DNA binding domain and subunit interactions of transcription factor IIIC revealed by dissection with poliovirus 3C protease.

Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.

Inhibition of host cell transcription by poliovirus: cleavage of transcription factor CREB by poliovirus-encoded protease 3Cpro.

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. Previous studies from our laboratory showed that activated transcription from a cyclic AMP-responsive element (CRE)-containing promoter was severely inhibited in extracts prepared from poliovirus-infected HeLa cells compared to those from mock-infected cells. Here we demonstrate that the CRE-binding protein, CREB, is specifically cleaved by the poliovirus-encoded protease 3Cpro both in vitro and in virus-infected cells. The proteolytic cleavage of CREB leads to a significant loss of its DNA binding as well as transcriptional activity. Additionally, we demonstrate that the phosphorylated, transcriptionally active form of CREB is cleaved by the viral protease in vitro. The results presented here suggest that a direct cleavage of CREB by the viral protease 3Cpro leads to inhibition of CREB-activated transcription in poliovirus-infected HeLa cells.

Poliovirus-encoded protease 2APro cleaves the TATA-binding protein but does not inhibit host cell RNA polymerase II transcription in vitro.

Transient expression of the poliovirus-encoded protease 2APro in eukaryotic cells results in inhibition of both cellular transcription and translation. The inhibition of transcription observed in cells expressing 2APro could be due to a primary effect or secondary effect caused by inhibition of translation. Because transcriptional activity of the TATA-binding protein (TBP) is drastically reduced in poliovirus-infected cells, we determined if 2APro is able to cleave TBP in vitro. We demonstrate here that 2APro directly cleaves the single tyrosine-glycine bond at position 34 of TBP. This cleavage is also seen in poliovirus-infected HeLa cells. Surprisingly, despite TBP cleavage 2APro was unable to inhibit RNA polymerase II transcription in vitro. Under similar conditions, however, 2APro inhibited translation of a capped cellular mRNA in vitro. Thus, cleavage of TBP at position 34 does not alter its transcriptional activity in vitro. These results suggest that inhibition of host cell RNA polymerase II transcription seen in cells transiently transfected with 2APro is due to host cell translational shutoff.

Cleavage of transcriptional activator Oct-1 by poliovirus encoded protease 3Cpro.

In HeLa cells, RNA polymerase II mediated transcription is severely inhibited by poliovirus infection. Both basal and activated transcription are affected to bring about a complete shutoff of host cell transcription. We demonstrate here that the octamer binding transcription factor, Oct-1, is cleaved in HeLa cells infected with poliovirus. Incubation of Oct-1 with the purified, recombinant 3Cpro results in the generation of the cleaved Oct-1 product seen in virus infected cells. Poliovirus infection leads to the formation of altered Oct-1 DNA complexes that can also be generated by incubation of Oct-1 with purified 3Cpro. We also show that Oct-1 cleaved by 3Cpro loses its ability to inhibit transcriptional activation by the SV40 B enhancer. These results suggest that cleavage of Oct-1 in poliovirus infected cells leads to the loss of its activity.

Inhibition of basal transcription by poliovirus: a virus- encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro.

Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient-transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences.

Folding and function of I domain-deleted Mac-1 and lymphocyte function-associated antigen-1.

In those integrins that contain it, the I domain is a major ligand recognition site. The I domain is inserted between beta-sheets 2 and 3 of the predicted beta-propeller domain of the integrin alpha subunit. We deleted the I domain from the integrin alpha(M) and alpha(L) subunits to give I-less Mac-1 and lymphocyte function-associated antigen-1 (LFA-1), respectively. The I-less alpha(M) and alpha(L) subunits were expressed in association with the wild-type beta(2) subunit on the surface of transfected cells and bound to all the monoclonal antibodies mapped to the putative beta-propeller and C-terminal regions of the alpha(M) and alpha(L) subunits, suggesting that the folding of these domains is independent of the I domain. I-less Mac-1 bound to the ligands iC3b and factor X, but this binding was reduced compared with wild-type Mac-1. In contrast, I-less Mac-1 did not bind to fibrinogen or denatured bovine serum albumin. Binding to iC3b and factor X by I-less Mac-1 was inhibited by the function-blocking antibody CBRM1/32, which binds to the beta-propeller domain of the alpha(M) subunit. I-less LFA-1 did not bind its ligands intercellular adhesion molecule-1 and -3. Thus, the I domain is not essential for the folding, heterodimer formation, and surface expression of Mac-1 and LFA-1 and is required for binding to some ligands, but not others.

Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits.

Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

Testicular torsion: a review.

Torsion of the testis, also referred to as torsion of the spermatic cord, is a subject of debate among physicians and surgeons. Testicular torsion is an acute vascular event causing the rotation of the vascular pedicle of the testis, thereby impeding the blood flow to the testis and the scrotal contents. It could be either within or outside the tunica vaginalis. Testicular torsion causes immediate circulatory changes and long-term sequelae such as testicular function and fertility. It is considered a surgical emergency, as a delay causes irreversible testicular damage. The diagnosis and treatment of testicular torsion are discussed in this review, which also illustrates an algorithm and a scoring system for the diagnosis and management of this condition based on current literature.

Poliovirus 3C protease-mediated degradation of transcriptional activator p53 requires a cellular activity.

Infection of HeLa cells with poliovirus leads to rapid shut-off of host cell transcription by RNA polymerase II. Previous results have suggested that both the basal transcription factor TBP (TATA-binding protein) and transcription activator proteins such as CREB (cyclic AMP-responsive element-binding protein) and Oct-1 (the octamer-binding factor) are cleaved by the viral-encoded protease, 3C(Pro). Here we demonstrate that the transcriptional activator (and tumor suppressor) p53 is degraded by the viral protease 3C both in vivo and in vitro. Unlike other transcription factors that are directly cleaved by 3C(pro), degradation of p53 requires a HeLa cell activity in addition to 3C(Pro). The degradation of p53 by 3C(Pro) does not appear to involve the ubiquitin pathway of protein degradation. Vaccinia virus infection of HeLa cells leads to inactivation of the cellular activity required for 3C(Pro)-mediated degradation of p53. The vaccinia-encoded protein (CrmA) is known to inhibit caspase I (ICE protease) that converts inactive IL-1beta to an active secreted form. Incubation of HeLa cells with caspase I inhibitor Z-VAD-fmk does not interfere with 3C(Pro)-mediated degradation of p53. The cellular activity present in extracts of HeLa cells can be fractionated through phosphocellulose. A partially purified fraction that elutes at 0.6 M KCl from phosphocellulose contains the activity that degrades p53 in a 3C(Pro)-dependent manner. These results suggest that both poliovirus-encoded protease 3C(Pro) and a cellular activity are required for the degradation of p53 observed in cells infected with poliovirus.