The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990 -3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferaseNS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919 -2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.

Hepatitis B virus X protein (HBx) transactivates viral and cellular genes through a wide variety of cis-elements. However, the mechanism is still obscure. Our finding that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common subunit of RNA polymerases, implies that HBx directly modulates the function of RNA polymerase (Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 142-150). In this context, we examined the possibility that HBx and RPB5 interact with other general transcription factors. HBx and RPB5 specifically bound to transcription factor IIB (TFIIB) in vitro, both of which were detected by either far-Western blotting or the glutathione S-transferaseresin pull-down assay. Delineation of the binding regions of these three proteins revealed that HBx, RPB5, and TFIIB each has two binding regions for the other two proteins. Co-immunoprecipitation using HepG2 cell lysates that express HBx demonstrated trimeric interaction in vivo. Some HBx substitution mutants, which had severely impaired transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB. These results indicated that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.

To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). In eukaryotes, nuclear RNA polymerases I, II, and III are highly conserved multisubunit enzymes involved in the synthesis of rRNAs, mRNAs, and tRNAs, respectively (48). All these nuclear RNA polymerases share the function of RNA synthesis but utilize different promoters and require different transcription factors. For example, promoter-specific transcription initiation from protein-coding genes requires the concerted action of a complex array of factors involving RNA polymerase II and general transcriptional factors (TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) (14,46). Transcriptional activators and repressors bind distal elements of a promoter and modulate transcription through communication with components of a preinitiation complex, such as TATA-binding protein (TBP) and its binding proteins, TFIIB, TFIIA, and TFIIH (18,19,24,42). Recently, another group of proteins, cofactors (also called mediators), have been demonstrated to affect transcription positively or negatively by communicating with promoterspecific regulatory factors and the transcriptional machinery. These proteins include global coactivator p300/CBP (20, 25, 31, 34), nuclear silence mediators (2, 12, 52), and a large number of mediator proteins, or SRBs (10,28,39,47,54). RNA polymerase subunits may be additional targets for transcriptional regulators, because RNA polymerases are the ultimate target of transcriptional modulation. In this context, several subunits have been recently reported to interact with the regulators (8, 9), in addition to the well-documented regulatory role of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (28, 30).Hepatitis B virus (HBV) X protein (HBx) is essential for HBV infection and plays an important role in HBV-associated hepatocellular carcinoma (11,17,26,51). Many reports have shown that HBx transactivates viral and cellular genes through a wide variety of cis-acting elements. However, the mechanism of this effect has not been well elucidated (3,5,7,15,16,22,23,50). It has been previously demonstrated that HBx directly interacts with RNA polymerase II subunit 5 (RPB5), a common RNA polymerase subunit (13), that both RPB5 and HBx communicate with TFIIB but through different sites (21), and that the trimeric interaction of these three factors is involved in HBx t...

Gastrointestinal symptoms, such as abdominal pain and diarrhea, are frequently observed in patients with Plasmodium falciparum malaria. However, the correlation between malaria intestinal pathology and intestinal microbiota has not been investigated. In the present study, infection of C57BL/6 mice with P. berghei ANKA (PbA) caused intestinal pathological changes, such as detachment of epithelia in the small intestines and increased intestinal permeability, which correlated with development with experimental cerebral malaria (ECM). Notably, an apparent dysbiosis occurred, characterized by a reduction of Firmicutes and an increase in Proteobacteria. Furthermore, some genera of microbiota correlated with parasite growth and/or ECM development. By contrast, BALB/c mice are resistant to ECM and exhibit milder intestinal pathology and dysbiosis. These results indicate that the severity of cerebral and intestinal pathology coincides with the degree of alteration in microbiota. This is the first report demonstrating that malaria affects intestinal microbiota and causes dysbiosis.

HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication, which has the "palm and fingers" substructure. We recently identified five novel residues critical for RdRP activity (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728 -737). Among them, GLU-18 and His-502, far from the catalytic center, may be involved in conformational change(s) for RdRP activity as addressed in some palm and fingers enzymes. We examined the possibility that NS5B is oligomerized, and we could detect the interaction between two different tagged NS5B proteins in vitro and transiently expressed in mammalian cells. By scanning 27 clustered and then point alanine substitutions in vivo and in vitro, Glu-18 and His-502 were found to be critical for the homomeric interaction in vivo and in vitro, strongly suggesting a close relationship between the oligomerization and RdRP activity of NS5B. All mutants with substitutions at these two residues failed to bind wild type NS5B, however E18H interacted with H502E in vitro and in vivo. Interestingly, the NS5B protein with E18H or H502E did not exhibit RdRP activity, but a mixture of the two mutant proteins did. These results clearly indicate that two residues of HCV NS5B are critical for the oligomerization that is prerequisite to RdRP activity. Hepatitis C Virus (HCV)1 is the major causative agent of parenterally transmitted hepatitis (1-3). HCV infection frequently leads to chronic hepatitis, liver cirrhosis, and eventually hepatocellular carcinoma (4,5). In the case of HCV-associated hepatocellular carcinoma, there is often prolonged active inflammation manifested by high alanine aminotransferase level generally associated with high virus load. Therefore, eradication of replication of HCV would be expected to reduce or even prevent incidence of hepatocellular carcinoma. HCV has a positive-sense single-stranded RNA genome of approximately 9.6 kb, which contains a large open reading frame encoding a polyprotein of ϳ3,000 amino acid residues and two highly conserved untranslated regions flanking the 5Ј and 3Ј ends of the genome. The viral encoded polypeptide precursor is cotranslationally or posttranslationally processed by host and viral proteases into at least 10 distinct products: NH 2 -C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (6 and the references therein). The non-structural proteins NS2-5B are thought to be required for viral genome replication.The exact mechanism of replication of the HCV genome remains to be elucidated (7,8), but NS5B is an RNA-dependent RNA polymerase (RdRP), a core enzyme for HCV replication. NS5B belongs to a large family of nucleic acid-dependent nucleic acid polymerases having the palm and fingers substructure and harboring the motifs (ABCDEF) conserved among RdRPs. Several residues of NS5B within the conserved motifs have been identified as critical for RdRP activity by introducing mutations within the motifs (7-14). In addition, NS5B has several unique structural characteristics...