WO2001057073A2 - Clone infectieux genetiquement modifie du genome du virus de l'hepatite b (vhe), production et utilisations de ce dernier - Google Patents

Clone infectieux genetiquement modifie du genome du virus de l'hepatite b (vhe), production et utilisations de ce dernier Download PDF

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WO2001057073A2
WO2001057073A2 PCT/IN2001/000015 IN0100015W WO0157073A2 WO 2001057073 A2 WO2001057073 A2 WO 2001057073A2 IN 0100015 W IN0100015 W IN 0100015W WO 0157073 A2 WO0157073 A2 WO 0157073A2
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hev
rna
genome
clone
virus
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PCT/IN2001/000015
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WO2001057073A3 (fr
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Subrat Kumar Panda
Israrul Haque Ansari
Shripa Agrawal
Hemlata Durgapal
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Department Of Science And Technology
All India Institute Of Medical Sciences
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Priority to AU44516/01A priority patent/AU4451601A/en
Publication of WO2001057073A2 publication Critical patent/WO2001057073A2/fr
Publication of WO2001057073A3 publication Critical patent/WO2001057073A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/28011—Hepeviridae
    • C12N2770/28111—Hepevirus, e.g. hepatitis E virus
    • C12N2770/28121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/28011—Hepeviridae
    • C12N2770/28111—Hepevirus, e.g. hepatitis E virus
    • C12N2770/28161—Methods of inactivation or attenuation
    • C12N2770/28162—Methods of inactivation or attenuation by genetic engineering

Definitions

  • the present invention relates to a unique genetically engineered nucleic acid cDNA clone of hepatitis E virus (HEV) genome found to be infectious in cell culture and to a process for the in-vitro synthesization of such a cDNA clone of HEV. Also included within the scope of the invention is the expression from said clone of viral proteins adapted for use in the detection of antibodies to HEV in biological samples and a diagnostic test kit for the presence of HEV infection essentially comprising the expressed proteins.
  • HEV hepatitis E virus
  • hepatitis A and hepatitis B Two types of the hepatitis virus were initially identified which were referred to as hepatitis A and hepatitis B. Other types of this virus which were known but until they were positively identified were lumped collectively under the identity "non A - non B" (NANB) hepatitis virus. Subsequently, as a result of epidemiological studies and animal transmission, the existence of two or more viruses within the so-called entity NANB hepatitis viruses were suspected. One type of these viruses was found to be parenterally transmittable and this was identified as a flavivirus referred to as hepatitis C. -[Alter et al., Lancet 1, 459-463 (1978); Hollinger et al., Intervirology 10, 60-68 ( 1978); Tabor et al., Lancet 1, 463-466 (1978)].
  • HEV hepatitis E virus which is conveniently abbreviated as HEV. It is HEV which forms the study by the applicants and the subject of the present invention. HEV is now established as an etiological agent of both the epidemic and sporadic forms of water-borne hepatitis which is endemic to the Indian sub-continent and prevalent in most parts of the developing world. In fact, infection due to HEV accounts for one third of the sporadic acute viral hepatitis and almost all the described epidemics in the Indian subcontinent [26, 39]. It is a major health problem in tropical and sub- tropical parts of the world.
  • HEV has been provisionally classified as the prototype member of the group of hepatitis-E viruses. HEV has been found to have a positive strand polyadenvlated ribonucleic acid (RNA) as genome with a size of -7.2 kb and possessing three open reading frames identified as ORF 1, ORF 2 and ORF 3.
  • RNA polyadenvlated ribonucleic acid
  • ORF 1 encodes the putative non-structural polyprotein including domains representative of (a) a viral methyltransferase, (b) a papain-like cysteine protease, (c) a RNA helicase, and (d) a viral RNA dependent RNA polymerase.
  • ORF 2 encodes an -88 kDa glycoprotein that is the major viral capsid protein.
  • ORF 3 encodes a 13.5 kDa phosphoprotein of unknown function.
  • NCR's non-coding regions
  • ORF1 is the largest in size, starts 27 nucleotides downstream of the 5' end and terminates at position 5079, resulting in a protein of 1683 amino acids.
  • ORF 2 begins 37 nucleotides downstream of ORF1 and codes for a protein of 660 amino acids.
  • the third ORF (ORF3) is the smallest in size, encoding a protein o( 123 amino acids and overlaps with ORF1 at its terminal base [55].
  • Non-structural ORF 1 is believed to code for a putative polyprotein which has different motifs, such as those for a viral methyltransferase, a papain-like cysteine protease, a RNA helicase and a RNA dependant RNA polymerase [28]. However none of these putative functional regions have been characterized so far.
  • the structural ORFs have been expressed in prokaryotic and eukaryotic systems and immunogenicity of the resulting proteins have been reported in a number of studies [20, 34, 40].
  • the inventors have earlier expressed ORF2 and ORF3 in animal cells.
  • the ORF2 protein (pORF2) is an 88 kDa glycoprotein that is expressed intracellularly as w ell as on the cell surface [23].
  • the ORF3 protein (pORF3) is a 13.5 kDa phosphoprotein. which is phosphorylated by the cellular mitogen activated protein kinase and associates with the cytoskeleton [64].
  • the hepatitis E virus has been cloned and sequenced from several countries, among them India, Pakistan, Sri Lanka, China and Mexico. However, only partial expression of the viral structural proteins has been described so far by other workers.
