An Infectious Rous Sarcoma Virus Gag Mutant That Is Defective in Nuclear Cycling.

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. "Complex" retroviruses, such as human immunodeficiency virus (HIV), use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. "Simple" retroviruses, such as Mason-Pfizer monkey virus (MPMV) and murine leukemia virus (MLV), exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous sarcoma virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling-deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to wild-type (WT) virus and surprisingly is replication competent, albeit with a slower rate of spread than observed in WT virus. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. IMPORTANCE While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. Rous sarcoma virus (RSV) Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.

Crystal structure of the Rous sarcoma virus intasome.

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase-DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

Retroviral host range extension is coupled with Env-activating mutations resulting in receptor-independent entry.

The extent of virus transmission among individuals and species is generally determined by the presence of specific membrane-embedded virus receptors required for virus entry. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor is the first and crucial step in determining host specificity. Using a well-established retroviral model-avian Rous sarcoma virus (RSV)-we analyzed changes in an RSV variant that had repeatedly been able to infect rodents. By envelope gene (env) sequencing, we identified eight mutations that do not match the already described mutations influencing the host range. Two of these mutations-one at the beginning (D32G) of the surface Env subunit (SU) and the other at the end of the fusion peptide region (L378S)-were found to be of critical importance, ensuring transmission to rodent, human, and chicken cells lacking the appropriate receptor. Furthermore, we carried out assays to examine the virus entry mechanism and concluded that these two mutations cause conformational changes in the Env variant and that these changes lead to an activated, or primed, state of Env (normally induced after Env interaction with the receptor). In summary, our results indicate that retroviral host range extension is caused by spontaneous Env activation, which circumvents the need for original cell receptor. This activation is, in turn, caused by mutations in various env regions.

Sequence-Modified Antibiotic Resistance Genes Provide Sustained Plasmid-Mediated Transgene Expression in Mammals.

Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo even in quiescent mammalian tissues because the transgene expression cassette is silenced. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Increasing numbers of An/Tn sequences increased sustained transcription of both backbone sequences and adjacent expression cassettes. In order to recapitulate these expression profiles in compact and portable plasmid DNA backbones, we engineered the standard kanamycin or ampicillin antibiotic resistance genes, optimizing the number of An/Tn sequence without altering the encoded amino acids. The resulting vector backbones yield sustained transgene expression from mouse liver, providing generic DNA vectors capable of sustained transgene expression without additional genes or mammalian regulatory elements.

Development of Rous sarcoma Virus-like Particles Displaying hCC49 scFv for Specific Targeted Drug Delivery to Human Colon Carcinoma Cells.

PURPOSE: Virus-like particles (VLPs) have been used as drug carriers for drug delivery systems. In this study, hCC49 single chain fragment variable (scFv)-displaying Rous sarcoma virus-like particles (RSV VLPs) were produced in silkworm larvae to be a specific carrier of an anti-cancer drug. METHOD: RSV VLPs displaying hCC49 scFv were created by the fusion of the transmembrane and cytoplasmic domains of hemagglutinin from influenza A (H1N1) virus and produced in silkworm larvae. The display of hCC49 scFv on the surface of RSV VLPs was confirmed by enzyme-linked immunosorbent assay using tumor-associated glycoprotein-72 (TAG-72), fluorescent microscopy, and immunoelectron microscopy. Fluorescein isothiocyanate (FITC) or doxorubicin (DOX) was incorporated into hCC49 scFv-displaying RSV VLPs by electroporation and specific targeting of these VLPs was investigated by fluorescent microscopy and cytotoxicity assay using LS174T cells. RESULTS: FITC was delivered to LS174T human colon adenocarcinoma cells by hCC49 scFv-displaying RSV VLPs, but not by RSV VLPs. This indicated that hCC49 scFv allowed FITC-loaded RSV VLPs to be delivered to LS174T cells. DOX, which is an anti-cancer drug with intrinsic red fluorescence, was also loaded into hCC49 scFv-displaying RSV VLPs by electroporation; the DOX-loaded hCC49 scFv-displaying RSV VLPs killed LS174T cells via the specific delivery of DOX that was mediated by hCC49 scFv. HEK293 cells were alive even though in the presence of DOX-loaded hCC49 scFv-displaying RSV VLPs. CONCLUSION: These results showed that hCC49 scFv-displaying RSV VLPs from silkworm larvae offered specific drug delivery to colon carcinoma cells in vitro. This scFv-displaying enveloped VLP system could be applied to drug and gene delivery to other target cells.

Effect of multimerization on membrane association of Rous sarcoma virus and HIV-1 matrix domain proteins.

In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.

A two-pronged structural analysis of retroviral maturation indicates that core formation proceeds by a disassembly-reassembly pathway rather than a displacive transition.

Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C-terminal domain.

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C-terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y-CTD, F167Y-CTD) and suppressor mutations (R185W-CTD, I190V-CTD). The stabilities of three double mutant proteins (D155Y/R185W-CTD, F167Y/R185W-CTD, and F167Y/I190V-CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT-CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation.

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA.

