High Affinity Nucleocapsid Protein Binding to the μΨ RNA Packaging Signal of Rous Sarcoma Virus

ABSTRACT In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging. We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation. In this study, using both site-directed mutagenesis and in vivo random screening in the yeast three-hybrid binding assay, we have examined the amino acids in NC required for genomic RNA binding. We found that we could delete either of the two Cys-His boxes without greatly abrogating either RNA binding or packaging, although the two Cys-His boxes are likely to be required for efficient viral assembly and release. In contrast, substitutions for the Zn-coordinating residues within the boxes did prevent RNA binding, suggesting changes in the overall conformation of the protein. In the basic region between the two Cys-His boxes, three positively charged residues, as well as basic residues flanking the two boxes, were necessary for both binding and packaging. Our results suggest that the stretches of positively charged residues within NC that need to be in a proper conformation appear to be responsible for selective recognition and binding to the packaging signal (Ψ)-containing RNAs.

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Transfer of defective avian tumor virus genomes by a Rous sarcoma virus RNA packaging mutant.

Journal of Virology ◽

10.1128/jvi.38.1.380-382.1981 ◽

1981 ◽

Vol 38 (1) ◽

pp. 380-382 ◽

Cited By ~ 4

Author(s):

M Linial

Keyword(s):

Rous Sarcoma Virus ◽

Rna Packaging ◽

Rous Sarcoma ◽

Virus Rna ◽

Tumor Virus ◽

Sarcoma Virus ◽

Virus Genomes

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Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants.

Journal of Virology ◽

10.1128/jvi.68.9.5969-5981.1994 ◽

1994 ◽

Vol 68 (9) ◽

pp. 5969-5981 ◽

Cited By ~ 26

Author(s):

M Sakalian ◽

J W Wills ◽

V M Vogt

Keyword(s):

Rous Sarcoma Virus ◽

Deletion Mutants ◽

Rna Packaging ◽

Rous Sarcoma ◽

Sarcoma Virus

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Characterization of the DNA-Binding Domain of the Avian Y-Box Protein, chkYB-2, and Mutational Analysis of Its Single-Strand Binding Motif in the Rous Sarcoma Virus Enhancer

Journal of Virology ◽

10.1128/jvi.72.2.900-909.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 900-909 ◽

Cited By ~ 10

Author(s):

Ashok Nambiar ◽

S. K. Swamynathan ◽

Jagannadha C. Kandala ◽

Ramareddy V. Guntaka

Keyword(s):

Dna Binding ◽

Rous Sarcoma Virus ◽

Mutational Analysis ◽

Binding Domain ◽

Single Strand ◽

Dna Binding Domain ◽

Single Stranded Dna ◽

High Affinity ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT chkYB-2 is a sequence-specific, single-stranded DNA binding chicken Y-box protein that promotes Rous sarcoma virus long terminal repeat (RSV LTR)-driven transcription in avian fibroblasts. The DNA-binding domain of chkYB-2 has been mapped by characterizing the DNA binding properties of purified recombinant chkYB-2 mutant polypeptides. The data indicate that the invariant cold shock domain (CSD) is necessary but not sufficient for association with DNA and suggest that another conserved region, adjacent to the carboxyl boundary of the CSD, plays a role in high-affinity DNA binding. chkYB-2 binds to a tandem repeat of the 5′-GTACCACC-3′ motif on the RSV LTR. Mutational analysis of this recognition sequence revealed the requirement of an essentially unaltered template for both high-affinity binding by chkYB-2 as well as maximal transcriptional activity of the RSV LTR in vivo. The single-stranded DNA binding activity of chkYB-2 is augmented by Mg2+. The possible significance of this finding for transactivation by a single-strand DNA binding protein is discussed.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4769-4783.1992 ◽

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.

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