  • the inventors have focussed on two major areas: a De eloping a complete virus genome clone which can be used to infect cells and thereby provide a valuable in-vitro system for designing and analysing the effects of drugs and other inhibitors and vaccines which can help in the piev ention 01 cure of HEV associated liver disease and or severe complications arising therefrom, and b Studying the potential of the 3' NCR of HEV in serving as the replication initiation site for HEV RNA genome
  • the present invention provides a unique genetically engineered nucleic acid clone of hepatitis E virus (HEV) genome w hich comprises the complete structural and non-structural genes I e the complete coding sequence of HEV flanked by the 5' and 3' non-coding regions, along w ith a 3 polyadenylated tail to the 3' non-codmg region said clone being capable of infecting cultured vei cells and replicating, transcribing and assembling the HEV theiein
  • the nucleic acid clone of the invention comprises a DNA which is complementary to the hepatitis E virus genome Con eniently, this clone is deriv ed using multiple PCR amplification followed by assembly strategy
  • the complete coding sequence (Seq ID No 1 ) of the nucleic acid clone of HEV genome of the present invention is as follows SEQ ID No.l : atg gaggcccatc agtttctcaa ggctcccggc atcactactg ctgttgagca ggctgctcta gccacggcca actctgccct ggcgaatgct gtggtagtta ggccttttct ttctcaccag cagattgaga ttctcattaa cctaatgcaa cctcgccagc ttgttttccg ccccgaggttttctggaatc aacccatcca gcgtgtcatt cataacgagc tggagcttta ctgcgcttctggaacccatcca g
  • the coding sequence in the HEV genome is flanked by 5' and 3' non- coding regions (NCRs) which are 27 and 68 nucleotides long, respectively [55]. These non-coding regions are projected as having the potential to play an important role as cis- acting elements for the genomic RNA replication of HEV as has been reported for other positive strand RNA viruses [21, 30, 49, 52, 56, 62].
  • the 5' non-coding region of the nucleic acid clone of HEV genome of the present invention is homologous or complementary to an RNA sequence (Seq. ID No. 2) selected from:
  • the 3' non-coding region of the nucleic acid clone of HEV genome of the present invention is homologous or complementary to an RNA sequence (Seq. ID No. 3) selected from:
  • the complete sequence (Seq. ID No. 4) of the nucleic acid clone of HEV genome of the present invention comprises:
  • the non-structural gene (ORF1) of the nucleic acid clone of HEV genome codes for the polyprotein having the following amino acid sequence Seq. ID No. 5:
  • ORF1 MEAHQFLKAPGITTAVEQAALATANSALANAVVVRPFLSHQQIEILINLM
  • the nucleic acid clone of HEV genome of the present invention comprises a DNA which is complementary to the hepatitis E virus genome.
  • this DNA complementary to the hepatitis E genome is a DNA from an epidemic strain derived from Abrahampatnam, Andhra Pradesh, India.
  • the inventors have analyzed the simulated structure of the 3' end and its interaction with the viral RdRp and cellular proteins, which w ere partially characterized by RNase protection and UV-crosslinking assays. Also analyzed were the structural aspects of the HEV RdRp by comparison with results from computer predictions of secondary and tertiary structures of various representative RdRps [38]. with special reference to the crystal structure of the poliovirus RdRp [19]. This in itself can serve as a target for drug development.
  • the 3' non-coding region of the nucleic acid clone of HEV genome of the present invention is characterized by the presence of cis-acting regulatory elements capable of interacting with recombinant viral RNA-dependent RNA polymerase (RdRp) and cellular proteins for virus grow th, said elements being thus usable as targets for inhibitors of virus replication.
  • inhibitors of virus replication include anti-sense ribonucleic acid (RNA). ribozymes. analogues and inhibitors of the viral replicase.
  • the cis-acting regulatory elements of the 3' end which are usable as targets for inhibitors of virus replication possess the following sequence Seq ID No. 8: SEQ ID No. 8:
  • the cis-acting regulatory elements of the 3' end which are usable as targets for inhibitors of virus replication possess the structure shown in Figure 8A of the accompanying drawings.
  • the 3' polyadenylated tail of the 3' non-coding region preferably comprises at least 3 adenosine residues.
  • a unique genetic engineered nucleic acid clone of hepatitis E virus genome of the invention which can be transcribed to produce infectious transcripts, has a sequence number AF076239.
  • HEV RNA was used to study the replication of the virus v ia gene transfer.
  • HEV genome was cloned downstream of the T7 promoter. transcribed in-vitro and transcripts were characterized.
  • a process for the production of a unique genetic engineered nucleic acid clone of hepatitis E virus (HEV) genome which comprises:
  • the full-length cDNA clone or the coding components thereof may be cloned in an expression vector system to induce therefrom the expression of viral proteins.
  • the viral proteins thus expressed from the full-length cDNA clone or its coding components find use in the detection of antibodies to HEV in biological samples such as blood, serum, plasma, blood cells, lymphocytes, bile and liver tissue biopsy.
  • the invention also includes within its scope an in-vitro method for detecting antibodies to HEV in a biological sample obtained from a subject, which method comprises:
  • the present invention also includes a diagnostic test kit for the presence of HEV infection essentially comprising viral proteins expressed from the unique genetically engineered full-length cDNA clone of HEV genome of the invention.
  • the unique nucleic acid clone of hepatitis E virus (HEV) genome can be mutated to produce non-infectious HEV particles. These non-infectious HEV particles so produced can be combined with a pharmaceutically acceptable adjuvant to produce an HEV vaccine.
  • HEV hepatitis E virus
  • the invention also envisages a high throughput assay system for rapid anti-HEY drug testing which comprises the nucleic acid clone of hepatitis E virus (HEV) genome described herein modified by the insertion therein of a reporter gene for the analysis o ⁇ ⁇ replication inhibition.
  • HEV hepatitis E virus
  • along with the non-codmg regions were cloned using sub-genomic PCR amplification followed by reconstruction strategy [2, 23]
  • the cDNA clone complementary to the hepatitis E v irus genome was derived from an Indian isolate of HEV from an epidemic strain from Abrahampatnam, A P , India
  • the clone in question was generated using multiple PCR amplification followed by assembly strategv and possess the following sequence Seq ID No 4 SEQ ID No. 4:
  • RNA was resolved on a 0.8% formaldehyde denaturing agarose gel and transferred to nylon membrane (Hybond, Amersham Intl.. UK) m the presence of 20X SSC.
  • the membrane was washed with 10X SSC solution, air-dried and subjected to UV-crosslinking in an ultraviolet crosslinker (Stratagene, Germany)
  • the membrane was put in prehybridization solution (6X SSC, 5X Denhardt's solution, 0.5% Sodium dodecyl sulfate (SDS), 100 ⁇ g calf thymus DNA per ml of the solution) and incubated at 68°C foi 6 hours in hybridization oven (Shel Lab, Model 1004, USA).
