Identification of novel components of Trypanosoma brucei editosomes

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought

Hepatitis G virus in multitransfused thalassaemics from India

Hepatitis G virus (HGV)/GB virus-C (GBV-C) has been identified as a blood-borne agent with disputed pathogenicity. This virus belongs to the flaviviridae with a distant relationship to hepatitis C virus (HCV). Genetically divergent HGV isolates have been reported from different parts of the world. This study describes the prevalence of HGV in multitransfused thalassaemic children in India and genomic sequence variations in 11 HGV isolates from the same geographical location. Hepatitis G virus RNA was detected in 39.7% multitransfused thalassaemic children. The seroprevalence of hepatitis B virus (HBV) and HCV was 23.8% and 17.1%, respectively, and 11.4% had dual infection. The nucleotide sequence of a 166 bp HGV genomic segment from the putative capsid-envelope region (nucleotide; nt 578-743) from 11 Indian isolates was compared to the sequences available in the nucleotide databases. The isolates from India were 81.3-94.5% homologous to the isolates from other parts of the world. On phylogenetic analysis, it was observed that HGV isolates from India may belong to two genetically divergent types

Genotype determination of hepatitis C virus from Northern India: identification of a new subtype

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the nonstructural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India

TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

The uridine insertion/deletion RNA editing of kinetoplastid mitochondrial transcripts is performed by complex machinery involving a number of proteins and multiple protein complexes. Here we describe the effect of silencing of TbRGG1 gene by RNA interference on RNA editing in procyclic stage of Trypanosoma brucei. TbRGG1 is an essential protein for cell growth, the absence of which results in an overall decline of edited mRNAs, while the levels of never-edited RNAs remain unaltered. Repression of TbRGG1 expression has no effect on the 20S editosome and MRP1/2 complex. TAP-tag purification of TbRGG1 coisolated a novel multiprotein complex, and its association was further verified by TAP-tag analyses of two other components of the complex. TbRGG1 interaction with this complex appears to be mediated by RNA. Our results suggest that the TbRGG1 protein functions in stabilizing edited RNAs or editing efficiency and that the associated novel complex may have a role in mitochondrial RNA metabolism. We provisionally name it putative mitochondrial RNA-binding complex 1 (put-MRB complex 1)

Etiological role of hepatitis E virus in sporadic fulminant hepatitis

Non-A, non-B hepatitis viruses have been implicated as the etiological agent(s) in up to 60% of patients with fulminant hepatitis. These agents are reported to induce a higher mortality than other causes of fulminant hepatitis. Hepatitis E virus (HEV) and hepatitis C virus (HCV) at present constitute the major identifiable non-A, non-B hepatitis agents. Of these, HEV has been established as the sole cause of epidemic hepatitis in Afro-Asian countries, and fulminant hepatitis has been recorded during such epidemics. However, in sporadic cases, the etiological role of HEV in fulminant hepatitis has remained uncertain. The role of HCV in acute liver disease and fulminant hepatitis remains unclear. The present study was undertaken to investigate the association of HEV and HCV in patients with fulminant hepatitis by direct detection of the viral genome using reverse transcription-polymerase chain reaction (RT-PCR). Serum samples from 50 sero-logically identified non-A, non-B fulminant hepatitis cases negative for cryptic hepatitis B virus (HBV) infection examined via PCR were tested for HEV and HCV RNA using RT-PCR. For HEV primers from the nonstructural region (ORF-1) were used, and for HCV primers from the highly conserved 5' untranslated regions were used. The products were analysed using agarose gel electrophoresis and confirmed by hybridisation with radiolabelled internal oligonucleotide probes. HEV was detected in 31 (62%) of the 50 fulminant non-A, non-B hepatitis cases. In 18 (36%) cases, HCV RNA was detected. In 11 (22%) of the HCV cases, the HEV genome was also amplified. In 20 (40%) cases, HEV was detected alone. Twelve (24%) patients were negative for all viral hepatitis markers. This study establishes HEV as one of the major agents associated with sporadic fulminant hepatitis in a geographical region where epidemics of hepatitis caused by HEV are frequent

Diagnosis of hepatitis C virus-associated chronic liver disease in India: comparison of HCV antibody assay with a polymerase chain reaction for the 5' noncoding region

The relative value of an anti-hepatitis C virus (HCV) serological assay and reverse transcriptase-nested polymerase chain reaction assays (RT-PCR) were investigated for the constant 5' putative noncoding region of HCV for the diagnosis of HCV-associated chronic liver diseases in India. One hundred fifteen patients with biopsy proven chronic active hepatitis and 140 cases of cirrhosis of the liver were investigated for anti HCV antibody using a second generation commercial enzyme-linked immunosorbent assay (ELISA). A proportion of these patients: 42 with chronic hepatitis and 27 with cirrhosis of the liver were analysed further for HCV RNA in the serum using RT-nested PCR assay. Thirty-three (12.9%) of the 255 patients were positive for anti-HCV antibody and 23 of 69 (33.3%) patients were positive for HCV RNA in serum. Fifteen of the 33 (45.5%) anti-HCV positive patients had HCV RNA in the serum. Eight of 36 (22.2%) HCV seronegative patients tested were found with HCV RNA. This indicates that the diagnosis of HCV infection is not possible if it is based solely on the available serodiagnostic tests. Inclusion of both assays improved the diagnostic efficiency, 18.8% (13/69) were negative for all virological markers associated with HBV and HCV infection. Since a majority of the chronic liver disease patients (143/255 [56%]) were seronegative for either HBV or HCV infection, it is significant that HCV RNA was detected in 38% (8/21) of a randomly selected group from these patients. The antibody assay and PCR were compared using interclass correlation (kappa statistics). Both these tests were found to be independent of each other. Considering the wide strain variation throughout the world, it is suggested that the current commercial anti-HCV testing is inadequate for the diagnosis of all HCV infections in all geographical regions