Cellular Transcription Factor Sp1 Recruits Simian Virus 40 Capsid Proteins to the Viral Packaging Signal, ses (original) (raw)
Related papers
Journal of Molecular Biology, 1998
Chromatin structure and protein± protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found signi®cant cooperativity between Sp1 and VP3 in speci®c DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These ®ndings have led us to conclude that the novel host ± viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.
Assemblages of simian virus 40 capsid proteins and viral DNA visualized by electron microscopy
Biochemical and Biophysical Research Communications, 2007
SV40 assembles in the nucleus by addition of capsid proteins to the minichromosome. The VP1 5 VP2/3 capsomer is composed of a pentamer of the major protein VP1 complexed with a monomer of a minor protein, VP2 or VP3. In the capsid, the capsomers are bound together via their flexible carboxy-terminal arms. Our previous studies suggested that the capsomers are recruited to the packaging signal ses via avid interaction with Sp1. During assembly Sp1 is displaced, allowing chromatin compaction. Here we investigated the interactions in vitro of VP1 5 VP2/3 capsomers with the entire SV40 genome, using mutant VP1 deleted in the carboxy-arm that cannot assemble, but retains DNA-binding capacity. EM revealed that VP1 5 VP2/3 complexes bind non-specifically at random locations around the DNA. Sp1 was absent from mature virions. The findings suggest that multiple capsomers attach simultaneously to the viral genome, increasing their local concentration, facilitating rapid, concerted assembly reaction and removal of Sp1.
Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA
Proceedings of the National Academy of Sciences, 1996
All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood. The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was not observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of prote...
Identification of a DNA binding domain in simian virus 40 capsid proteins Vp2 and Vp3
Journal of Biological Chemistry, 1993
We have identified both biochemically and genetically a protein domain within the simian virus 40 virion protein Vp3, and within Vp2 since its carboxyl two-thirds are identical to the full-length Vp3, that binds DNA in a sequence nonspecific manner. Both the Vp2 and Vp3 (Vp2/3) components of SV40 and mutant SV40101~ bound either SV40 or pBR322 DNA equally well. Wild type and mutant Vp2/3 proteins, expressed as fusion proteins with glutathione S-transferase (GST), were tested for their ability to bind DNA. GST-Vp3 bound DNA at physiological salt concentrations with an apparent Kd of 2.5 % M and also bound RNA with 4-fold higher affinity. Over 90% of the nucleic acid binding, and all of the activity, was lost upon removal of the carboxyl-terminal 13 and 35 residues, respectively. The DNA binding domain was shown to be distinct and separable from the Vp2/3 nuclear transport signal since mutations within the nuclear transport signal that reduce or abolish nuclear localization of Vp2/3 had no effect on the DNA binding activity of mutant Vp2/3 fusion proteins. The carboxyl-terminal 40 residues of Vp2/3 in the form of a &galactosidase fusion protein, F6, are sufficient for DNA binding and may cause compaction of the DNA. The significance of this DNA binding and possible compaction are discussed in relation to the assembly of virion particles.
Journal of Virology, 2001
A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1⌬C58 pentamer bound DNA with a K d of 1.8 ؋ 10 ؊9 M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1⌬C17 had comparable apparent K d s of 5.3 ؋ 10 ؊9 to 7.3 ؋ 10 ؊9 M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1⌬C58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 ⅐ 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1⌬C58 level for NLSm1 ⅐ 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209-8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlaping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1 ⅐ 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1 ⅐ 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.
Journal of Virology
The early proteins of simian virus 40 (SV40) large T and small t antigen (T/t antigen) can each cause the transcriptional activation of a variety of cellular and viral promoters. We showed previously that simian cellular DNA-binding factors (the Band A factors) bind to sequences within the SV40 late promoter which are important for transcriptional activation in the presence of the SV40 early proteins. Band A factors isolated from simian cells which produce T/t antigen (COS cells or SV40-infected CV-1 cells) have altered binding properties in comparison with the factors from normal simian cells (CV-1). This suggests that the transcriptional activation mediated by T/t antigen may be due to either modification of existing factors or induction of new members of a family of factors. We have purified the Band A factors from both COS and CV-1 cells and have determined the binding site by methylation interference and DNase protection footprinting. The COS cell factors have altered chromatog...
DNA-binding properties of the major structural protein of simian virus 40
1986
We investigated whether the VPl protein of simian virus 40 binds to DNA. In vitro DNA-binding experiments clearly indicate that VP1 bound strongly to double-stranded and single-stranded DNA, with a higher affinity for the latter; additional experiments show that VP1 did not bind to a specific sequence of simian virus 40 DNA.
Essential role of the Vp2 and Vp3 DNA-binding domain in simian virus 40 morphogenesis
Journal of virology, 1995
Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a Kd of 3 x 10(-8) identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4- to 7-fold or 20- to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA b...
Proceedings of the National Academy of Sciences, 1985
A 440-base-pair fragment of African green monkey genomic DNA shares homology with the transcriptional regulatory region of simian virus 40 (SV40) and has been reported to direct transcription in vivo. We find that two regions within this fragment bind the promoter-specific cellular transcription factor Spl and are protected in DNase protection ("footprinting") experiments. As in SV40, binding occurs in regions containing multiple copies of the sequence GGGCGG. These regions, when fused to the proximal, or "TATA box," element ofthe herpes simplex virus thymidine kinase promoter, are able to direct Spl-dependent transcription in vitro. The finding that Spl is capable of productive interaction with sequences taken from a cellular promoter supports the idea that Abbreviations: SV40, simian virus 40; tk, thymidine kinase; bp, base pair(s). Present address: The Jackson Laboratory, Bar Harbor, ME 04609.
Journal of Molecular Biology, 1996
Using the experimental system of simian virus 40 (SV40) pseudovirions we Department of Hematology The Hebrew University have previously shown that SV40 requires a specific DNA element for Hadassah Medical School packaging, ses, which was mapped to the SV40 regulatory region. ses was Jerusalem, Israel, 91120 previously found to play a role in facilitating the nucleosomal rearrangement required for chromatin condensation and viral packaging.