MeA) where CRH neurons were more dispersed (Fig. 3d), the . 29, 2017; 9 stimulated animal, with 104 CRH neurons being activated out of 1018 total 184 CRH neurons and 569 total activated neurons (Fig. 3d,e, Supplementary 1...

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Section: Methodsmentioning

confidence: 99%

Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution

Wang

Zhu

Ding

et al. 2017

Preprint

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“…Using a well‐established VSV minireplicon assay, we found that three triple‐amino acid substitutions (TVK4‐6A3, RII7‐9A3, and VIV13‐15A3) and one single‐amino acid substitution (R7A) within the N‐terminal 21 amino acids of the NC resulted in RNA synthesis loss, whereas all the mutants maintained the ability to oligomerize and encapsidate viral RNA. Furthermore, Northern blotting analysis of nuclease sensitivity of gRNAs encapsidated by the NC or mutants showed that all the mutants failed to protect viral RNAs from nuclease digestion, suggesting that NCs are also involved in the protection of viral gRNA from nuclease digestion for the formation of a functional viral RNA template . Similarly, a recent study showed that the positively charged and polar amino acids in the surface cleft of the NC of tomato spotted wilt virus are critical for viral RNA template function, and a subsequent study found that the NC of tomato spotted wilt virus was indeed able to protect the RNA from the degradation of RNase; the mutants of NC, R94A/R95A and K183A/Y184A, which lost the RNA‐binding activity, were no longer able to protect the RNA from RNase degradation …”

Section: The Function Of Nc In Virus Life Cyclementioning

confidence: 97%

Nucleocapsid proteins: roles beyond viral RNA packaging

2016

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Viral nucleocapsid proteins (NCs) enwrap the RNA genomes of viruses to form NC-RNA complexes, which act as a template and are essential for viral replication and transcription. Beyond packaging viral RNA, NCs also play important roles in virus replication, transcription, assembly, and budding by interacting with viral and host cellular proteins. Additionally, NCs can inhibit interferon signaling response and function in cell stress response, such as inducing apoptosis. Finally, NCs can be the target of vaccines, benefiting from their conserved gene sequences. Here, we summarize important findings regarding the additional functions of NCs as much more than structural RNA-binding proteins, with specific emphasis on (1) their association with the viral life cycle, (2) their association with host cells, and (3) as ideal candidates for vaccine development.

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“…But these VSVs have high cytotoxicity that can lead to rapid death of mice, which limits long-term structural observation and gene manipulation or function research, restricting their applications in neuroscience [23]. Several mutants have been obtained by mutating the N gene related to the replication of VSV [24][25][26][27]. After mutating the seventh amino acid of the N protein (VSV-N R7A ), the replication speed of the virus decreased by 2-3 orders of magnitude within 24 h [24,25], which might be used as a better tracer, but whether it still has the ability of anterograde trans-synaptic labeling is still unknown.…”

Section: Introductionmentioning

confidence: 99%

A mutant vesicular stomatitis virus with reduced cytotoxicity and enhanced anterograde trans-synaptic efficiency

Lin,

Zhong,

Ying

et al. 2020

Mol Brain

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Understanding the connecting structure of brain network is the basis to reveal the principle of the brain function and elucidate the mechanism of brain diseases. Trans-synaptic tracing with neurotropic viruses has become one of the most effective technologies to dissect the neural circuits. Although the retrograde trans-synaptic tracing for analyzing the input neural networks with recombinant rabies and pseudorabies virus has been broadly applied in neuroscience, viral tools for analyzing the output neural networks are still lacking. The recombinant vesicular stomatitis virus (VSV) has been used for the mapping of synaptic outputs. However, several drawbacks, including high neurotoxicity and rapid lethality in experimental animals, hinder its application in long-term studies of the structure and function of neural networks. To overcome these limitations, we generated a recombinant VSV with replication-related N gene mutation, VSV-N R7A , and examined its cytotoxicity and efficiency of trans-synaptic spreading. We found that by comparison with the wild-type tracer of VSV, the N R7A mutation endowed the virus lower rate of propagation and cytotoxicity in vitro, as well as significantly reduced neural inflammatory responses in vivo and much longer animal survival when it was injected into the nucleus of the mice brain. Besides, the spreading of the attenuated VSV was delayed when injected into the VTA. Importantly, with the reduced toxicity and extended animal survival, the number of brain regions that was trans-synaptically labeled by the mutant VSV was more than that of the wild-type VSV. These results indicated that the VSV-N R7A , could be a promising anterograde tracer that enables researchers to explore more downstream connections of a given brain region, and observe the anatomical structure and the function of the downstream circuits over a longer time window. Our work could provide an improved tool for structural and functional studies of neurocircuit.

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Several residues within the N-terminal arm of vesicular stomatitis virus nucleoprotein play a critical role in protecting viral RNA from nuclease digestion

Cited by 11 publications

References 40 publications

Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution

Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution

Nucleocapsid proteins: roles beyond viral RNA packaging

A mutant vesicular stomatitis virus with reduced cytotoxicity and enhanced anterograde trans-synaptic efficiency

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