Structure of the Capsid Size-Determining Scaffold of "Satellite" Bacteriophage P4.

P4 is a mobile genetic element (MGE) that can exist as a plasmid or integrated into its Escherichia coli host genome, but becomes packaged into phage particles by a helper bacteriophage, such as P2. P4 is the original example of what we have termed "molecular piracy", the process by which one MGE usurps the life cycle of another for its own propagation. The P2 helper provides most of the structural gene products for assembly of the P4 virion. However, when P4 is mobilized by P2, the resulting capsids are smaller than those normally formed by P2 alone. The P4-encoded protein responsible for this size change is called Sid, which forms an external scaffolding cage around the P4 procapsids. We have determined the high-resolution structure of P4 procapsids, allowing us to build an atomic model for Sid as well as the gpN capsid protein. Sixty copies of Sid form an intertwined dodecahedral cage around the T = 4 procapsid, making contact with only one out of the four symmetrically non-equivalent copies of gpN. Our structure provides a basis for understanding the sir mutants in gpN that prevent small capsid formation, as well as the nms "super-sid" mutations that counteract the effect of the sir mutations, and suggests a model for capsid size redirection by Sid.

The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference.

SaPIs are molecular pirates that exploit helper bacteriophages for their own high frequency mobilization. One striking feature of helper exploitation by SaPIs is redirection of the phage capsid assembly pathway to produce smaller phage-like particles with T=4 icosahedral symmetry rather than T=7 bacteriophage capsids. Small capsids can accommodate the SaPI genome but not that of the helper phage, leading to interference with helper propagation. Previous studies identified two proteins encoded by the prototype element SaPI1, gp6 and gp7, in SaPI1 procapsids but not in mature SaPI1 particles. Dimers of gp6 form an internal scaffold, aiding fidelity of small capsid assembly. Here we show that both SaPI1 gp6 (CpmB) and gp7 (CpmA) are necessary and sufficient to direct small capsid formation. Surprisingly, failure to form small capsids did not restore wild-type levels of helper phage growth, suggesting an additional role for these SaPI1 proteins in phage interference.

Capsid size determination by Staphylococcus aureus pathogenicity island SaPI1 involves specific incorporation of SaPI1 proteins into procapsids.

The Staphylococcus aureus pathogenicity island SaPI1 carries the gene for the toxic shock syndrome toxin (TSST-1) and can be mobilized by infection with S. aureus helper phage 80alpha. SaPI1 depends on the helper phage for excision, replication and genome packaging. The SaPI1-transducing particles comprise proteins encoded by the helper phage, but have a smaller capsid commensurate with the smaller size of the SaPI1 genome. Previous studies identified only 80alpha-encoded proteins in mature SaPI1 virions, implying that the presumptive SaPI1 capsid size determination function(s) must act transiently during capsid assembly or maturation. In this study, 80alpha and SaPI1 procapsids were produced by induction of phage mutants lacking functional 80alpha or SaPI1 small terminase subunits. By cryo-electron microscopy, these procapsids were found to have a round shape and an internal scaffolding core. Mass spectrometry was used to identify all 80alpha-encoded structural proteins in 80alpha and SaPI1 procapsids, including several that had not previously been found in the mature capsids. In addition, SaPI1 procapsids contained at least one SaPI1-encoded protein that has been implicated genetically in capsid size determination. Mass spectrometry on full-length phage proteins showed that the major capsid protein and the scaffolding protein are N-terminally processed in both 80alpha and SaPI1 procapsids.

Transducing particles of Staphylococcus aureus pathogenicity island SaPI1 are comprised of helper phage-encoded proteins.

The relationship between the composition of SaPI1 transducing particles and those of helper phage 80alpha was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80alpha and SaPI1 virions, and all were encoded by 80alpha. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.

The capsid protein of satellite Panicum mosaic virus contributes to systemic invasion and interacts with its helper virus.

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5'-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5'-untranslated region (5'-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5'-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5'-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.

Osteogenesis in rats induced by a novel recombinant helper-dependent bone morphogenetic protein-9 (BMP-9) adenovirus.

