We succeeded in producing human gamma interferon (IFN-gamma) in tobacco protoplasts in quantity using genetically engineered brome mosaic virus (BMV strain ATCC66). This strain of BMV produces two types of coat protein, a full-length coat protein (20K) and a truncated coat protein (19K) which are translated from the first and second initiation codons, respectively. We replaced the truncated coa...

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete...

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fix...

RNA challenge phages are derivatives of bacteriophage P22 that enable direct genetic selection for a specific RNA-protein interaction. The bacteriophage P22 R17 encodes a wild-type R17 operator site and undergoes lysogenic development following infection of susceptible bacterial strains that express the R17/MS2 coat protein. A P22 R17 derivative with an OcRNA site (P22 R17 [A(-10)U]) develops l...

Abstract Members of the p24 protein family form a highly conserved family of type I transmembrane proteins that are abundant components of the early secretory pathway. Topologically, the proteins have a large luminal domain and a short cytoplasmic domain that allows for targeting to both coat protein complex II and coat protein complex I vesicles, and thus these proteins cycle between the endop...

Assembly of bacteriophage P22 procapsids has long served as a model for assembly of spherical viruses. Historically, assembly of viruses has been viewed as a non-equilibrium process. Recently alternative models have been developed that treat spherical virus assembly as an equilibrium process. Here we have investigated whether P22 procapsid assembly reactions achieve equilibrium or are irreversi...

Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3' untranslated regions of AMV and ilarviru...

Some capsid proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 coat protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to impr...

Icosahedral capsid assembly is an example of a reaction controlled solely by the interactions of the proteins involved. Bacteriophage P22 procapsids can be assembled in vitro by mixing coat and scaffolding proteins in a nucleation-limited reaction, where scaffolding protein directs the proper assembly of coat protein. Here, we investigated the effect of the buffer composition on the interaction...

The proteins initiated in vitro by nascent bacteriophage f2 RNA strands attached to isolated replicating structures have been analyzed. The observations confirm that coat protein is the major product initiated and completed. Nascent strands direct the initiation of viral maturation protein in amounts similar to the maximum levels observed in vivo; this synthesis is independent of translation of...