  • the hybridization was subsequently carried out in fresh pre-hyb ⁇ dization solution containing 1x10 cpm of an alpha J ⁇ P dCTP labelled probe generated from the full-length ORF2 clone of HEV [39].
  • the probe was prepared by using a commercial random priming kit (Prime-it, Stratagene, Germany) as per manufacturer's protocol.
  • the membrane was washed as follow s: a) 2x SSC, 0.1% SDS for 5 minutes at room temperature, b) 0 2x SSC, 0.1% SDS for 5 minutes at room temperature twice, c) 0 SSC, 0.1 % SDS for 15 minutes at 42°C, and d) 0 l x SSC, 0.1% SDS for 15 minutes at 68°C
  • the membrane was wrapped in Saran Wrap and exposed for autoradiography using Kodak X-Omat AR film with Du-Pont intensifying screens (Du-Pont, USA).
  • lane 1 shows the RNA on staining the gel w ith ethidium bromide
  • Lane 2 shows the RNA as detected by northern hybridization using HEV specific probe
  • Lane M shows the molecular size RNA markei in kilobases 01/57073 23
  • HEV transcripts The majority of the HEV transcripts were bout 7.2 Kb in size corresponding to complete genome w hen compared with standard RNA molecular weight marker ( Life Technologies, USA).
  • HepG2 cells were maintained in Dulbecco's modified Eagle's Medium (D EM ) containing 10% fetal calf serum (Life Technologies, USA). Cells at about -50% confluency were used for transfection of HEV RNA. Twenty microgram of in-vitro produced RNA was transfected by liposome induced method (Lipofectamine. Life Technologies, USA) as per the manufacturer's guidelines. The plasmid vector (pSGI) served as a control for the transfection. For each 60-mm diameter culture dish. 20 ⁇ g of the HEY RNA and 10 ⁇ l of lipofectamine were diluted in 1.5 ml of serum-free medium.
  • D EM Dulbecco's modified Eagle's Medium
  • pSGI plasmid vector
  • the mixture was kept at room temperature for 30 minutes and gently overlaid on to the monolayer.
  • Fresh medium with 10% fetal calf serum was replaced after 6 hours and the cells were kept in an atmosphere of 5% CO2- After 72 hours the cells were harvested for extraction of total RNA.
  • Transfected cells were pulse labelled with (100 ⁇ Ci/ ml/ 60 mm plate) ⁇ " S methionine-cysteine (Promix, Amersham Intl., UK) for four hours at 72 hours post transfection in methionine-cysteine deficient DMEM (Sigma, USA).
  • the metabolically labelled cells were harvested and proteins were immunoprecipitated using HEV speci fic polyclonal antibodies. Similar labelling experiments were also carried out at 12. 24. 36. 72 and 96 hours to determine the expression kinetics of the viral RNA dependent RN.A Polymerase (RdRp).
  • a batch of the HEV RNA transfected cells were maintained in the culture and allowed to grow for next 45 days (8 passages). These cells w ere analyzed at 3. 7, 15, 33 and 45 days post transfection for the presence of anti-sense RNA replicativ e intermediates using strand-specific PCR.
  • RNA transfected cells were positive for sense as well as anti-sense strands of the HEV genome at 3, 7, 15 and 33 days.
  • RNA extracted at 72 hours (3 days) was carried out to determine an approximate ratio of sense and anti-sense strands.
  • sense strand detection the HepG2 cells transfected with plasmid pSGI and serum from HEV infected monkey with viremia served as negative and positive controls respectively.
  • anti-sense detection RNA isolated from HEV infected monkey liver containing anti- sense replicative intermediate served as positive control whereas the bile fluid or serum from the same viremic animal served as negative control.
  • the reverse transcription was carried out using either sense or anti-sense primer.
  • RNA in the reaction mix was degraded by digestion with RNase H (2 units) and RNase A (1 ⁇ g) (Promega, USA). Following RNase treatment the reaction mix was extracted once with phenol/ chloroform and ethanol precipitated. The precipitated cDNA was used for PCR amplification using both sense and anti-sense primers.
  • Figure 3A in this figure represents detection of the negative-sense strand of HEV RNA and Figure 3B represents detection of the positive-sense strand of HEV RNA. Explanation of the other integers shown in Figure 3 are as follows: Figure 3A: Detection of negative strand of HEV RNA:
  • RNA ( l ⁇ g) extracted from HepG2 cells transfected w ith in-vitw synthesized HEV RNA at 72 houis (Neat) 2 through 9 Log dilution of the transfected HepG2 cell RNA from 10 through 10
  • total RNA (30 ⁇ g) extracted from the transfected cells was immobilized on a nylon membrane (Amersham Intl , UK) using a Hyb ⁇ slot manifold (Life Technologies, USA) RNA from cells transfected with plasmid pSGI was used as negative control
  • Sense and anti-sense specific ⁇ boprobes were prepared by transcription with T7 and SP6 polymerases (Ribopiobc system-T7 and Riboprobe system-SP6, Promega, USA) using direct and rev eise onented clones of a HEV cDNA encompassing nucleotide 1-457 in pCR-Scnpt SK (-) (Stratagene, Germany) and pGEM-T (Promega, USA) vectors respectiv elv
  • the transcription leaction was carried out in the presence of alpha 3 p UTP (2500 Ci mmol Ameisham Intl , UK) Prior to m-vi o transcription, the template DNA as lineaiized bv restriction enzyme digestion at the end of the fragment The reaction mixtuie w as tieated w ith DNasel. extracted with phenol/ chloroform and ethanol piecrpitated as
  • Panel A of Figure 4 shows hybridisation with alpha 33 P UTP labelled riboprobe of anti-sense polarity.
  • Slots 1, 2, 3 and 4 represent the following:
  • Panel B of Figure 4 shows hybridisation with alpha " J P UTP riboprobe of sense polarity.
