Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A) tails at the 3' end.

Abstract

We have determined the nucleotide sequence at the extreme 5' and 3' termini of the hepatitis C virus (HCV) genome. Our analyses ofthese sequences show (t) the nucleotide sequence in the 5' untranslated region is highly conserved among HCV isolates of widely varying geographical orign, (i) within this region, there are blocks of nucleotide sequence homology with pestiviruses but not with other viruses, (ii) the relative position of short open reading frames present in the same region of the HCV genome is similar to that of the pestiviral genome, (iv) RNAs truncated at the 5' and 3' ends are found, but the origin and functions of these RNAs are unknown, and (v) poly(A) tails appear to be present on 3' subgenomic RNAs. (ORFs). The HCV genome, however, displays singular characteristics at each terminus. We detect a hairpin structure at the 5' end of the genome as well as 5' and 3' subgenomic RNAs, the latter of which are polyadenylylated. These are consistent with a polyadenylylated 3' terminus of the viral genome and perhaps of functional subgenomic RNAs. Our data provide insights into the organization of the HCV genome, which may have important ramifications regarding the replication strategy and evolution of the virus. MATERIALS AND METHODSRNA was extracted from a high-titer plasma of an experimentally infected chimpanzee (6) and plasma (or serum) from HCV-positive or negative blood donors by a low-temperature guanidinium thiocyanate method (7). Poly(A)+ RNA was isolated from the liver ofthe same infected chimpanzee by the guanidinium thiocyanate/urea method (8). cDNA was synthesized from RNA according to Han and Rutter (9) and amplified by polymerase chain reaction (PCR) according to Saiki et al. (10). Briefly, RNA isolated from about 500 Al of plasma or S Ag of poly(A)+ liver RNA was converted into single-stranded cDNA by reverse transcriptase (BRL) using 150 pmol of the appropriate cDNA primer. For 5' end characterization by primer extension (7), first-strand cDNA was precipitated by spermine (11) and tailed with dA (9). Tailed or untailed cDNA was converted into double-stranded cDNA using a second-strand cDNA primer (9). This doublestranded cDNA was amplified using the indicated HCVspecific PCR primers for 35 cycles (940C, 1.5 min; 60'C, 2 min; 720C, 3 min). PCR without cDNA template was routinely performed to check for possible contamination during PCR. The PCR product was analyzed by Southern blot hybridization using a 32P-labeled oligonucleotide probe. The sequences and locations in the HCV genome of various cDNA and PCR primers are shown in Figs. 2 and 5. Most primers were designed to contain a Not I site for subsequent cloning of the PCR products into pUC18S, which contains a polylinker derived from Agt22 (9). DNA sequence was obtained by the supercoil sequencing (12) or the direct PCR sequencing method (13). RESULTSBased on the sequence of the major part of the HCV genome determined from overlapping A clones (2), we devised a directed strategy for obtaining clones representing the remaini...

The Particle Size Of Hepatitis C Virus Estimated By Filtration Through Microporous Regenerated Cellulose Fibre

“…HCV contains a positive-stranded RNA molecule of about 9400 nucleotides which contains a single large open reading frame encoding a viral polyprotein precursor of up to 3011 amino acids (aa) . The nucleotide and deduced aa sequences of the genomes of different isolates have shown that HCV is distantly related to flaviviruses and pestiviruses Miller & Purcell, 1990;Takeuchi et al, 1990;Kato et al, 1990;Takamizawa et al, 1991 ;Choo et al, 1991 ;Han et al, 1991). These characteristics, as well as the gene organization of HCV, are consistent with the theory that at least one of the causative agents of post-transfusion non-A, non-B hepatitis is a small enveloped virus, such as a togavirus or flavivirus (Bradley et al, 1983).…”

“…Thus, the diameter of HCV particles estimated in this study was smaller than had been thought previously. However, the 5' structural gene region of HCV is much smaller than that of flaviviruses and is more like that of pestiviruses (Takeuchi et al, 1990;Han et al, 1991), which also have smaller diameters and relatively lower buoyant densities than flaviviruses (Mayr et al, 1967;Horzinek, 1973). Another explanation may be that particle diameter is altered by the treatment of virus samples for electron microscopy or the pretreatment of the filter to minimize the adsorption of virus to the membrane during filtration (Kamide et al, 1982).…”

Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection

“…Conversely, asymptomatic HCV carriers with normal serum ALT levels (P õ .01). E2/NS1 antibody titers were also lower 8 of the 13 patients with low viremia of õ10 6 prevalence was estimated to be very low at 10 to 17%…”

Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses†

“…The putative organization of the HCV genome (ordered from the 5Ј-end) is as follows: the 5Ј-UTR; three or four structural proteins (core, E1, E2/p7); six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B); and the 3Ј-UTR. [5][6][7] However, the effects of these proteins on the functions of HCV-infected cells are not fully understood.…”