The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

Investigation of Murine Norovirus Replication in RAW264.7 Cells by Strand-specific RT-PCR

Murine norovirus (MNV) is a non-enveloped virus with a positive-sense RNA genome and causes lethal infection in mice. MNV has been used as a model virus for human norovirus (NV) whose in vitro cell culture system has not been available to date since MNV and NV are genetically related. In this study, the genome replication of MNV was investigated using strand-specific RT-PCR in RAW264.7 cells. Reverse transcription (RT) using a sense primer followed by PCR showed that negative-sense RNAs were first detected in RAW264.7 cells between 6 and 9 [3 and 6] hours post infection (h.p.i.). However, these negative-sense RNAs were not detected when cells were treated with a translation inhibitor cycloheximide. Then, RT with an antisense primer followed by PCR was performed to detect positive-sense RNAs. RT-PCR results revealed that the amount of positive-sense RNAs began to increase from 9 [6] h.p.i., indicating the accumulation of the newly synthesized (+)RNA genome. Furthermore, cycloheximide abrogated the increase of newly made RNAs during MNV infection. In conclusion, strand-specific RT-PCR using a sense or antisense primer, in combination with cycloheximide treatment, enabled us to detect positive-sense and negative-sense RNAs selectively and provided a useful tool to understand the replication cycle of MNV.

The mode of inhibition of the biosynthesis of phenylalanine ammonia lyase by its product cinnamic acid in aging potato parenchyma tissue.

Excised potato parenchyma tissue upon aging in air and light, showed synthesis of RNA, as assessed by the incorporation of [3H]-uridine, reaching a maximum in 18 h. The increase in RNA synthesis was in parallel with the increase in phenylalanine ammonia lyase synthesis. The treatment of the tissue, initially with actinomycin D at 15 μg/g tissue, or with trans-cinnamate at 3·5 mM, caused 65% and 50% inhibition of RNA synthesis ,respectively. The inhibition was reduced to 20% by delaying the treatment of trans-cinnamate to the ninth hour. A comparative study on the nuclear fractions, isolated from trans-cinnamate treated and untreated aged parenchyma tissues showed that trans-cinnamate treatment inhibited the in vitro RNA synthesis by 38%. Studies on the in vivo synthesis of PAL and other proteins showed that trans-cinnamate treatment mainly impaired the de novo synthesis of PAL. The total RNA, isolated from trans-cinnamate treated parenchyma, was 66% less efficient in the translation of PAL, in cell free wheat germ protein synthesizing system. The translation product, purified by affinity chroma tography on phenylalanine conjugated sepharose-4B was found to be homogeneous and showed a single radioactive peak corresponding to protein band on polyacrylamide gel electrophoresis.

RNA synthesis in maturing avian erythrocyte nuclei.

The rate of RNA synthesis and its inhibition by α-amanitin in the nuclei of mature and immature avian erythrocytes are increased with the increase in ionic strength of incubation medium. Polyacrylamide gel electrophoresis indicates that heterogeneous species of RNAs are synthesised in the mature and immature erythrocyte nuclei. However, a large number of high molecular weight RNAs are synthesised in the nuclei of immature erythrocytes. Elution profiles on poly(U)- sepharose chromatography indicate that the RNAs synthesised in the nuclei of two types of cell contain poly(A) segments. Sixteen per cent of mature erythrocyte nuclear RNA syntbesised are polyadenylated, while it is 13% in immature erythrocyte nuclei. However, the total RNA synthesised is 2–3 fold higher in immature erythrocyte nuclei than that in mature erythrocyte nuclei.

Effect of phytohormones on nuclear RNA synthesis in germinating seeds of Trigonella foenumgraeceum and its callus.

Treatment of Trigonella foenumgraeceum (fenugreek) seedlings with naphthalene acetic acid plus gibberellic acid enhanced the RNA synthesising capacity of nuclei isolated from the hypocotyl and cotyledonary regions. This increase was more pronounced in the nuclei from the hypocotyl region than from the cotyledonary region. In vitro addition of these phytohormones did not stimulate RNA synthesis by nuclei. The RNA synthesis by mitochondria was not affected by preincubating the seedlings with the hormones. The nuclei isolated from callus cultures of fenugreek hypocotyl treated with the hormone also showed increased RNA synthesis.

Characterisation of rinderpest virus RNA and the action of actinomycin D on its replication.

RNA extracted from purified rinderpest virus was characterised by sucrose gradient sedimentation and polyacrylamide gel electrophoresis. The predominant virion RNA species had a sedimentation constant of 46S and its estimated molecular weight was 4·8 × 106 daltons. Consistently high amounts of UMP and AMP were detected. The melting-temperature profile of the virion RNA suggested absence of secondary structure. The effect of actionomycin D on the replication of rinderpest virus in Vero cells was studied by following the viral RNA synthesis using labelled uridine as well as by infectivity titration. The viral RNA synthesis was not affected until 12 h following infection and was inhibited thereafter between 18 and 48 h to an extent of 25% at 5 and 10 μg levels of the drug. A 100 to 1000-fold reduction in the infectivity titres was observed in the presence of the drug. These results suggest that actinomycin D inhibits rinderpest viral RNA replication. Sedimentation analysis of viral RNA extracted from drug-treated cultures showed inhibition of the genome RNA of rinderpest virus.

Uterine RNA synthesis during protein deficiency and steroid-maintained pregnancy in rats.

Some aspects of uterine RNA synthesis including [3H]-uridine incorpo ration into RNA, activities of RNA polymerases and ribonucleases were studied. It was observed that both normal and pregnant animals, kept on protein-free diet for 15 and 20 days, showed a significant increase in in vivo uptake of [3H]-uridine into total RNA. Activities of RNA polymerase I and polymerase III increased two-fold in animals kept on a protein-free diet; however, RNA polymerase II activity was unaffected by protein restriction. In animals kept on protein-free diet where pregnancy was maintained by exogenous estrogen and progesterone, specific activity of nuclear RNA was further increased and the activities of RNA polymerases I, II and III markedly increased. Levels of RNase were also increased significantly during protein deficiency, thus showing a rapid turn-over of uterine RNA. These observations indicate that during protein restriction, uterine RNA synthesis is regulated at transcriptional level by a selective stimulation of RNA polymerase and RNase also plays an important role.