  • Slots 1, 2, 3 and 4 represent the following: 1. In-vitro synthesized HEV RNA of negative sense polarity (Positive control)
  • RNA isolated from HepG2 cells transfected with pSGI vector as control In-vitro synthesized sense and anti-sense HEV RNA were used to validate the specificity of hybridisation method in detecting the strands. The presence of anti-sense HEV RNA was reconfirmed by hybridisation.
  • the cells transfected with HEV RNA according to the procedure of Example 3 were lysed in 750 ⁇ l of RIP A buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl. 5 mM Iodoacetamide, 0.5% Triton X-100, 1 % SDS. 1% sodium deoxycholate. 2 mM phenymethylsulfonyl fluoride).
  • the clarified lysate was incubated with 7 ⁇ l of anti- ORF2.
  • anti-ORF3 or anti-ORFl putative anti-Methyl transferase (anti-met).
  • putativ e anti-Helicase anti-hel
  • putative anti-RNA dependent RNA Polymerase RdRp
  • the polyclonal antibodies were raised in rabbits against the structural proteins (pORF2, pORF3) and components of non-structural polyprotein pORFl (putative methyl transferase, helicase and RdRp regions) as described elsewhere [2. 40].
  • the antigen-antibody complexes formed were further incubated w ith 100 ⁇ l 10% suspension of pre-swollen Protein A Sepharose-4B (Pharmacia. Upssala. Sweden) and the reaction mixture was kept at 4°C with slow end to end shaking.
  • reaction mixture was centrifuged for one minute at 10,000 rpm in a refrigerated microcentrifuge (Hermle, Germany). Supernatant was discarded as radioactive waste and the beads were washed thrice with 1 ml of RIPA buffer for 10 minutes at 4°C with shaking.
  • the complex was boiled with 50 ⁇ l of 2X SDS-PAGE sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% 2-Beta mercaptoethanol. 0.1 % bromophenol blue) and analyzed on a SDS 6-15% gradient PAGE. The gel was treated with 0.5 M sodium salicylate, washed, dried and exposed for autoradiography as described above.
  • HEV ORF2 and ORF3 proteins were detected by immunoprecipitation with their corresponding specific antibodies.
  • An autoradiograph showin ⁇ immunoprecipitation of the structural proteins from the HEV RNA transfected HepG2 cells is depicted in Figure 5A of the drawings.
  • the arrow marked signals in the lane 2 and 4 represent the pORF2 (-72 kDa) and pORF3 (13.5 kDa) respectively.
  • the pSGI transfected cells were immunoprecipitated with anti-ORF2 and anti-ORF3 antibodies ( Figure 5A, lanes 1 and 3) to serve as control.
  • the immunoprecipitates were analyzed on SDS 6-15% gradient PAGE and visualized by fluorography. The molecular sizes of C labelled markers (in kilodaltons) are indicated on the right (Amersham Int.. UK).
  • the signals corresponding to putative domains of methyl transferase (-35 kDa) and helicase (-38 kDa) could be detected by immunoprecipitation. These are autoradiographically depicted in Figures 5B of the drawings. The putative helicase and methyl transferase were detected in samples prepared at 72 hours post transfection.
  • the autoradiograph of Figure 5B shows immunoprecipitation of putative domains of non- structural polyprotein corresponding to methyl transferase and helicase from the HEV RNA transfected HepG2 cells.
  • the arrow marked signals in lanes 2 and 3 represent the signals corresponding to putative methyl transferase (-35 kDa) and helicase (-38 kDa). respectively.
  • the mock transfected pSGI cells were immunoprecipitated with anti-met and anti-hel antibodies ( Figure 5B, lanes 1 and 4) to serve as control.
  • the immunoprecipitates were analyzed on SDS 6-15% gradient PAGE and visualized by fluorography. Molecular size markers (in kilodaltons) are indicated on the right (Rainbow markers. Amersham Int.. UK).
  • RdRp RNA dependent RNA polymerase
  • the autoradiograph of Figure 5C shows immunoprecipitation of putative domains of non- structural polyprotein corresponding to RNA dependent RNA polymerase (RdRp) from the HEV RNA transfected HepG2 cells.
  • the immunoprecipitation was carried out using anti-RdRp antibodies at the stated 12, 24 and 36 hours post transfection.
  • the HepG2 cells were transfected with pSGI vector and immunoprecipitated at 36 hours with the same antibody.
  • the immunoprecipitates were analyzed on SDS 6-15% gradient PAGE and visualized by fluorography. Molecular size markers (in kilodaltons) are indicated on the right (Rainbow markers, Amersham Int., UK). Putative RdRp could not be detected in transfected cells at 72 and 96 hours (Data not shown).
  • transfection was carried out on cells grow n on cover slips (30 mm diameter). After 72 hours, the cells on coverslips were washed tvv ice O 01/5
  • H and K of Figure 6 represent the lmmunofluorescent staining of the HEV transfected HepG2 cells with ant ⁇ -pORF2, ant ⁇ -pORF3, anti-met, anti-hel and anti-RdRp antibodies, respectively
  • Panels A, C, E, G and J represent the immunostaining of the control HepG2 cells w ith the same antibodies corresponding to the test panel All the immunostaining w as earned out at 72 houi post tiansfection except that with the anti-RdRp antibodies w hich w as performed on the 24 hours post transfection
  • HEV RNA transcribed according to Example 2
  • RNAse inhibitor Promega, USA
  • HEV antibodies w ere detected by m-house ELISA system using recombinant HEV proteins (pORF2.
  • HEV RNA was observ ed w ith the help of RT-PCR in the sera collected between days 24 to 37. During this period (24-37 days) the AST and ALT values increased to between 1.5 to 2.5 (53-100 IL liter) times normal level.
  • ORF2 and ORF3 viral proteins were detected after 4 weeks and persisted for the next 14 davs
  • the animals (M-1927. M-2197) which received in-vitro produced HEV RNA as well as the control monkey (M-1761) remained normal with no rise in ALT and AST v alues and no seroconv ersion for antibodies were observed. They also remained negative foi HEV RNA in semm (viremia) throughout the follow up period.
