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Transmission of Hepatitis C by Intrahepatic Inoculation with Transcribed RNA

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Science  25 Jul 1997:
Vol. 277, Issue 5325, pp. 570-574
DOI: 10.1126/science.277.5325.570

Abstract

More than 1% of the world's population is chronically infected with hepatitis C virus (HCV). HCV infection can result in acute hepatitis, chronic hepatitis, and cirrhosis, which is strongly associated with development of hepatocellular carcinoma. Genetic studies of HCV replication have been hampered by lack of a bona fide infectious molecular clone. Full-length functional clones of HCV complementary DNA were constructed. RNA transcripts from the clones were found to be infectious and to cause disease in chimpanzees after direct intrahepatic inoculation. This work defines the structure of a functional HCV genome RNA and proves that HCV alone is sufficient to cause disease.

In 1989, HCV, the viral agent believed to be responsible for most posttransfusion non-A, non-B hepatitis, was molecularly cloned (1). HCV is an enveloped positive-strand RNA virus classified in the family Flaviviridae (2). Characterization of HCV genome organization and expression has progressed rapidly since its discovery (3). The HCV genome RNA is ∼9.6 kb and consists of a 5′ nontranslated region (NTR) that functions as an internal ribosome entry site, a long open reading frame (ORF) encoding a polyprotein of >3000 amino acids, and a 3′ NTR. The genome RNA was originally thought to terminate with polyadenylate [poly(A)] or polyuridylate [poly(U)] tracts, but recent studies have revealed the presence of an internal poly(U)/polypyrimidine [poly(U/UC)] tract followed by a highly conserved 98-base sequence (4, 5). The HCV polyprotein is processed by host signal peptidase and two viral proteinases to yield at least 10 different structural and nonstructural (NS) proteins (Fig.1). Properties of many of the HCV-encoded replication enzymes, such as the serine proteinase, RNA helicase, and polymerase have begun to emerge as part of intensive efforts to develop new and more effective treatments for chronically infected HCV carriers. These efforts have been hampered, however, by poor replication in cell culture, the restricted host range of the virus (the chimpanzee, Pan troglodytes, is the only known nonhuman host), and the lack of an infectious molecular clone for genetic analysis of HCV functions.

Figure 1

Features of the 10 full-length consensus clone derivatives tested in chimpanzees. (Top) Schematic of HCV H77 cDNA consensus RNA. The 5′ and 3′ NTRs (solid lines) and the consensus ORF (open box) are indicated with the approximate locations of the polyprotein cleavage products shown below. (Bottom) The 10 RNA transcripts used for chimpanzee inoculation are diagrammed. Additional 5′ nucleotides and 75-base versus 133-base poly(U/UC) tracts are indicated. All clones and transcripts included two silent nucleotide substitutions: position 899 (C instead of U, asterisks); position 5936 (C instead of A, open circles) (nucleotide positions refer to the HCV H sequence). The substitution at position 5936 inactivated an internal Bsm I site in the H77 cDNA sequence so that an engineered Bsm I site could be used for production of runoff RNA transcripts with the exact 3′ terminus of HCV genome RNA (15). Clones with additional 5′ bases contained a silent marker mutation inactivating the Xho I site at position 514 (solid triangles). Clones with 75-base versus 133-base poly(U/UC) tracts were distinguished by A (solid circles) versus G at position 8054, respectively. The 10 derivatives were created by standard recombinant DNA methods and their structures were verified by restriction digestion and sequence analysis.

