NS1-Binding protein (NS1-BP): a novel human protein that interacts with the influenza A virus nonstructural NS1 protein is relocalized in the nuclei of infected cells

We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathione S-transferase-NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of approximately 50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.     

BCL6 gene rearrangement and other cytogenetic abnormalities in diffuse large cell lymphoma

Evidence for rearrangement of the BCL6 gene at 3q27 has been documented in 20-30% diffuse lymphomas with a large cell component (DLLC), and was found to be of prognostic significance at the time of diagnosis. To incorporate these observations into current cytogenetic and clinical prognostic models, 76 cases of DLLC with known BCL6 status were analyzed. Cytogenetic indicators of progression, including trisomy 7, trisomy 12, del(6)(q21q25), and structural alterations of 17p were less frequent in BCL6 rearranged DLLC compared to BCL6 germline tumors. Despite a 93% overall survival at median follow-up of 30 months, a trend for continued relapse resulted in a projected freedom from progression for the BCL6 rearranged cohort of 66% at 4 years, compared to 39% for the BCL6 germline cohort. Six cases among the BCL6 rearranged group lacked additional cytogenetic indicators of progression and remained free of disease at follow-up in excess of 7 years, whereas BCL6 rearranged cases with increasing numbers of cytogenetic aberrations showed decreased intervals free from progression of disease. These results suggest that BCL6 rearrangement should be combined with other known clinical and cytogenetic indicators in prognostic analyses of patients with DLLC.     

NRP/B, a novel nuclear matrix protein, associates with p110(RB) and is involved in neuronal differentiation

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted NH2 terminus, and a "kelch motif" in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110(RB)) is found to be associated with the nuclear matrix and overexpression of p110(RB) induces neuronal differentiation, we investigated whether NRP/B is associated with p110(RB). Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110(RB) during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110(RB) and participates in the regulation of neuronal process formation.     

Rearrangements of the BCL6 gene and chromosome aberrations affecting 3q27 in 54 patients with non-Hodgkin's lymphoma

Chromosome aberrations affecting 3q27 are among the most frequent non-random abnormalities in non-Hodgkin's lymphomas (NHL), especially the diffuse, large cell type. Recently, an association between BCL6 rearrangement and frequent extranodal lesions, rare bone marrow infiltration and a favorable clinical outcome was reported. We performed molecular studies of the BCL6 gene in 54 patients with NHL. Twelve patients (22%) with rearranged BCL6 genes were selected for histological, clinical, molecular, and cytogenetic studies. Ten of these cases were diffuse, large cell type lymphoma, one a follicular lymphoma, and one a mantle cell lymphoma (MCL). All cases were of the B-cell type and this is the first time a rearranged BCL6 gene has been found in an MCL. Cytogenetic data for 10 cases were available and the partner sites of the 3q27 translocation were determined in 7 of 10 patients. These locations were variable, including 6p21.3, 9p22, and 14q11 in addition to the immunoglobulin loci 14q32 (IGH), 2p12 (IGK), and 22q11 (IGL). The heterogeneity in partner sites is distinct from other lymphoma subgroups and may suggest that the genetic events are not uniform among patients with BCL6 rearrangements.     

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.