  • the IgG anti-HEV antibodies w eie detected in the infected monkey (M-1690) 3 months after the inoculation
  • Figure 7 of the drawings represents a photograph of 2% agarose gel depicting the RT-nested PCR products (343bp) for HEV genome amplified from the sei uin of infected rhesus Macaca mulata (M-1690)
  • Lane P indicates Positive control.
  • Lane N indicates the control monkey (M-1761)
  • Lane M represents 100 bp DNA laddei ( Li fe Technologies, USA)
  • Lanes 1 , 2, 3, 4 and 5 represent the serum collected from the monkev M-1690 on days 24, 28. 33, 37 and 43, respectively, post injection
  • the arrow indicates amplified fragments from HEV genome EXAMPLE 7
  • Stem-loop structures 1 and 2 (SL1, SL2) comprise nucleotides 7173-7194 and 7089- 7163, respectively, separated by a single strand region (SS) of nt 7164-7172.
  • the SL 1 structure involves the poly(A) stretch at its 3' end in base-pairing.
  • PCR-based strategy was used to generate the cDNA clones for the production of RNA transcripts corresponding to the 3' end of the HEV genome and its various mutant forms.
  • a schematic representation and description of the constructs is shown in Figure 9 of the drawings.
  • Forward and reverse primers were designed using the OLIGO 4.0 software and synthesized on an automated DNA synthesizer (Model 392, Applied Biosystems). These primers are identified in Table I at the end of this description.
  • Such primers were used to amplify regions of the 3' end using a HEV cDNA 6046-7194 nt pCRScript clone [40] as a template [which was later used for the construction of an infectious HEV cDNA clone pSGl-HEV(I) of the present invention] and a combination of Tag (Promega) and Pfu (Stratagene) DNA polymerases.
  • the forward primer contained the T7 promoter sequence immediately upstream of the HEV sequence.
  • the amplified products were gel purified, polished with Klenow fragment of DNA polymerase (Amersham International) and phosphorylated with polynucleotide kinase (Amersham International).
  • plasmids were linearized by cutting with the appropriate restriction enzyme at the 3' end of the insert.
  • Xlw I restriction enzyme site present in the 3' end primer was used to linearize the clones [3'(+)A ] and [s3'(+)A ] and Bam HI site of the vector MCS was used for other mutants (Table 1 ).
  • Correct production of RNA transcripts was ensured by linearizing the clones with restriction enzymes giving rise to 5' overhangs and not 3' overhang, which might otherwise lead to the transcription of the wrong strand of DNA.
  • In-vitro transcription reaction was performed with T7 RNA polymerase (Life Technologies Inc., USA) in the presence of 500 ⁇ M each of ATP.
  • RNA synthesized [43]. Upto 60% incorporation of the [ J ⁇ P] UTP was routinely achieved resulting in labelled transcripts with a specific activity of typically 10 8 cpm/ ⁇ g.
  • the transcription reaction generated expected size transcripts as visualized on a 8% denaturing polyacrylamide gel. Unlabelled RNA w as similarly transcribed using 500 ⁇ M of each of NTPs, phenol: chloroform extracted and ethanol precipitated.
  • RNA transcript of the 3' end with a poly(A) stretch [3'(+)A n ] was performed.
  • the in-vitro transcribed RNA from the Hind III linearised clone [3'(+)A ] was treated with calf intestinal alkaline phosphatase (Promega) and purified on 8% polyacrylamide/ 8 M urea gel.
  • the RNA bands were visualized by UY shadowing using fluorescent TLC plate, cut and eluted overnight in a buffer containing 1% SDS and 2.5 M ammonium acetate.
  • the transcripts were ethanol precipitated and 5' end labelled using T4 polynucleotide kinase (Promega) and [ ⁇ - " P] ATP (5000 Ci. mmol: BARC, India) and further purified on 8% polyacrylamide / 8 M urea gel to remove the unincorporated nucleotides. Autoradiographic exposure of 1 min enabled the visualization of the labelled bands.
  • the 32 P-labeled RNA transcripts Prior to reaction, the 32 P-labeled RNA transcripts were supplemented with carrier tRNA (Calbiochem) to a final concentration of 8 ⁇ M and subjected to a denaturation / renaturation procedure by heating the samples at 65"C for 3 min and slow cooling to 25°C over 60 min.
  • the renaturation step was performed in the buffer containing 10 mM Tris/HCl, pH 7.2, 10 mM MgCl 2 and 40 mM NaCl for lead ion induced cleavage and in the respective enzyme buffers for enzymatic cleavage reaction. Subsequently, Pb(OAc)? (Sigma) solution was added to the final concentration of 2.5 mM and 5 mM, and the digestion was conducted at 25°C for 15 min. For enzymatic cleavage.
  • Ribonuclease I (0.1 U) (Promega), SI nuclease (0.4125 U) (Amersham International) and Mung Bean nuclease (0.1 U) (Amersham International) were independently added and the reaction proceeded at 25°C for 10 min. All the reactions were quenched by adding equal volume of 7M urea, 10 mM EDTA solution and loaded on 15 % polyacrylamide 7 M urea gel at -10 4 cpm / well. Electrophoresis was performed in 0.5X TBE buffer at 2000Y for -1.5 - 4 h for short and long runs, respectively. Autoradiograp v w as performed at -80°C with an intensifying screen (DuPont).
  • RNA cleavage products were run along with the products of alkaline RNA hydrolysis and limited ribonuclease Tl (Calbiochem) digestion of the same RNA.
  • Alkaline hydrolysis ladder was generated by incubating the RNA with formamide in boiling water for 15 min.
  • Partial Tl digestion w as performed in denaturing conditions (50 mM sodium citrate, pH 4.5, 7 M urea) with 0.05 U of enzyme at 55°C for 10 min. The above mentioned procedure was carried out after the standardization of reaction, gel electrophoresis and autoradiography conditions.
  • Figure 8B shows the nucleotides within the native RNA that were determined to be susceptible to Pb (II) and RNases. These are labelled p-Pb 2+ ; o-Ribonuclease ONE: s- S I nuclease; m-Mung Bean nuclease. Nucleotides prone to Pb(II) hydrolysis in the RN.A- RdRp complex are shown in brackets [ ].
