Reproduction in vitro of a quasispecies from a hepatitis C patient and determination of factors influencing selection of a dominant species

word count: 230 Text Word count: 3520 Molecular Hepatitis and Hepatitis Viruses Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. 2 Current address: Gastroenterology,Kanazawa University, Graduate School of Medical Science,Kanazawa,Japan Corresponding author: Dr. Suzanne U. Emerson, Molecular Hepatitis Section, Bld 50/Rm 6537, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. E-mail: [email protected]; phone: 301-496-2787, fax: 301-496-0524. Abstract Hepatitis C virus infections proceed to chronicity in the majority of cases. In patients,Hepatitis C virus infections proceed to chronicity in the majority of cases. In patients, hepatitis C viruses exist as a dynamic and complex quasispecies. The dominant species at any one time arises in response to host immune pressure and other, incompletely understood factors. It is critical to understand all the mechanisms by which dominance is achieved but this is difficult to study in vivo. Therefore, it would be useful to develop a cell culture system in which naturally occurring quasispecies could be studied. Hepatitis C virus glycoprotein genes E1 and E2 were PCR-amplified as a cassette from the plasma of a chronically infected patient and shotgun cloned into a modified 1a/JFH1 infectious cDNA clone. Following transformation of bacteria, plasmids were batch-harvested, transcribed, and transfected into Huh 7.5 cells to produce a quasispecies of the hypervariable region 1 (HVR1) that mimicked that circulating in vivo. Serial passage of the quasispecies in vitro resulted in replacement of the initially dominant species with a new HVR1 species co-existing with selected growth-enhancing mutations located outside of HVR1. Antibody raised against one HVR1 sequence neutralized virus with the homologous HVR1 and cross-neutralized virus with a different sequence. Reciprocal swapping of the HVR1 regions between the two dominating species demonstrated that the HVR1 sequence affects the efficiency of replication and of neutralization by anti-HVR1 but that both processes are strongly influenced by regions outside of HVR1. Hepatitis C virus is a small, enveloped RNA virus with a positive sense genome that encodes non-structural proteins required for replication and/or assembly, a capsid or core protein, and two glycoproteins, E1 and E2, that form heterodimers which comprise the virus receptor. The virus is a member of the Flaviviridae family, genus Hepacivirus (7) and is a significant human pathogen which is transmitted mainly through blood or blood products. 170 million people worldwide are infected by HCV and it is the major cause of chronic hepatitis: severe sequelae include liver cirrhosis, hepatocellular carcinoma and death (10). Presently, antiviral therapy includes interferon and ribavirin treatment (13,17) but the efficacy of these drugs varies depending on the genotype of HCV, host factors, and treatment dose and schedule (5,8). Unfortunately, side effects of drug treatment can be severe. For these reasons, vaccine alternatives to drug treatment have been vigorously pursued, but with limited success (15). Vaccine development has been stymied by the extreme quasispecies diversity of HCV (2). Antibodies to the glycoproteins usually arise in the chronic phase of infection and virus persists even in the presence of neutralizing antibodies, presumably because neutralizationescape mutants are selected from the vast quasispecies. The hypervariable region 1 (HVR1) located at the N-terminus of E2 is the most variable domain in HCV and may play a role in immune evasion (3,14). The HVR1 region is thought to be involved in cell attachment through interaction with scavenger receptor class B type 1 (12) ; although antibodies to HVR1 could neutralize virus post-attachment to cultured hepatoma cells, they did not inhibit binding to CD81, another HCV receptor (18) A deletion mutant lacking the HVR1 region was viable, although attenuated, in chimpanzees (4). Antibodies directed against the HVR1 can neutralize the virus and it has been proposed to serve as a decoy to divert the humoral immune response away from more critical regions. Recent studies of JFH1-based recombinant viruses lacking HVR1 suggested that HVR1 shields important conserved neutralization epitopes (9). Until recently, studies of HCV replication and neutralization had to be performed in chimpanzees but the development of the HCV pseudoparticle system provided a more widely available system with which to study virus-cell interactions and neutralization (1). Even more useful was the subsequent development of the JFH1 cell culture system since this genotype 2a virus strain could