Infectious Bursal Disease Virus: Ribonucleoprotein Complexes of a Double-Stranded RNA Virus

Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some double-stranded DNA (dsDNA) viruses, and the nucleocapsid proteins of single-stranded RNA viruses. dsRNA viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T = 2 core involved in the metabolism and insulation of the dsRNA genome. The birnaviruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T = 13 capsid only. Moreover, as in many negative single-stranded RNA viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease virus, the best characterized member of the Birnaviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at ∼ 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNase III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA viruses.     

重组质粒转染蟑螂对黑胸大蠊浓核病毒的拯救

用Glassmilk法纯化PfDNV dsDNA,在其3′端和PstⅠ切开的pUC119质粒的3′端分别加dG和dC尾,连接、转化、筛选得到分子量为8.7kb的重组质粒.通过酶切证明插入DNA为PfDNV全基因组DNA.利用DEAE-dextran转染技术,将此重组质粒导入黑胸大蠊幼虫体内,此重组质粒能使虫体发病死亡.电镜超薄切片观察发现虫体细胞内存在大量的病毒粒子.同样,在免疫扩散实验中虫体PBS浸出液能与抗PfDNV的抗体产生沉淀线.用重组质粒感染致死的虫体喂食健康黑胸大蠊,也能使其发病死亡,通过电镜可以观察到在细胞内增殖的病毒粒子.     

重组质粒转染蟑螂对黑胸大蠊浓核病毒的拯救

用Ｇｌａｓｓｍｉｌｋ法纯化ＰｆＤＮＶｄｓＤＮＡ,在其３′端和ＰｓｔⅠ切开的ｐＵＣ１１９质粒的３′端分别加ｄＧ和ｄＣ尾,连接、转化、筛选得到分子量为８．７ｋｂ的重组质粒。通过酶切证明插入ＤＮＡ为ＰｆＤＮＶ全基因组ＤＮＡ。利用ＤＥＡＥｄｅｘｔｒａｎ转染技术,将此重组质粒导入黑胸大蠊幼虫体内,此重组质粒能使虫体发病死亡。电镜超薄切片观察发现虫体细胞内存在大量的病毒粒子。同样,在免疫扩散实验中虫体ＰＢＳ浸出液能与抗ＰｆＤＮＶ的抗体产生沉淀线。用重组质粒感染致死的虫体喂食健康黑胸大蠊,也能使其发病死亡,通过电镜可以观察到在细胞内增殖的病毒粒子。     

High-Efficiency Rescue of Equine Infectious Anemia Virus from a CMV-Driven Infectious Clone

<正>Dear Editor,Equine infectious anemia virus (EIAV) belongs to the macrophage-tropic lentiviruses family and infects mainly equines, including horses, donkeys and mules. EIAV shares many similar characteristics in its viral biology and hostvirus immune regulation with other lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), and has been accepted as a     

Rescue of Influenza A Virus from Recombinant DNA

We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.     

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)¹, and the negative staining has been performed by Wu et al. (2007).²Open in a separate windowClick here to view.^{(60M, flv)}     

Isolation of an Infectious Ribonucleoprotein from Vesicular Stomatitis Virus Containing an Active RNA Transcriptase

A ribonucleoprotein complex (TNP) containing an active RNA polymerase was isolated from purified vesicular stomatitis virus particles. The TNP sedimented through a sucrose gradient as a single band and appeared under the electron microscope as discrete long filaments in a spiral configuration. TNP contained one major and two minor polypeptides, but not the polypeptides associated with the outer coat of vesicular stomatitis virus. BHK-21 clone 13 cells could be infected with TNP, yielding infectious virus particles.     

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes

Genome editing using RNA‐guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5′‐NNGRRT‐3′, compared with Streptococcus pyogenes Cas9 (SpCas9) of 5′‐NGG‐3′ PAM. Here, SaCas9‐RNP‐mediated genome editing is reported in human cells. The SaCas9‐RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9‐RNP is successfully implemented to correct a pathogenic RS1 mutation for X‐linked juvenile retinoschisis. It is also shown that off‐target effects triggered by SaCas9‐RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9‐RNP‐mediated genome editing in human cells, which could facilitate genome‐editing‐based therapy.     

Systems of Delivery of CRISPR/Cas9 Ribonucleoprotein Complexes for Genome Editing

Russian Journal of Bioorganic Chemistry - The discovery of RNA-guided nucleases have enabled to leap forward in genome editing of cells and organisms. These nucleases can be delivered into cells as...     

Identification of A Ribonucleoprotein Gene from Rice Seedlings

The mixed oligonucleotides derived from partial amino acid sequence of RNP-CS I and RNP-CS I were used as primers for PCR amplification from cDNA of rice (Oryza sativa L. "Guangluai No. 4” ) etiolated seedling and a 300 bp fragment was obtained. Sequence analysis revealed that this gene fragment contains two partial RNP-CS-type RNA binding domains with space region which is typical of ribonucleoprotein in feature.