Evolution of naturally occurring 5' non-translated region variants of hepatitis C virus genotype 1b in selectable replicons

Quasispecies shifts are essential for the development of persistent hepatitis C virus (HCV) infection. Naturally occurring sequence variations in the 5' non-translated region (NTR) of the virus could lead to changes in protein expression levels, reflecting selective forces on the virus. The extreme 5' end of the virus' genome, containing signals essential for replication, is followed by an internal ribosomal entry site (IRES) essential for protein translation as well as replication. The 5' NTR is highly conserved and has a complex RNA secondary structure consisting of several stem-loops. This report analyses the quasispecies distribution of the 5' NTR of an HCV genotype 1b clinical isolate and found a number of sequences differing from the consensus sequence. The consensus sequence, as well as a major variant located in stem-loop IIIa of the IRES, was investigated using self-replicating HCV RNA molecules in human hepatoma cells. The stem-loop IIIa mutation, which is predicted to disrupt the stem structure, showed slightly lower translation efficiency but was severely impaired in the colony formation of selectable HCV replicons. Interestingly, during selection of colonies supporting autonomous replication, mutations emerged that restored the base pairing in the stem-loop. Recloning of these altered IRESs confirmed that these second site revertants were more efficient in colony formation. In conclusion, naturally occurring variants in the HCV 5' NTR can lead to changes in their replication ability. Furthermore, IRES quasispecies evolution was observed in vitro under the selective pressure of the replicon system

Organ tropism of murine coronavirus does not correlate with the expression levels of the membrane-anchored or secreted isoforms of the carcinoembryonic antigen-related cell adhesion molecule 1 receptor

Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Characterization of defective interfering RNAs of Berne virus

were generated by serial undiluted passaging of the virus in embryonic mule skin cells. Two DI RNAs of 1.0 and 1.4 kb (designated DI1000 and DI1400) were characterized in more detail. Isokinetic sucrose gradi-ent analysis howed that these DI RNAs are specifical-ly packaged into particles with smaller S values than standard virions. Both DI RNAs were cloned and sequenced. Three genomic DNA clones were identi-fied using probes complementary to the 5 ' end of a DI RNA, which are thought to be derived from the 5'-terminal region of the BEV genome. A non-translated region of about 700 nt and the 5 ' end of the putative BEV replicase gene were identified in the consensus nucleotide sequence. Both DI RNAs were shown to contain sequences from the 5 ' and 3 ' ends of the BEV genome. A conserved sequence motif, which has been postulated to be involved in sub-genomic RNA tran-scription, was also identified just downstream of the extreme 5 ' ends of DI1000 and DI1400

The human collagen beta(1-O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein

Background: Glycosyl transferases transfer glycosyl groups onto their substrate. Localization partially defines their function. Glycosyl transferase 25 domain 1 (GLT25D1) was recently shown to have galactosyltransferase activity towards collagens and another well known substrate, mannose binding lectin (MBL). To gain more insight in the role of galactosylation of lysines in the Gly-X-Lys repeats of collagenous proteins, we investigated the subcellular localization of GLT25D1. Results: Immunofluorescence analysis of GLT25D1 expressed in the human hepatoma cell line (Huh7), revealed a perinuclear lattice like staining, resembling localization to the endoplasmic reticulum (ER). Possible targeting signals, an N-terminal signal sequence and a C-terminal ER-retention signal, were identified using prediction programs. These signals were then investigated by constructing a series of epitope-tagged forms of GLT25D1 that were analyzed by immunofluorescence and western blotting. In agreement with the predictions our results show that GLT25D1 is directed to the ER lumen as a soluble protein and retained there. Moreover, using two endoglycosidase enzymes EndoH and EndoF, we demonstrate that the putative bi-functional glycosyl transferase itself is a glycoprotein. Additionally we examined co-localization of GLT25D1 with MBL and lysyl hydroxylase 3 (LH3, PLOD3), which is a protein able to catalyze hydroxylation of lysine residues before they can be glycosylated. We demonstrate overlapping localization patterns of GLT25D1, MBL and LH3. Conclusions: Taken together our data indicate that galactosylation of collagenous proteins by the soluble GLT25D1 occurs in the early secretory pathway.Medical Microbiolog

Equine arteritis virus (EAV) induced polypeptide synthesis

Intracellular virus-specific proteins induced by equine arteritis virus (EAV) have been compared with in vitro translation products of virion and intracellular EAV RNAs. In infected BHK-21 cells, the two major virion proteins (C and El) an