  • cleavage products obtained by hydrolysis of 5' end labeled RNA transcripts with Pb 2+ ions and digestion with single strand specific RNases were separated on 15% polyacrylamide gel and given short and long runs.
  • RNases Single strand specific RNases
  • the labeled RN.A transcripts were supplemented with tRNA and brought to a concentration of 8 ⁇ .VI and subjected to denaturation/ renaturation procedure.
  • the results according to lanes shown in Figure 10 were as follows:
  • Lane A control incubation of the RNA without Pb 2+ ions or RNases.
  • Lane B limited RNase Tl hydrolysis under denaturing conditions: cutting at cv erv G residue. Some of the numbers of G residues in the RNA sequence are depicted on the left.
  • Lane C formamide ladder. Lanes D & E: Pb (II) induced hydrolysis at 2.5 and 5 mM Pb(OAc) 2 .
  • Lane F same as 5 but with the omission of the denaturation/ renaturation procedure.
  • Lanes G. H & I digestion with Ribonuclease ONE. SI nuclease and Mung Bean nuclease. respectively.
  • Pb " and RNases including SI nuclease and Mung Bean nuclease. cut at ev ery single nucleotide from nt 7164 to 7172 which forms a single strand region (SS ) in the computer predicted structure model. Occasional cuts within this region were observed by Ribonuclease ONE enzyme too.
  • Pb 2+ ions could identify and thus confirm the loops and bulge regions in SL1 and SL2, the reactivities being shown by the nt 7177. 7182-83.
  • HEV genome [28] was amplified as a larger fragment (3493-5163 nt) by PCR using a combination of Taq (Promega) and Pfu (Stratagene) DNA polymerases from a HEX ' cDNA clone comprising nucleotides 2346-5163 in the pCRScript vector [2] [which w s later used for the reconstruction of full-length HEV cDNA infectious clone pSGl -HEY(I) of the present invention].
  • the primers used were: Forward primer.
  • ACAGctcgagcccgggGCATGATTCAGTCG nucleotides 3493 to 3523 (Genbank AF076239); Reverse primer, GCGaagcttCg taccTGGTCGCGAACCCATGG nucleotides 5163 to 5131 (Genbank. AF076239).
  • the forward primer sequence was modified at the 5' end to create the rest ⁇ ction enzyme sites for Xlio I and Snui I (low er case nt in the primer sequence) and the start codon ATG.
  • the reverse primer was modified at the 5' end to incorporate the restriction enzyme sites for Kpn I and Hind III (lower case nt in the primer sequence).
  • the amplified fragment was cloned into pGEM-T vector (Promega) and sequenced as described earlier. It was then subcloned into the expression vector pRSET (Invitrogen) as a Xlw 1-Kpn I fragment.
  • the recombinant pRSET C-RdRp clone was transformed into E.coli BL21-DE3 (Stratagene) and its overnight culture (O N) grown in Luria-Bertani (LB) medium.
  • the overnight culture w as inoculated at 1% in NZ-Amine medium supplemented with 0.4% Glucose, IX M9 salts, 1 mM MgSO ⁇ and 50 ⁇ g/ml Ampicillin and the culture incubated at 37°C with shaking.
  • Iso- propyl ⁇ -D thiogalactopyranoside (IPTG) was added at 1 mM concentration when the culture attained an optical density of 0.5.
  • the culture was further incubated at 37°C with shaking for 3 hours. Cells were pelleted, solubilised in 2X Laemmili sample buffer ( 100 mM Tris-HCl, pH 6.8, 2% ⁇ -mercaptoethanol, 4% SDS, 0.2% bromophenol blue.
  • the protein was refolded while immobilized on the column to avoid the formation of misfolded aggregates. Renaturation was carried out over a period of 1.5 hours using a linear 6 M-l M urea gradient in 500 mM NaCl, 20% glycerol. Tris/HCl pH 7.4, containing phenyl-methyl-sulfonyl-fluoride (PMSF) (Sigma) as a protease inhibitor. After refolding, the bound protein was eluted by the addition of 250 mM imidazole
  • E.coli BL21(DE3) cells w ere transformed w ith pRSET C vector containing HEV RdRp coding sequence (nt 3493-5163 ) in- frame and induced with 1 mM IPTG.
  • P-lane show ing the band of purified RdRp protein.
  • the 63 kDa band is composed of -59 kDa RdRp and - 4 kDa His tag from the vector sequence.
  • Figure 12B shows the western blot of E.coli expressed recombinant HEV RdRp with patient sera and rabbit sera raised against RdRp.
  • Lanes 1 & 2 immunoblot ith patient sera of the uninduced and induced lanes of RdRp, respectively.
  • Lanes 3 & 4. immunoblot with preimmune rabbit sera of the un-induced and induced RdRp lanes. respectively.
  • Lanes 5 & 6 immunoblot with rabbit sera raised against RdRp [2] of the uninduced and induced lanes of RdRp, respectively.
  • HepG2, Cos-7 and Hep2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies Inc., USA) supplemented w ith 10% fetal bov ine serum (Life Technologies Inc., USA) in a 5% CO2 ⁇ atmosphere.
  • DMEM Dulbecco's Modified Eagle's Medium
  • HepG2 cells grown in 25 cm " flasks were washed twice with ice cold phosphate buffered saline (PBS), then harvested with a rubber policeman and pelleted by cent ⁇ fugation at
  • the cells were lysed w ith three cv cles of freezing and thawing at -80°C followed by incubation on ice, respecti ely.
  • the cell debris w as removed by centrifugation at 12,000 rpm for 10 mm at 4°C in 220.87YO1 rotor
  • Hermle 323K centrifuge Germany
  • the supernatant with a protein concentration 5- 10 ⁇ g/ ⁇ l was used as the cell lysate.
  • EMS A was standardized for protein concentration, buffei conditions leaction tempei dtui e and time, type of gel and electrophoresis conditions
  • binding assav 10 ⁇ g of whole cell extract protein or 5 ⁇ g of purified RdRp and -1 ng of [ P] UTP labelled RNA probe (-10" cpm) were incubated at 28°C foi 20 mm in a buf fei containing 10 mM Hepes (pH 7 6), 0 3 mM MgC .