Problems with constructing a functional HCV cDNA clone include the highly variable “quasi-species” nature of this RNA virus, the small quantities of viral RNA present in clinical samples, which necessitate in vitro amplification before cloning, and the lack of a simple and verified transfection assay for infectivity. As starting material, we used serum obtained from a patient in the early phase of infection (designated H77) (6). This high specific–infectivity serum contains ∼108 RNA molecules and ∼106.5 chimpanzee infectious doses per milliliter. A combinatorial library with a complexity of >105full-length HCV cDNAs was constructed by high-fidelity assembly reverse transcription–polymerase chain reaction (RT-PCR) followed by subcloning into a recipient plasmid vector containing the 5′- and 3′-terminal HCV consensus sequences (7). A flanking T7 promoter and an engineered specific restriction site allowed for production of runoff RNA transcripts; 233 of the clones were prescreened by restriction analysis, polyprotein processing, and production of the COOH-terminal NS5B protein (the HCV RNA-dependent RNA polymerase). In an unsuccessful experiment, RNAs transcribed from 34 clones that passed these analyses were assayed for infectivity by direct intrahepatic injection in two HCV-naı̈ve chimpanzees (8). As a negative control, a third animal received similar injections with transcripts from a clone containing a 20–amino acid in-frame deletion encompassing the NS5B polymerase active site. Serum samples were collected for 2 months after inoculation and analyzed for evidence of HCV replication. Neither experimental animal nor the negative control animal exhibited signs of productive infection (circulating HCV RNA, increased liver enzymes, histopathology, or seroconversion) (9). Of note was the absence of detectable circulating HCV RNA 2 days after inoculation.

Missing terminal sequences, low RNA transfection efficiencies in vivo, and errors introduced during cDNA synthesis or PCR amplification might account for these negative results. Because the latter concern was readily addressed, we sequenced six clones from the combinatorial library, as well as uncloned RT-PCR products, to determine a consensus sequence for the H77 isolate (10). From this analysis, it became apparent that each of the six clones sequenced contained numerous non–consensus sequence changes scattered throughout the genome, which could be deleterious and would explain the negative results. We used this information to direct the assembly of a full-length clone reflecting the H77 consensus sequence (11). Aside from the sporadic changes corrected in this consensus clone, even greater sequence heterogeneity was noted in three regions of the HCV genome. During analysis of the extreme 5′ terminus, we found a substantial number of clones that contained one or more bases in addition to the reported 5′-terminal sequence (5′-GCCAG … -3′) (12). In the structural protein coding region, the NH2-terminus of the E2 virion envelope glycoprotein was highly variable, but a predominant sequence was identified (13). Near the 3′ terminus, the poly(U/UC) tract was variable in length and composition (14). Because these differences might be functionally significant, variants of the consensus clone with additional bases at the 5′ terminus and two different poly(U/UC) tracts were constructed (Fig. 1). Silent nucleotide substitutions were engineered as markers so that virus recovered from transfected animals could be sequenced to identify which clones were infectious.

From each of the 10 clones, full-length uncapped RNAs were transcribed from linearized template DNAs (15) and used for inoculation of two chimpanzees that were seronegative for all known hepatitis viruses, were negative for HCV RNA by nested RT-PCR, and had normal baseline amounts of liver enzymes. Animals were inoculated by direct intrahepatic injection at multiple sites (16). Serum samples and liver biopsies were taken before inoculation and at weekly intervals thereafter. For 3 months after inoculation, serum samples were assayed for liver enzymes, antibodies to HCV, and viremia, as assessed by quantifying circulating HCV RNA by branched DNA (bDNA) and quantitative-competitive (QC) RT-PCR (5).

Chimpanzee 1535 had increased concentrations of serum alanine aminotransferase (ALT), which is a marker for liver damage, at week 2 after inoculation; ALT gradually declined to the preinoculation baseline amount and then increased again in weeks 10 to 12 (Fig.2A). HCV RNA titers were undetectable before inoculation, increased to 0.45 Meq/ml by week 1, decreased slightly at week 2 (0.28 Meq/ml), and then continued to climb steadily, reaching 8.3 Meq/ml by week 13. This animal became clearly HCV seropositive at week 13 (Fig. 2C) (17). Chimpanzee 1536 showed no evidence of early liver damage (Fig. 2B). At week 10, ALT concentrations increased sharply and peaked at week 12, declining rapidly thereafter. At week 1, the HCV RNA titer was undetectable by bDNA (detection limit, 0.2 Meq/ml or ∼2 × 105 RNA molecules per milliliter), but 104 RNA molecules per milliliter were measured by QC RT-PCR. The circulating HCV RNA titer peaked at week 10 (5.8 Meq/ml) and then declined to quantities undetectable by bDNA by week 13. Chimpanzee 1536 had seroconverted by week 10 (Fig. 2C). Histologic changes in liver biopsies were typical for hepatitis C in chimpanzees (18) and were characterized predominantly by portal inflammation and focal necrosis. Hepatocyte alterations included multinucleated hepatocytes, cytoplasmic clumping, and vacuolization probably representing fat droplets (9). The severity of histologic lesions appeared generally to parallel the ALT elevations. These pathogenesis profiles are strikingly reminiscent of those obtained in chimpanzees inoculated with the infectious H77 material or other HCV-containing samples (19).

