Capsid

Haploid leaf tissue of tobacco cultivars K326 and K149 was transformed with several transgenes containing cDNA of the potato virus Y (PVY) coat protein (CP) open reading frame (ORF). The various transgenes containing the PVY CP ORF sequence produced (1) the expected mRNA and CP product, (2) an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense RNA that was untranslatable as a result of the incorrect orientation of the PVY CP ORF behind the transcriptional promoter. Homozygous doubled haploid (DH) (diploid) plants were generated, and selfed progeny from these plants were examined. Resistance was virus specific, functioning only against PVY. An inverse correlation between transgene-derived PVY transcript steady state levels and resistance was generally noted with lines expressing the untranslatable sense version of the PVY CP ORF. A collection of DH lines, derived from a single transformation event of a common haplo...

The structure of the bacteriophage phi X174 was examined in a 2.7 A resolution map and refined, using 6.0 A to 3.0 A resolution data with F > or = 5 sigma (F). The final R-factor was 20.9% and the root-mean-square deviation from idealized bond lengths was 0.021 A. The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement icosahedral averaging procedure. The mature phage capsid consists of 60 copies of the F protein with 426 amino acids, the G protein with 175 amino acids and the J protein with 37 amino acids, as well as 12 copies of the H protein with 328 amino acids. The entire polypeptide chain of the F and G protein, all but the first N-terminal residue of the J protein, and 178 solvent molecules were included in the refinement calculations. The secondary structural features of the F, G and J proteins and their interactions with each other are described. The majority of the protein-protein interactions are between the icosahedral 5-fold related interfaces of the F and of the G proteins. These pentameric units of the F and G proteins form the 9S and 6S assembly intermediates, respectively. The J protein lacks any secondary structure and acts as a linking arm between the icosahedral 5-fold related F proteins. Water molecules were introduced only after phase extension to 2.7 A resolution had been completed. The F protein is associated with lower "thermal" parameters and exhibits greater water order in its environment than the G and J proteins. The largest thermal parameters occur in residues on the viral surface. The solvent contributes to the interactions between the proteins. There is an interface of solvent molecules between the F and the G pentamers which stabilizes the pentameric G protein spikes in a crater centered at each of the icosahedral 5-fold vertices of the F protein capsid. Sequence alignments of the F, G and J amino acid sequences for the homologous bacteriophages G4, alpha 3, phi K and phi X174 with respect to the phi X174 structure demonstrated the conservation of functionally important residues on the viral surface.

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90° light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound ant...

With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750 – 800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a Mr of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3′ end of the viral genome encompassing the 3′ non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3′ non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested.

The production of recombinant therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry, particularly monoclonal antibodies and Fc-fusion proteins. Currently, mammalian cells are the dominant production system for these proteins because they can perform complex post-translational modifications that are often required for efficient secretion, drug efficacy, and stability. These protein modifications include misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, variable glycosylation, and proteolysis. Such modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications and aggregation can lead to an immune response to the therapeutic protein. This mini-review describes examples analytical and preventative advances in the fields of protein oxidation, deamidation, misfolding and aggregation (glycosylation is covered in other articles in this issue). The feasibility of partially replacing traditional analytical methods such as peptide mapping with high-throughput screens and their use in clone and media selection are evaluated. This review also discusses how further technical advances could improve the manufacturability, potency, and safety of biotherapeutics.