BACKGROUND: Although recombinant first-generation BMP adenoviruses can induce ectopic bone formation in immune deficient animals, the osteoinductive activity of these BMP vectors is reduced in immune competent animals. Helper-dependent adenoviral vectors have been developed to decrease the immune response and, therefore, increase gene expression in immune competent animals compared with first-generation vectors. In the present study, the osteoinductive activity of a helper-dependent GFP and BMP-9 adenoviral vector (ADGBMP9) was evaluated in vitro and in vivo. METHODS: Initially, purified ADGBMP9 was used to transduce human mesenchymal stem cells (hMSCs) and the alkaline phosphatase activity was determined as a measure of osteogenic activity. The vector was then injected into the thigh muscle of athymic nude and Sprague-Dawley rats, and CT scans and histology were subsequently used to assess bone formation. RESULTS: In vitro, ADGBMP9 was capable of inducing alkaline phosphatase expression in hMSCs. In vivo in athymic nude and Sprague Dawley rats, ADGBMP9 initiated the process of bone formation 3 days after percutaneous injection into the thigh musculature. The rats demonstrated intramuscular ectopic ossification in CT scans as early as day 9 post viral injection and ultimately formed significant amounts of ectopic bone. Histologically, the induced bone was formed via normal endochondral mechanisms. CONCLUSIONS: A helper-dependent adenoviral vector containing the BMP-9 and GFP genes has significant osteoinductive activity in both athymic nude and immune competent rats. Additional direct and ex vivo BMP gene therapy studies are required to assess the vector's activity in more animal models.

Gene transfer into baboon repopulating cells: A comparison of Flt-3 Ligand and megakaryocyte growth and development factor versus IL-3 during ex vivo transduction.

Oncoretroviral vectors require division of target cells for successful transduction. In the case of hematopoietic repopulating cells this can be achieved by cytokine stimulation using growth factor combinations which facilitate gene transfer and maintain engraftment. Interleukin-3 (IL-3) has been widely used in growth factor combinations, although more recent data in the mouse showed reduced engraftment in the presence of IL-3. Here, we used a competitive repopulation assay to study the influence of IL-3 and the early acting cytokines megakaryocyte growth and development factor (MGDF) and Flt3-ligand (Flt3-L) on gene transfer efficiency during ex vivo transduction of hematopoietic repopulating cells. In a direct comparison, baboon CD34-enriched cells were transduced on CH-296 fibronectin fragment in the presence of either IL-6, stem cell factor (SCF), Flt3-L, and MGDF or IL-3, IL-6, and SCF. Animals were followed for up to 55 weeks, and analysis of peripheral blood leukocytes by semiquantitative polymerase chain reaction showed that both cytokine combinations achieved marking of repopulating cells. A trend toward increased gene marking, especially early after transplant (P = 0.06), was seen with the combination of IL-6, SCF, Flt3-L, and MGDF. However, the highest gene marking was achieved when IL-3 was combined with early acting cytokines, suggesting that the difference observed in this study was probably due to the addition of MGDF and Flt3-L and not due to a negative effect of IL-3 on engraftment.

Double-helical RNA in satellite tobacco mosaic virus.

Satellite tobacco mosaic virus (STMV) is the spherical satellite to an obligatory rod-shaped helper tobacco mosaic virus (TMV), which is required for replication. STMV has 60 protein subunits of M(r) 17,500 on a T = 1 icosahedral capsid containing a single-stranded RNA genome of 1,059 bases. STMV appears similar to another virus, STNV, but is approximately 20 per cent smaller. It shows no amino-acid homology or immunological cross-reactivity with either STNV or its host TMV. Here we report the X-ray crystal structure of STMV, which shows that the coat protein of STMV contains a 'Swiss roll' beta-barrel. An amino-terminal strand extends more than 60A and is primarily responsible for quaternary interactions. Each capsid dimer is associated with a segment of genomic RNA double helix comprising seven base pairs. The dyad of each protein dimer is coincident with that of the central base pair of the associated RNA segment whose helix axis is directed along an icosahedral edge. Protein-nucleic acid interactions are extensive. The RNA helices, which have additional stacked bases at their 3' termini, differ significantly from canonical nucleic acid helical forms.