Figure 2

Viremia, hepatitis, and serologic response in chimpanzees inoculated with transcribed RNA. Before and after inoculation, animals were monitored weekly for the amount of circulating HCV RNA, liver transaminases, and HCV-specific antibodies. RNA and liver enzyme data are shown for chimpanzees 1535 (A) and 1536 (B). RNA was quantified by the bDNA assay (Chiron Corp.) and is reported as mega equivalents of RNA per milliliter (Meq/ml) (detection limit is 0.2 Meq/ml). Liver enzymes measured included serum alanine aminotransferase (ALT) (indicated in IU/liter) and γ-glutamyltransferase (GGT) (IU/liter). Normal ALT and GGT concentrations range from 18 to 45 and from 0 to 60 IU/liter, respectively. (C) HCV-specific serologic responses (+) were detected by commercial enzyme immunoassay (EIA) and recombinant immunoblot assay (RIBA) (17) as well as by immune precipitation of metabolically labeled HCV antigens (23). Only EIA-positive samples were analyzed by RIBA (ND, not determined). For the immunoprecipitation analyses, radioactively labeled HCV antigens were produced by infection of BHK-21 cells with a vaccinia recombinant expressing the entire HCV H77 consensus polyprotein (vvHCV 1-3011con) [(9); see also (23)]. Cells were lysed in TNA [0.5% Triton X-100, 200 mM NaCl, 50 mM tris-HCl (pH, 7.4), 1 mM EDTA, 1 mg of bovine serum albumin per milliliter, and 20 μg of phenylmethylsulfonyl fluoride per milliliter], clarified, and incubated with 2.5 μl of chimpanzee serum followed by 3 μl of rabbit antibody to human immunoglobulin. Immunoprecipitates were collected, washed, and separated by electrophoresis on a SDS–12% polyacrylamide gel. HCV NS3 was present in the 70-kD species immunoprecipitated by the chimpanzee sera (arrow) as shown by solubilizing the immunoprecipitates (in 2% SDS, 1% 2-mercaptoethanol, and 20 mM DTT, and heating at 80°C for 20 min), diluting the eluted proteins with 20 volumes of TNA, and reprecipitating with an HCV NS3-specific rabbit polyclonal antiserum (9, 23).

To prove that the signals detected in the bDNA and QC RT-PCR assays were not due to residual template DNA from the transcription reactions, we assayed all the samples by PCR without reverse transcription. No products were obtained, which demonstrated that the signals detected were due to HCV RNA (Fig. 3A). If the HCV RNA detected in serum were from replicating virus, RNAs should be packaged in enveloped virions and resistant to degradation by ribonuclease (RNase), whereas residual transcript RNA should be RNase- sensitive. Indeed, the HCV RNA in samples of chimpanzee serum was resistant to RNase digestion under conditions that completely degraded excess naked competitor RNA (Fig. 3B). Moreover, there was no correlation between the level of apparent viremia and the amount of inoculated RNA. The total amount of transcript RNA used for inoculation of chimpanzee 1535 was ∼3000 μg but it was only ∼22 μg for chimpanzee 1536. Despite this ∼150-fold difference, similar levels of viremia were observed (Fig. 2). Finally, in two previous animal experiments (a total of six animals), circulating HCV-specific nucleic acid was never detected, even as early as 2 days after inoculation (9). These experiments suggested that circulating RNA was a result of authentic virus replication rather than release of inoculated nucleic acid.

