Further purification and characterization of scrapie prions

https://mmbr.asm.org/content/mmbr/44/2/175.full.pdf

5'-terminal cap structure in eucaryotic messenger ribonucleic acids.

The Plant Viruses

The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does not interact with a catalytically inactive kinase mutant. Using mutant AtSERK1 proteins in which Thr 462, Thr 463, and Thr 468 in the A-loop of the AtSERK1 kinase domain were replaced by alanines, we show that phosphorylation status of the receptor is involved in interaction with KAPP. KAPP and AtSERK1 cDNAs were fused to two different variants of green fluorescent protein (GFP), the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP). Both KAPP and AtSERK1 proteins are found at the plasma membrane. Our results show that AtSERK1-CFP becomes sequestered into intracellular vesicles when transiently coexpressed with KAPP-YFP proteins. AtSERK1T463A-CFP and AtSERK13T→A-CFP proteins were partially sequestered intracellularly in the absence of KAPP-YFP protein, suggesting an active role for KAPP dephosphorylation of threonine residues in the AtSERK1 A-loop in receptor internalization. The interaction between the KAPP-CFP/YFP and AtSERK1-CFP/YFP fusion proteins was investigated with fluorescence spectral imaging microscopy (FSPIM). Our results show that AtSERK1-CFP and KAPP-YFP proteins are colocalized at the plasma membrane but only show fluorescence energy transfer (FRET) indicative of physical interaction in intracellular vesicles. These results suggest that KAPP is an integral part of the AtSERK1 endocytosis mechanism.

/pdf/the-arabidopsis-kinase-associated-protein-phosphatase-58zzgm41yn.pdf

The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1

Interaction of the Arabidopsis Polycomb group proteins FIE and MEA mediates their common phenotypes

Evidence is presented for the presence of a protein covalently bound to the 5' termini of both M and B RNA components of CPMV. The protein is found to be linked in both cases to the 5' phosphate of the dinucleotide pUpAp, derived by ribonuclease digestion of the RNA. The intact protein is not required for infectivity or for in vitro translation of the RNA in cell-free extracts.

A protein linked to the 5' termini of both RNA components of the cowpea mosaic virus genome

The inhibition of cowpea mosaic virus replication by actinomycin D.

Summary
A study was made of the proteins synthesized in cowpea protoplasts infected with cowpea mosaic virus (CPMV). Protoplasts were labelled with 35S-methionine at different times during the infection cycle and the proteins synthesized were analysed by SDS-polyacrylamide slab gel electrophoresis of different subcellular fractions. Since no shut-off of host protein synthesis occurred, virus-specific protein synthesis had to be distinguished from a high background of protoplast proteins. Eleven polypeptides with mol. wt. of 170, 130, 112, 110, 87, 84, 68, 37, 30, 24 and 23 (all × 103) were detected whose synthesis was either induced or stimulated by CPMV infection. No differences were observed between the electrophoretic patterns of samples from uninfected and CPMV-infected protoplasts during the first 8 h of infection (latent phase). The 170 × 103 and 30 × 103 species were the first virus-related polypeptides detectable by labelling between 9 and 15 h after inoculation. From about 16 h after inoculation, all other virus-related proteins appeared to be synthesized at increasing rates. The polypeptides with mol. wt. of 170, 130, 112, 110, 68, 37 and 23 (all × 103) could be designated as virus-induced; the others may well be host-coded proteins, the synthesis of which is stimulated by CPMV infection. Although CPMV replication is known to occur in close association with virus-induced cytopathic membrane structures the 112 × 103 and 68 × 103 mol. wt. polypeptides were the only virus-related proteins specifically found in the particulate fractions of the protoplasts. From the 11 virus-related proteins, the 37 × 103, 24 × 103 and 23 × 103 mol. wt. proteins were identified as virus coat proteins. The 170 × 103 and 30 × 103 mol. wt. polypeptides also appeared to be virus-specific and to be coded for by CPMV bottom component RNA.

Protein Synthesis in cowpea mosaic virus-infected cowpea protoplasts: Detection of virus-related proteins

Summary
Inoculation of cowpea protoplasts with purified middle component of cowpea mosaic virus (CPMV) did not result in the production of newly induced virus-related proteins. In contrast, infection of protoplasts with purified bottom component produced virus-related polypeptides with mol. wt. 170×103, 112 × 103, 110 × 103, 84 × 103 and 30 × 103, but not the virus capsid proteins. These results suggest that CPMV B-RNA specifies one or more early functions, one of which is the replicase function, while M-RNA encodes the virus coat proteins. Attempts to elucidate the mechanism by which the virus-specific proteins are synthesized were inconclusive. No precursor-product relations could be demonstrated and virus subgenomic messengers were not observed. Some kind of processing was nonetheless found to occur with the small virus capsid protein:several discrete oligopeptides were successively cleaved from this protein after assembly into the virion particle.

P. J. M. Rottier

Papers

A protein linked to the 5' termini of both RNA components of the cowpea mosaic virus genome

The inhibition of cowpea mosaic virus replication by actinomycin D.

Protein Synthesis in cowpea mosaic virus-infected cowpea protoplasts: Detection of virus-related proteins

Protein synthesis in cowpea mosaic virus-infected cowpea protoplasts: Further characterization of virus-related protein synthesis