Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus. NS5b is an RNA-dependent RNA polymerase that polymerizes the newly synthesized RNA. HCV likely uses host proteins for its replication, similar to other RNA viruses. To identify the cellular factors involved in HCV replication, we searched for cellular proteins that interact with the NS5b protein. HnRNP A1 and septin 6 proteins were identified by coimmunoprecipitation and yeast two-hybrid screening, respectively. Interestingly, septin 6 protein also interacts with hnRNP A1. Moreover, hnRNP A1 interacts with the 5-nontranslated region (5 NTR) and the 3 NTR of HCV RNA containing the cis-acting elements required for replication. Knockdown of hnRNP A1 and overexpression of C-terminally truncated hnRNP A1 reduced HCV replication. In addition, knockdown of septin 6 and overexpression of N-terminally truncated septin 6 inhibited HCV replication. These results indicate that the host proteins hnRNP A1 and septin 6 play important roles in the replication of HCV through RNA-protein and protein-protein interactions.Approximately 170 million people worldwide are persistently infected with hepatitis C virus (HCV), and these individuals account for most cases of chronic liver disease, such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (45,70).HCV has a single-stranded RNA genome of positive polarity that contains a single long open reading frame flanked by nontranslated regions (NTRs) at its 5Ј and 3Ј ends. Nearly the entire 5Ј NTR is needed for efficient RNA amplification, although the minimal replication element is within the first 120 nucleotides (20,35,58). In addition, the 5Ј NTR has been found to contain an internal ribosomal entry site (IRES), which is required for the translation of polyprotein (29,67,75). The 3Ј NTR is composed of a nonessential variable region, a poly(U)/poly(U ⅐ C) tract, and a highly conserved and essential 3Ј X domain (19,40,74). The viral proteins are translated as a single large polyprotein of 3,010 to 3,040 amino acids, which is cotranslationally and/or posttranslationally processed by cellular and viral proteases into mature structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) viral proteins (57). NS5b is an RNA-dependent RNA polymerase that produces complementary RNAs from template RNAs (3, 66).Subgenomic HCV RNA replicons have been developed to mimic the replication of a viral RNA infecting a host cell. These replicons are composed of the HCV 5Ј NTR; a selection marker, such as neomycin phosphotransferase; the IRES of encephalomyocarditis virus; HCV nonstructural proteins NS3, -4a, -4b, -5a, and -5b; and the HCV 3Ј NTR. When introduced into human hepatoma (Huh 7) cells, the HCV replicon RNA replicates autonomously (47). Using this replicon system, several groups have reported that HCV replication occurs in a distinct replication complex, which comprises viral RNA and HCV proteins (1,12,23,54). The replication complex is formed on intracellular membranes with vesic...
The GINS complex mediates the assembly of the MCM2-7 (minichromosome maintenance) complex with proteins in a replisome progression complex. The eukaryotic GINS complex is composed of Sld5, Psf1, Psf2, and Psf3, which must be assembled for cell proliferation. We determined the crystal structure of the human GINS complex: GINS forms an elliptical shape with a small central channel. The structures of Sld5 and Psf2 resemble those of Psf1 and Psf3, respectively. In addition, the N-terminal and C-terminal domains of Sld5/ Psf1 are permuted in Psf2/Psf3, which suggests that the four proteins have evolved from a common ancestor. Using a structure-based mutational analysis, we identified the functionally critical surface regions of the GINS complex.Supplemental material is available at http://www.genesdev.org.
It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin 3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin 3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin 3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin 3 gene. Human karyopherin 3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin 3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin 3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis (1,8,38). Chronic infection with HCV results in liver cirrhosis and hepatocellular carcinoma (7,45). HCV belongs to the family Flaviviridae, having a positive-sense RNA genome (32,42,47). The RNA encodes a polyprotein (ϳ3,010 amino acids) with the following gene order: 5Ј-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3Ј. During and/or after translation, the polyprotein is processed into functional proteins by host-and virus-encoded proteases. Core (C) and envelope (E1 and E2) proteins are believed to compose the structural elements of the virion particle. Nonstructural protein 2 (NS2), NS3, and NS4A are involved in the proteolytic processing of the HCV polyprotein (4,5,15,18,25,26,27,28,30,40,50,52). RNA-dependent RNA polymerase and RNA helicase activities are assigned to NS5B and the C-terminal two-thirds of NS3, respectively (6,36). No function has yet been assigned to NS4B.NS5A exists in two different forms (p56 and p58) in cells. The proteins differ in their phosphorylation status (2, 34, 51). NS4A or NS3-4A-4B augments hyperphosphorylation of NS5A (43, 51). The sequence around the middle part of NS5A (amino acids 2209 to 2248) is termed the interferon sensitivity-determining region, since it correlates with interferon sensitivity of the HCV genotype 1b (16,17,39). The sequence in the interferon sensitivity-determining region was shown to play a key role in the inhibition of the protein kinase PKR, a mediator of interferon-induced resistance, through protein-protein interaction (20, 21). NS5A was also shown to interact with a SNARE-like protein (53). The C-terminal region of NS5A contains a potential transcriptional activation domain, but the role of its activity ...
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