Figure 3

Circulating HCV nucleic acid is RNA and is RNase resistant. (A) Serum samples from inoculated animals do not contain carryover template DNA. The analysis shown is for chimpanzee 1535, which received the largest amount of inoculated HCV RNA and where the template DNA had not been degraded by digestion with DNase I (16). Duplicate RNA samples (from 10 μl of serum) from the indicated weeks after inoculation without (lane 1) or with competitor RNA (cRNA) (102 molecules in lanes 2 to 7; 103 molecules in lanes 8 to 14) were amplified by RT-PCR with (+) or without (−) enzyme in the RT step (5). The cRNA, which contained an internal 29-base deletion, was added at 1/10 to 1/30 the previously determined HCV nucleic acid concentrations to serve as an internal RT-PCR control. An equal portion of each reaction mixture was separated by electrophoresis on a 6% polyacrylamide gel and bands were visualized by staining with ethidium bromide. In the exposure shown, a weak band corresponding to the cRNA product can be seen in some lanes (such as lane 8). No specific PCR band was detected in the absence of cDNA synthesis, indicating that the HCV-specific nucleic acid signal was due to RNA. (B) Circulating HCV RNA from inoculated animals is protected from RNase. The sample analyzed was week 6 serum from chimpanzee 1536, which had an HCV RNA titer of 6 × 106 molecules per milliliter by QC RT-PCR. Extracted RNA samples were subjected to nested RT-PCR (5) and an equal portion of each reaction mixture was separated by electrophoresis on a 6% polyacrylamide gel. Lane 1, 10 μl of serum (containing ∼6 × 104 HCV RNA molecules) was mixed with excess cRNA (3 × 105 molecules), digested with 0.5 μg of RNase A for 15 min at room temperature, and extracted with RNAzol. Lane 2, 10 μl of serum was first extracted with RNAzol and then mixed with 3 × 105 molecules of cRNA. Lane 3, 10 μl of serum without added cRNA was predigested with RNase A and then extracted with RNAzol. Lane 4, negative control for RT-PCR. The RT-PCR products corresponding to HCV genomic and cRNA are indicated. In lane 2, the relative intensity of the genomic and cRNA products reflects the approximately fivefold difference in the molar amounts of these RNAs in this competitive RT-PCR reaction. The additional band of higher apparent molecular mass represents a heteroduplex of the genomic and cRNA PCR products, see (5).

Restriction enzyme digestion and sequence analysis of recovered viral RNA revealed the presence of engineered markers, proving that these infections stemmed from the inoculated transcript RNAs. Two silent mutations at position 899 (C instead of T) and at position 5936 (C instead of A, which ablated the internal Bsm I site at position 5934), marked all the transfected RNAs (Fig. 1). For the nucleotide (nt) 899 marker, the region between nt 466 and nt 950 was amplified by nested RT-PCR, sequenced directly, and found to have the expected H77 consensus sequence except for the engineered C marker at nt 899 (9). The region from nt 5801 to 6257 was also amplified by nested RT-PCR and was found to be resistant to digestion with Bsm I but not to four other enzymes known to cleave in that region (9). These analyses were conducted for both chimpanzee 1535 (week 5) and chimpanzee 1536 (week 6).

Additional silent markers were analyzed to identify the 5′-terminal sequence(s) and the length(s) of poly(U/UC) tract required or preferred for initiating infection (20, 21). Although it is not possible to draw firm quantitative conclusions about differences in specific infectivity, the results clearly demonstrated that the RNA transcripts without any additional 5′ nucleotides were infectious (20). Transcripts with additional 5′ nucleotides could also initiate infection, although our analysis did not allow us to distinguish among the various derivatives tested (Fig. 1). Transcripts containing either the 75-base or the 133-base poly(U/UC) tracts were infectious, but the 133-base poly(U/UC) tract was preferred (21).

The demonstration that RNA transcribed from cloned HCV cDNA can initiate infection and cause hepatitis in transfected chimpanzees provides formal proof (22) that HCV alone is the causative agent of this disease. With this model, HCV infection can now be launched from clonal derivatives to facilitate studies on virus evolution, pathogenesis, and host immune response relevant to understanding the factors that determine viral clearance versus chronic infection. Our experiments also define the elements of functional HCV genome RNA. Aside from a consensus genome sequence, no additional 5′ sequences were required for infectivity and 3′ poly(U/UC) tracts of variable length were tolerated. Infectious HCV RNAs can now be produced in unlimited quantities and used to identify and optimize cell-culture replication systems and to begin genetic analyses of HCV replication in vivo and in vitro.

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