  • RdRp-RNA complex in the binding mix was incubated w ith the l abbit seia laised against E coli expressed RdRp for 20 min at 30°C
  • the rabbit sei a w s eai liei v erified foi the presence of anti-RdRp antibodies by lmmunoblotting [2]
  • the RdRp- RNA complex was incubated with pre-immune sera also in a separate leaction mix to be used as a control
  • the reaction mix was then subjected to nondenatu ⁇ ng polyaciv lamide gel electrophoresis as described above
  • Quantitative estimation of binding was performed by measuring the latio of RN A probe bound to RdRp to the unbound probe under optimal binding conditions and a known RdRp concentration. This was accomplished by cutting the gel piece corresponding to the bound and unbound probe and measuring the counts by scintillation. Percent binding was determined as follows:
  • mutant [s3'(+)A n ] was deleted of nt 7084-7138 and did not form SL2. Both these mutants showed less than 5% of the binding observed with the w ild type 3' end containing poly(A) sequences. Dose response experiments further indicated that amounts of RdRp lower than the optimal level (5 ⁇ g) failed to bind to even such a low- degree to these mutants (data not shown). This clearly suggested that the integrity of the SL1 and SL2 domains, along with the presence of the poly(A) tail is necessary for binding of the viral RdRp to the 3' end of HEV genome.
  • a compensatory C ⁇ T point mutation at nucleotide 7176 in the [3'(+)M l ] background was introduced in mutant [3'(+)M2], which restored base-pairing.
  • This mutant showed a slightly increased binding efficiency of -60%.
  • the deletion mutant [3'(-)M3] lacking the interior loop showed only 13% binding, whereas the deletion mutant [3'(+)M4] lacking the hairpin loop still showed about 60% binding.
  • HEY RNA binding studies were performed with extracts prepared from liver as well as non-liv er cells.
  • the HepG2 cell line which has a human hepatoblastoma origin, was chosen as a representative liver cell and SV40-transformed monkey kidney Cos-7 and human epithelial Hep2 cells were used as representative non-liver cells.
  • Total cell extracts were allowed to bind to the m-vitro transcribed [ ⁇ " P] UTP labelled RNA probes corresponding to the 3' end with added poly(A) sequences [3'(+)A ] in the presence of an excess of tRNA to reduce non-specific binding.
  • the binding reaction was set up as described m Example 13 with 40 ⁇ g of cell extract protein and ⁇ 10 6 cpm of RNA probe.
  • the reaction mixture containing the RNA-protein complexes was UV irradiated on ice for 30 min in CL-1000 Ultraviolet Crosslinker (UVP Products limited, UK) at a distance of 5 cm from the UV source.
  • 20 ⁇ g of RNase A (Sigma. USA) and 20 U of RNase Tl (Calbiochem. USA) were added and the reaction mix was incubated at 37°C for 30 min to digest the unbound RNA completely.
  • UV-crosslinked products were boiled in 2X Laemmili sample buffer for 3 min and analysed on a SDS-12% polyacrylamide gel.
  • the 5' end labeled probe of [3'(+)A n ] was allowed to bind to RdRp as mentioned above and the complex was subjected to reaction with Pb(OAc) 2 at final concentration of 1.25. 2.5 and 5 mM at 25°C for 10 min.
  • the reaction was stopped by the addition of EDTA to a final concentration of 10 mM, extracted with w ater-saturated phenol and RNA piecipitated with ethanol
  • the samples were dissolved in 7 M urea/ 10 mM EDT A solution and loaded on the gel as desc ⁇ bed above.
  • the putative HEV RdRp sequence was aligned with the poliov irus RdRp sequence to screen for homology and the presence of "palm" domain motifs A. B. C. D and E [38].
  • the motif domains were also subjected to secondary structure predictions by the neural net method of Rost and Sander [50].
  • the sequence alignment, secondary structure predictions, structure-based sequence alignment and partial crystal structure of the poliovirus RdRp was used to build a core structure of the "palm" domain for HEY RdRp.
  • the mutations and loops were generated using Swiss PDB viewer [18] and the model was energy minimized with AMBER 5.0. [42].
  • TBSY p92, HCV NS5B, Q ⁇ replicase subunit II, poliovirus and HIV RT are shown in bold letters with asterisks, high similarity residues in underlined letters with double dots and low similarity residues are depicted in italics with single dots.
  • RdRp as is apparent from a superimposed picture of the two RdRps.
  • the core structure of polymerase palm domain is also characterized b its similarity to RNA lecognition motif (RRM) found in splicing proteins and sev eral iibosomal proteins
  • RRM RNA lecognition motif
  • Motif B also forms one of the two helices that pack beneath the four-stranded antiparallel ⁇ sheet of the polymerase core structure
  • the predicted extent of ⁇ helical region matches well with hepatitis C virus (HCV) NS5B [38]
  • Motif E ( Figure 19B) - Motif E is unique to RdRps and RTs, which forms a short ⁇ strand-turn- ⁇ strand and is positioned between the palm and thumb.
  • Ho ev er. the structure of motif E varies in different classes of polymerases and even in different crystals of HIV-RT. The hydrophobic residues in this motif seem to be important for the interaction with the palm core structure. The hydrogen bonding interactions with ⁇ strand of the thumb are thought to be crucial in proper positioning of the thumb on substrate binding.
  • the in-vitro produced viral RN.A transcripts have to mimic the virion RNA as closely as possible. This is because the v iral genome has to interact with several viral as well as cellular proteins. These interactions determine the efficiency of replication, transcription and translation.
  • the parameters, w hich affect infection by gene transfer are: heterogeneity of the transcript population, presence of point mutation and the sequences at 5' and 3 ' ends i.e. number of non-v iral nucleotides. presence of a cap structure at the 5' end and poly A tail at the 3 ' end.
  • the problem of heterogeneity in transcript population is mainly due to poor fidelity of RNA polymerase [1, 15]. As a result it may hamper the infectivity of the transcripts [5].
