scholarly journals Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation.

1989 ◽  
Vol 109 (2) ◽  
pp. 577-584 ◽  
Author(s):  
J Martin-Perez ◽  
D Bar-Zvi ◽  
D Branton ◽  
R L Erikson
Keyword(s):  
Heavy Chain ◽  
Rous Sarcoma Virus ◽  
Immunofluorescent Staining ◽  
Transformed Cells ◽  
Normal Cells ◽  
Clathrin Heavy Chain ◽  
Rous Sarcoma ◽  
Sarcoma Virus

We have shown that the heavy chain of clathrin is phosphorylated in chicken embryo fibroblast cells transformed by Rous sarcoma virus, but not in normal cells. Approximately 1 mol of phosphate is bound for every 5 mol of heavy chain in the maximally phosphorylated transformed cells. Two-thirds of the phosphate is on serine and one-third on tyrosine residues. Clathrin heavy chain is a substrate for pp60v-src in vitro. Cleveland analysis of the in vivo and in vitro clathrin heavy chain phosphopeptides, generated by protease V8 digestion, show labeled proteolytic fragments of similar molecular weight, suggesting that pp60v-src could be directly responsible for the in vivo phosphorylation of clathrin. Phosphate is equally incorporated into clathrin in both the unassembled and the assembled clathrin pools, whereas [35S]methionine is preferentially incorporated into the assembled pool. In normal cells, clathrin visualized by immunofluorescent staining appears in a punctate pattern along the membrane surface and concentrated around the nucleus; in transformed cells the perinuclear staining is completely absent. The phosphorylation of clathrin heavy chain in transformed cells may be linked to previously observed transformation-dependent alterations in receptor-mediated endocytosis of ligands such as EGF and thrombin.

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants

1984 ◽  
Vol 4 (8) ◽  
pp. 1508-1514
Author(s):  
A W Stoker ◽  
P J Enrietto ◽  
J A Wyke
Keyword(s):  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Temperature Sensitive ◽  
Tyrosine Kinase Activity ◽  
Rous Sarcoma ◽  
Heat Labile ◽  
Src Gene ◽  
Sarcoma Virus

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.

scholarly journals COMPARATIVE STUDIES IN ROUS SARCOMA WITH VIRUS, TUMOR CELLS, AND CHICK EMBRYO CELLS TRANSFORMED IN VITRO BY VIRUS

1962 ◽  
Vol 115 (1) ◽  
pp. 245-251 ◽  
Author(s):  
Robert M. Dougherty ◽  
Herbert R. Morgan
Keyword(s):  
Chick Embryo ◽  
Tumor Cells ◽  
Comparative Studies ◽  
Rous Sarcoma Virus ◽  
Rous Sarcoma ◽  
Transformed Fibroblasts ◽  
Embryo Cells ◽  
Sarcoma Virus

Chick embryo fibroblasts infected in vitro with Rous sarcoma virus have properties similar to tumor cells when injected into virus-immune chickens. When such virus-transformed fibroblasts are injected into normal chickens, they apparently participate in the production of tumors independent of their release of virus and are thus apparently malignant in vivo.

scholarly journals Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

2007 ◽  
Vol 82 (1) ◽  
pp. 503-512 ◽  
Author(s):  
Jangsuk Oh ◽  
Kevin W. Chang ◽  
Rafal Wierzchoslawski ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Rous Sarcoma Virus ◽  
In Vitro Assays ◽  
Viral Dna ◽  
Experimental Conditions ◽  
Linear Dna ◽  
Rous Sarcoma ◽  
Viral Dnas ◽  
Sarcoma Virus

ABSTRACT The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3′ end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5′ end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5′ end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo.

scholarly journals Subcellular Localization and Integration Activities of Rous Sarcoma Virus Reverse Transcriptase

2002 ◽  
Vol 76 (12) ◽  
pp. 6205-6212 ◽  
Author(s):  
Susanne Werner ◽  
Patrick Hindmarsh ◽  
Markus Napirei ◽  
Karin Vogel-Bachmayr ◽  
Birgitta M. Wöhrl
Keyword(s):  
Subcellular Localization ◽  
Rous Sarcoma Virus ◽  
Preintegration Complex ◽  
Lower Efficiency ◽  
Α Subunit ◽  
Proviral Dna ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT Reverse transcriptases (RTs) αβ and β from avian Rous sarcoma virus (RSV) harbor an integrase domain which is absent in nonavian retroviral RTs. RSV integrase contains a nuclear localization signal which enables the enzyme to enter the nucleus of the cell in order to perform integration of the proviral DNA into the host genome. In the present study we analyzed the subcellular localization of RSV RT, since previous results indicated that RSV finishes synthesis of the proviral DNA in the nucleus. Our results demonstrate that the heterodimeric RSV RT αβ and the β subunit, when expressed independently, can be detected in the nucleus, whereas the separate α subunit lacking the integrase domain is prevalent in the cytoplasm. These data suggest an involvement of RSV RT in the transport of the preintegration complex into the nucleus. In addition, to analyze whether the integrase domain, located at the carboxyl terminus of β, exhibits integration activities, we investigated the nicking and joining activities of heterodimeric RSV RT αβ with an oligodeoxynucleotide-based assay system and with a donor substrate containing the supF gene flanked by the viral long terminal repeats. Our data show that RSV RT αβ is able to perform the integration reaction in vitro; however, it does so with an estimated 30-fold lower efficiency than the free RSV integrase, indicating that RSV RT is not involved in integration in vivo. Integration with RSV RT αβ could be stimulated in the presence of human immunodeficiency virus type 1 nucleocapsid protein or HMG-I(Y).

scholarly journals Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

2020 ◽  
Author(s):  
Martin Obr ◽  
Clifton L. Ricana ◽  
Nadia Nikulin ◽  
Jon-Philip R. Feathers ◽  
Marco Klanschnig ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Electron Tomography ◽  
Structural Mechanism ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Subtomogram Averaging ◽  
Opposing Effects ◽  
Hiv 1

AbstractInositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 was ∼100-fold more potent at promoting RSV mature CA assembly than observed for HIV-1 and removal of IP6 in vivo reduced infectivity by 100-fold. By cryo-electron tomography and subtomogram averaging, mature virus-like particles (VLPs) showed an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 had opposing effects on CA in vitro assembly, inducing formation of T=1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles revealed that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

scholarly journals Biochemical Characterization of Rous Sarcoma Virus MA Protein Interaction with Membranes

2005 ◽  
Vol 79 (10) ◽  
pp. 6227-6238 ◽  
Author(s):  
Amanda K. Dalton ◽  
Paul S. Murray ◽  
Diana Murray ◽  
Volker M. Vogt
Keyword(s):  
Host Cell ◽  
Rous Sarcoma Virus ◽  
Biochemical Characterization ◽  
Virus Assembly ◽  
Membrane Association ◽  
Host Cell Membrane ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo.

Transcription of the Rous Sarcoma Virus Genome In Vitro and In Vivo

2015 ◽  
pp. 517-523
Author(s):  
J. M. Bishop ◽  
C. T. Deng ◽  
A. J. Faras ◽  
H. M. Goodman ◽  
R. R. Guntaka ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Virus Genome ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 568 ◽  
Author(s):  
Shuohui Liu ◽  
Rebecca Kaddis Maldonado ◽  
Tiffiny Rye-McCurdy ◽  
Christiana Binkley ◽  
Aissatou Bah ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rous Sarcoma Virus ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Gag Polyprotein ◽  
Interaction Sites ◽  
Rous Sarcoma ◽  
Sarcoma Virus

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions.

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