complete the entire replication cycle of HCV in vitro (19). Although the JFH1 virus remains the only strain to be successfully cultured, chimeric viruses which express the structural proteins of the other genotypes from the JFH1 backbone were subsequently developed and all are available as infectious cDNA clones (6,20). In the current proof-of-principle study, we have explored the possibility of utilizing the chimeric genotype 1a/JFH1 cDNA clone to reproduce and study a naturally occurring quasispecies of HVR1. Materials and Methods Shotgun cloning. Acute phase plasma (40 uL) from patient H, containing 10 6.5 50% chimpanzee infectious doses of HCV per mL was extracted with Trizol LS (Invitrogen, Carlsbad, CA) and HCV RNA was reverse transcribed with Superscript II RTase (Invitrogen). The first step of nested PCR was performed as long PCR (15) with KlenTaq enzyme (Clontech, Mountain View, CA) and primers in core and the 3’ UTR as described previously (10). Second step PCR used E1and E2specific primers. The sense primer ( 5'-TGCCCGCTTCAGCCTACCAAGTTCGAAATTCC-3')contained an introduced unique Bsp119I restriction site ( bold and underlined )at the 5’ end of E1 and the antisense primer (5'-GATGCTGCATTGAGTATTACGAGGTTCTCCAGCGCT-3') contained an introduced AfeI restriction site ( bold and underlined) at the 3’ end of E2: neither introduced restriction site altered the amino acid sequence. The backbone plasmid, a gift from S. Lemon, consisted of the 1a/2a intergenotypic chimera H77-NS2/NS3-JFH1/Q125L, a chimera that had been adapted to grow in cell culture (20). A naturally-occurring AfeI restriction site was abolished and Bsp119I and a new AfeI restriction site were introduced by standard methods at the beginning of E1 and the end of E2. Following digestion of the plasmid and the plasma PCR products with Bsp119I and AfeI , the two components were purified on agarose gels, ligated together and transformed into the JM109 strain (Promega, Fitchburg, WI) of Escherichia coli. Individual colonies were picked from one plate for sequencing to determine quasispecies distribution, whereas, colonies on a duplicate plate consisting of 115 well-separated colonies and about 50 overlapping colonies were batch-harvested as a mixture for production of a quasispecies. Primer sequences for plasmid construction or PCR amplification are available on request. Transfection of Huh7.5 cells. The mixture of quasispecies plasmids was linearized with XbaI and 50 ug of DNA was transcribed with T7 RNA polymerase (Promega) as described previously (10). The RNA reaction mixture was mixed with DMRIE-C transfection reagent (Invitrogen) and inoculated onto Huh7.5 cells. Transfected cells were grown at 37°C in the CO2 incubator and medium was harvested at the indicated times for HCV sequence analysis and serial passage. The whole experiment was performed twice with very similar results; therefore, only data from the first experiment are shown. Recovery of viral quasispecies and sequence analysis. HCV was extracted with Trizol LS from 100uL cell culture medium harvested at the indicated times and was amplified with the E1 and E2 primers described above. The product was digested with Bsp119I and AfeI, cloned into the modified 1a/2a vector, and sequenced for either HVR1 or the entire E1E2 sequence. At least 18 colonies were analyzed for each time point. Infectivity determinations. Medium from cultured cells was plated on Huh7.5 cells growing in Lab-Tek 8-well chamber slides (Thermo Fisher Scientific, Waltham, MA) and incubated at 37°C. Three days post infection, cells were fixed in 100% acetone and incubated for 20 min with mouse anti-core or chimpanzee 1530 anti-HCV plasma. After 20 min, slides were rinsed and incubated with Alexa 568 anti-mouse or Alexa 488 anti-human serum (Invitrogen) to detect core or core and E1E2 proteins respectively. Immunofluorescence microscopy was performed with an Axioskop 2-Plus Zeiss microscope and individual foci (focus forming units or FFU) were manually counted. Neutralization by anti-HVR1 antibody. LMF87 is a rabbit polyclonal antibody raised against a 21 amino acid peptide containing the HVR1 sequence of species A (3). Triplicate aliquots of 100uL each of virus in cell culture medium was mixed with control serum or anti-LMF87 serially diluted with complete DMEM medium, incubated at 37°C for 1 hr, and inoculated onto Huh7.5 cells growing in 8-well chamber slides. After 5 hrs, the inoculum was replaced with complete DMEM and incubation was continued for 2 days. Cells were stained for immunofluorescence microscopy as described above and the foci were counted. Mutagenesis. Backbone mutations were reverted or HVR1 regions were swapped by standard techniques. Primers and detailed procedures will be provided on request.