Retroviral Gag polyproteins coopt host factors to traffic from cytosolic ribosomes to the plasma membrane, where virions are released. Before membrane transport, the multidomain Gag protein of Rous sarcoma virus (RSV) undergoes importin-mediated nuclear import and CRM1-dependent nuclear export, an intrinsic step in the assembly pathway. Transient nuclear trafficking of Gag is required for efficient viral RNA (vRNA) encapsidation, suggesting that Gag:vRNA binding might occur in the nucleus. Here, we show that Gag is imported into the nucleus through direct interactions of the Gag NC domain with importin-alpha (imp-alpha) and the MA domain with importin-11 (imp-11). The vRNA packaging signal, known as psi, inhibited imp-alpha binding to Gag, indicating that the NC domain does not bind to imp-alpha and vRNA simultaneously. Unexpectedly, vRNA binding also prevented the association of imp-11 with both the MA domain alone and with Gag, suggesting that the MA domain may bind to the vRNA genome. In contrast, direct binding of Gag to the nuclear export factor CRM1, via the CRM1-RanGTP heterodimer, was stimulated by psiRNA. These findings suggest a model whereby the genomic vRNA serves as a switch to regulate the ordered association of host import/export factors that mediate Gag nucleocytoplasmic trafficking for virion assembly. The Gag:vRNA interaction appears to serve multiple critical roles in assembly: specific selection of the vRNA genome for packaging, stimulating the formation of Gag dimers, and triggering export of viral ribonucleoprotein complexes from the nucleus.

The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the intracellular phase of the virion assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice.

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.

Proton-linked dimerization of a retroviral capsid protein initiates capsid assembly.

In mature retroviral particles, the capsid protein (CA) forms a shell encasing the viral replication complex. Human immunodeficiency virus (HIV) CA dimerizes in solution, through its C-terminal domain (CTD), and this interaction is important for capsid assembly. In contrast, other retroviral capsid proteins, including that of Rous sarcoma virus (RSV), do not dimerize with measurable affinity. Here we show, using X-ray crystallography and other biophysical methods, that acidification causes RSV CA to dimerize in a fashion analogous to HIV CA, and that this drives capsid assembly in vitro. A pair of aspartic acid residues, located within the CTD dimer interface, explains why dimerization is linked to proton binding. Our results show that despite overarching structural similarities, the intermolecular forces responsible for forming and stabilizing the retroviral capsid differ markedly across retroviral genera. Our data further suggest that proton binding may regulate RSV capsid assembly, or modulate stability of the assembled capsid.

NMR relaxation studies of an RNA-binding segment of the rous sarcoma virus gag polyprotein in free and bound states: a model for autoinhibition of assembly.

Assembly of retrovirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein-protein contacts in the capsid domain (CA), followed by higher order assembly to form the immature virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394-577), spans the critical SP region and allows assessment of this key Gag-nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)(4), an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short to promote Gag dimerization. The results show that the CTD and NC domains tumble independently. In contrast, the two zinc finger domains within NC are rotationally coupled in both the unbound and bound states, even though only the first zinc finger appears to make direct contact with (GT)(4). In addition, the NMR data indicate that SP and flanking residues undergo a conformational exchange process that is slowed in the presence of (GT)(4). This region around SP where relaxation is strongly affected by (GT)(4) binding is nearly identical to the assembly domain defined previously by mutagenesis studies. Other changes in relaxation induced by (GT)(4) implicate conformational perturbations of helices 1 and 4 in CTD. On the basis of the combined data, we propose a model for the promotion of Gag dimerization by RNA association in which NC-RNA binding disrupts an assembly inhibitory, intramolecular interaction involving SP and CTD. Disruption of this intramolecular interaction is proposed to enhance the accessibility of the Gag dimer contact surface and release the assembly domain to promote intermolecular oligomerization.

Foreign glycoproteins can be actively recruited to virus assembly sites during pseudotyping.

Retroviruses like human immunodeficiency virus type 1 (HIV-1), as well as many other enveloped viruses, can efficiently produce infectious virus in the absence of their own surface glycoprotein if a suitable glycoprotein from a foreign virus is expressed in the same cell. This process of complementation, known as pseudotyping, often can occur even when the glycoprotein is from an unrelated virus. Although pseudotyping is widely used for engineering chimeric viruses, it has remained unknown whether a virus can actively recruit foreign glycoproteins to budding sites or, alternatively, if a virus obtains the glycoproteins through a passive mechanism. We have studied the specificity of glycoprotein recruitment by immunogold labeling viral glycoproteins and imaging their distribution on the host plasma membrane using scanning electron microscopy. Expressed alone, all tested viral glycoproteins were relatively randomly distributed on the plasma membrane. However, in the presence of budding HIV-1 or Rous sarcoma virus (RSV) particles, some glycoproteins, such as those encoded by murine leukemia virus and vesicular stomatitis virus, were dramatically redistributed to viral budding sites. In contrast, the RSV Env glycoprotein was robustly recruited only to the homologous RSV budding sites. These data demonstrate that viral glycoproteins are not in preformed membrane patches prior to viral assembly but rather that glycoproteins are actively recruited to certain viral assembly sites.

Genetic evidence for a connection between Rous sarcoma virus gag nuclear trafficking and genomic RNA packaging.

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal psi, at the 5' end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.

Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus.

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944-3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790-6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing.

Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.

Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation.

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.

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3'UTRs.

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3' untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3'UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3'UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3'UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5'-3' exonucleolytic (XRN1) function in knockdown experiments in human cells.

Evidence that a threshold of serine/arginine-rich (SR) proteins recruits CFIm to promote rous sarcoma virus mRNA 3' end formation.

The weak polyadenylation site (PAS) of Rous sarcoma virus (RSV) is activated by the juxtaposition of SR protein binding sites within the spatially separate negative regulator of splicing (NRS) element and the env RNA splicing enhancer (Env enhancer), which are far upstream of the PAS. Juxtaposition occurs by formation of the NRS - 3' ss splicing regulatory complex and is thought to provide a threshold of SR proteins that facilitate long-range stimulation of the PAS. We provide evidence for the threshold model by showing that greater than three synthetic SR protein binding sites are needed to substitute for the Env enhancer, that either the NRS or Env enhancer alone promotes polyadenylation when the distance to the PAS is decreased, and that SR protein binding sites promote binding of the polyadenylation factor cleavage factor I (CFIm) to the weak PAS. These observations may be relevant for cellular PASs.