Solution structure of the Rous sarcoma virus nucleocapsid protein: muPsi RNA packaging signal complex.
(1/62)
The 5'-untranslated region (5'-UTR) of retroviral genomes contains elements required for genome packaging during virus assembly. For many retroviruses, the packaging elements reside in non-contiguous segments that span most or all of the 5'-UTR. The Rous sarcoma virus (RSV) is an exception, in that its genome can be packaged efficiently by a relatively short, 82 nt segment of the 5'-UTR called muPsi. The RSV 5'-UTR also contains three translational start codons (AUG-1, AUG-2 and AUG-3) that have been controvertibly implicated in translation initiation and genome packaging, one of which (AUG-3) resides within the muPsi sequence. We demonstrated recently that muPsi is capable of binding to the cognate RSV nucleocapsid protein (NC) with high affinity (dissociation constant K(d) approximately 2 nM), and that residues of AUG-3 are essential for tight binding. We now report the solution structure of the NC:muPsi complex, determined using NMR data obtained for samples containing ((13)C,(15)N)-labeled NC and (2)H-enriched, nucleotide-specifically protonated RNAs. Upon NC binding, muPsi adopts a stable secondary structure that consists of three stem loops (SL-A, SL-B and SL-C) and an 8 bp stem (O3). Binding is mediated by the two zinc knuckle domains of NC. The N-terminal knuckle interacts with a conserved U(217)GCG tetraloop (a member of the UNCG family; N=A,U,G or C), and the C-terminal zinc knuckle binds to residues that flank SL-A, including residues of AUG-3. Mutations of critical nucleotides in these sequences compromise or abolish viral infectivity. Our studies reveal novel structural features important for NC:RNA binding, and support the hypothesis that AUG-3 is conserved for genome packaging rather than translational control. (+info)
Genetic Studies of the beta-hairpin loop of Rous sarcoma virus capsid protein.
(2/62)
The first few residues of the Rous sarcoma virus (RSV) CA protein comprise a structurally dynamic region that forms part of a Gag-Gag interface in immature virus particles. Dissociation of this interaction during maturation allows refolding and formation of a beta-hairpin structure important for assembly of CA monomers into the mature capsid shell. A consensus binding site for the cellular Ubc9 protein was previously identified within this region, suggesting that binding of Ubc9 and subsequent small ubiquitin-like modifier protein 1 (SUMO-1) modification of CA may play a role either in regulating the assembly activity of CA in immature particles or mature cores or in controlling postentry function(s) during the establishment of infection. In the present study, mutations designed to eliminate the consensus binding site were used to dissect the potentially overlapping functions of these residues. The resulting replication defects could not be traced to a failure to form particles of normal composition but, rather, to a deficit in genome replication. Genetic suppressors of two detrimental beta-hairpin mutations improved infectivity without restoring the consensus site or creating a novel one elsewhere. Optimal restoration of infectivity to a Lys-to-Arg mutant required a combination of secondary changes, one on the surface of each domain of CA. Rather than arguing for a critical role of Ubc9 and SUMO in RSV replication, these findings provide strong support for a structural role of the N-terminal residues and a particularly striking example of long-range interactions between regions of CA in achieving a functional core competent for genome replication. (+info)
Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation.
(3/62)
Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes. (+info)
Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA.
(4/62)
All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had a diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5. (+info)
Transcriptional profile of Rous Sarcoma Virus transformed chicken embryo fibroblasts reveals new signaling targets of viral-src.
(5/62)
Transformation of chicken fibroblasts in vitro by Rous Sarcoma Virus represents a model of cancer in which a single oncogene, viral src, uniformly and rapidly transforms primary cells in culture. We experimentally surveyed the transcriptional program affected by Rous Sarcoma Virus (RSV) in primary culture of chicken embryo fibroblasts. As a control, we used cells infected with non-transforming RSV mutant td106, in which the src gene was deleted. Using Affymetrix GeneChip Chicken Genome Arrays, we report 811 genes that were modulated more than 2.5 fold in the virus transformed cells. Among these, 409 genes were induced and 402 genes were repressed by viral src. From the repertoire of modulated genes, we selected 20 genes that were robustly changed. We then validated and quantified the transcriptional changes of most of the 20 selected genes by real-time PCR. The set of strongly induced genes contains vasoactive intestinal polypeptide, MAP kinase phosphatase 2 and follistatin, among others. The set of strongly repressed genes contains TGF beta 3, TGF beta-induced gene, and deiodinase. The function of several robustly modulated genes sheds new light on the molecular mechanism of oncogenic transformation. (+info)
Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation.
(6/62)
The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion. (+info)
Overlapping roles of the Rous sarcoma virus Gag p10 domain in nuclear export and virion core morphology.
(7/62)
Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology. (+info)
Serine/arginine-rich proteins contribute to negative regulator of splicing element-stimulated polyadenylation in rous sarcoma virus.
(8/62)
Rous sarcoma virus (RSV) requires large amounts of unspliced RNA for replication. Splicing and polyadenylation are coupled in the cells they infect, which raises the question of how viral RNA is efficiently polyadenylated in the absence of splicing. Optimal RSV polyadenylation requires a far-upstream splicing control element, the negative regulator of splicing (NRS), that binds SR proteins and U1/U11 snRNPs and functions as a pseudo-5' splice site that interacts with and sequesters 3' splice sites. We investigated a link between NRS-mediated splicing inhibition and efficient polyadenylation. In vitro, the NRS alone activated a model RSV polyadenylation substrate, and while the effect did not require the snRNP-binding sites or a downstream 3' splice site, SR proteins were sufficient to stimulate polyadenylation. Consistent with this, SELEX-binding sites for the SR proteins ASF/SF2, 9G8, and SRp20 were able to stimulate polyadenylation when placed upstream of the RSV poly(A) site. In vivo, however, the SELEX sites improved polyadenylation in proviral clones only when the NRS-3' splice site complex could form. Deletions that positioned the SR protein-binding sites closer to the poly(A) site eliminated the requirement for the NRS-3' splice site interaction. This indicates a novel role for SR proteins in promoting RSV polyadenylation in the context of the NRS-3' splice site complex, which is thought to bridge the long distance between the NRS and poly(A) site. The results further suggest a more general role for SR proteins in polyadenylation of cellular mRNAs. (+info)