scholarly journals Genetic Studies of the β-Hairpin Loop of Rous Sarcoma Virus Capsid Protein

2006 ◽  
Vol 81 (3) ◽  
pp. 1288-1296 ◽  
Author(s):  
Jared L. Spidel ◽  
Carol B. Wilson ◽  
Rebecca C. Craven ◽  
John W. Wills
Keyword(s):  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Critical Role ◽  
Strong Support ◽  
Genome Replication ◽  
Consensus Binding Site ◽  
Rous Sarcoma ◽  
Capsid Shell ◽  
Sarcoma Virus

ABSTRACT 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 β-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 β-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.

scholarly journals Rous Sarcoma Virus Gag Protein-Oligonucleotide Interaction Suggests a Critical Role for Protein Dimer Formation in Assembly

2002 ◽  
Vol 76 (11) ◽  
pp. 5452-5462 ◽  
Author(s):  
Yu May Ma ◽  
Volker M. Vogt
Keyword(s):  
Nucleic Acid ◽  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Free Form ◽  
Zinc Ions ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, ΔMBDΔPR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the ΔMBDΔPR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was 22 nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:2 ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag dimer, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus.

1984 ◽  
Vol 49 (2) ◽  
pp. 325-332 ◽  
Author(s):  
F Poirier ◽  
P Jullien ◽  
P Dezelee ◽  
G Dambrine ◽  
E Esnault ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Tumor Formation ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus.

1982 ◽  
Vol 42 (3) ◽  
pp. 780-789 ◽  
Author(s):  
F Poirier ◽  
G Calothy ◽  
R E Karess ◽  
E Erikson ◽  
H Hanafusa
Keyword(s):  
Cell Proliferation ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Rous Sarcoma ◽  
Sarcoma Virus

Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein

1992 ◽  
Vol 12 (10) ◽  
pp. 4769-4783
Author(s):  
A M Boulden ◽  
L J Sealy
Keyword(s):  
Long Terminal Repeat ◽  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Serum Response Factor ◽  
Response Factor ◽  
High Affinity ◽  
Rous Sarcoma ◽  
Binding Factor ◽  
Sarcoma Virus ◽  
Serum Response

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.

scholarly journals Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters.

1989 ◽  
Vol 109 (5) ◽  
pp. 2337-2344 ◽  
Author(s):  
G Marazzi ◽  
F Bard ◽  
M W Klymkowsky ◽  
L L Rubin
Keyword(s):  
Monoclonal Antibody ◽  
Acetylcholine Receptor ◽  
Rous Sarcoma Virus ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Muscle Cells ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Receptor Clusters

We have shown previously that chick muscle cells transformed with Rous sarcoma virus are unable to form clusters of acetylcholine receptors (AChRs) (Anthony, D. T., S. M. Schuetze, and L. L. Rubin. 1984. Proc. Natl. Acad. Sci. USA. 81:2265-2269) and are missing a 37-KD tropomyosin-like protein (TM-2) (Anthony, D. T., R. J. Jacobs-Cohen, G. Marazzi, and L. L. Rubin. 1988. J. Cell Biol. 106:1713-1721). In an attempt to clarify the role of TM-2 in the formation and/or maintenance of AChR clusters, we have microinjected a monoclonal antibody specific for TM-2 (D3-16) into normal chick muscle cells in culture. D3-16 injection blocks the formation of new clusters but does not affect the preexisting ones. In addition, TM-2 is concentrated at rat neuromuscular junctions. These data suggest that TM-2 may play an important role in promoting the formation of AChR clusters.

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