  • the HEV transcript used in the studies according to the present invention has 12 non- viral nucleotides at the 5' end in addition to the complete viral genome (Genbank Accession No. AF076239). These additional nucleotides in the transcript at the 5' (12 nucleotides) and 3' (1 nucleotide) ends did not abolish its competence for replication as observed in these studies. In recent studies by- other researchers, the presence of cap structure in HEV genome has been described [24]. However, the present invention demonstrates replication of HEV RNA w ithout a cap structure. Therefore, it may be presumed that the presence of cap structure is not obligatory for replication of HEV genome.
  • the negative strand of viral RNA usually serves as the replicative intermediate in most of the positive stranded RNA viruses. Such a species was demonstrated for HEV in the transfected HepG2 cells, indicating active viral replication.
  • the anti-sense strand was found to be in a lower amount than the sense strand like in other positive stranded virus systems [53]. While the sense strand was detected up to 10 " dilution of the template
  • RNA the anti-sense strand was detected upto a dilution of 10 " . This is possibly because anti-sense pregenome tends to get converted into sense strand faster. In addition some of the RN . A used for transfection may persist ev en after thorough washing and may lead to very high level of sense strand detected. In most of the positive stranded RNA v iruses, the positiv e and negative strands are synthesized in unequimolar ratio, i.e. the positi e strand is produced in excess of the negative strand [53].
  • the transfected viral genome was not only capable of replication but also expressed viral proteins in the transfected cells and released infectious v irus into the culture supernatant as evaluated by experimental infection into a Rhesus monkey Nearly
  • the metabohcally labelled viral proteins were immunoprecipitated from transfected cells using their respective antibodies derived from both structural and putativ e non-structural regions.
  • the components of non-structural polyprotein identified from the predicted homology to putative methyl transferase, helicase and RdRp domains w ere immunoprecipitated separately from the transfected cells.
  • the putative RdRp w as detected up to 36 hours of transfection. This is possible because it is an early protein and undergoes rapid degradation. Therefore, no signal corresponding to RdRp could be detected at 72 and 96 hours. However, the other proteins were detected at 72 hours post transfection. which include putative methyl transferase and helicase. This indicates that the protein product from the ORFl region undergoes processing.
  • the other viral proteins either directly or indirectly through cell dependent mechanisms may activate proteases responsible for such processing.
  • a putative protease domain has been identified in the ORFl gene based on sequence comparison [28], However this has not been characte ⁇ zed yet. The possibility of the viral protease needing activation cannot be ruled out.
  • the ORF3 protein is a phosphoprotein that binds to src homology domain III. It is phosphorylated by mitogen activated protein (M.AP) kinase. Therefore, this possibly can play a role in protein phosphorylation [64]. Whether this protein alters the activity of any cellular or viral protease to initiate the polyprotein processing needs further investigation.
  • alfalfa mosaic virus [17]
  • TCV turnip crinckle virus
  • human rhinovirus [57]
  • encephalomyocarditis virus [14]
  • AAMV alfalfa mosaic virus
  • TCV turnip crinckle virus
  • encephalomyocarditis virus [14]
  • AAMV alfalfa mosaic virus
  • TCV turnip crinckle virus
  • human rhinovirus human rhinovirus
  • encephalomyocarditis virus [14]
  • RNA dependent RNA polymerase enzyme RNA dependent RNA polymerase enzyme
  • hepatitis A virus RNA preumed to form a pseudoknot structure [31 ].
  • Sindbis virus [30].
  • west nile virus [4] and conserved 11 nucleotide sequence at the 3" end of coronavirus mouse hepatitis [63] bind to their respective host cell proteins.
  • the host cell proteins can also interact with the viral RNA replication complex, as in brome mosaic virus [45].
  • RNA genomes has not been clarified so far. Most such proteins have been found to be associated with cellular RNA-processing pathways or translation machinery [33]. Some of these cell proteins may aid in replication of the viral genome in conjunction w ith the RdRp-replicase complex.
  • the two major polypeptides interacting with the 3' end of HEV RNA genome were identified as doublets of -45 kDa and -95-105 kDa and 3-4 minor species represented by faint bands.
  • the 45 kDa protein might be an important specificit determining factor, as interaction with such a protein is absent with the Cos7 cell extract. Howev er, the Cos7 cell extract showed crosslinking of a smaller polypeptide.
  • w hich might suggest that this protein(s) is somewhat smaller in monkey cells compared to human cells, or in kidney cells as compared to liver cells. Proteins with similar molecular weights have been identified in other systems.
  • a host protein of 45 kDa bound to the RdRp was determined to be an analog of eIF-3 subunit p41 [45].
  • the 3' end of hepatitis A RNA is also known to bind to a 45 kDa host protein [31].
  • a 103 kDa cellular protein is reported to bind to the 3' end of mouse hepatitis virus [63] and another 105 kDa protein binds to sense and anti-sense strands of 3' non coding region of west nile virus [4].
  • the 3' end of rubella virus negative strand RNA binds to 97 kDa cellular protein whose intensity is increased in infected cells [36]
  • the doublet signals observed in the UV-crosslinking assays might be either due to RNA binding to different proteins of slightly different molecular weights, differentially modified forms of the same protein (e.g. glycosylated and the un-glycosylated forms) or due to differential digestion of the RNA after UV-crosslinking. Difference in the intensities of the detected bands do not necessarily exemplify the differences in the binding capabilities, but could also be due to the difference in their respective amounts in the cell extract or due to difference in the UV-crosslinking abilities.
  • HEV RdRp HEV RdRp structural analysis comparisons with other RNA polymerases reveal the presence of conserved structured domains with discrete functions. Cellular proteins also form complexes at SLl and SL2 sequence regions and the interaction is independent of the presence of poly(A) stretch.
  • T7 RNA polymeiase promotei sequence (TGTAATACGACTCACTATAGG ) 7084 and 7139 are the nucleotide positions in the HEV genome at which the foi w aid pi unei s begin REFERENCES
  • RNA-protein interactions involvement of NS3, NS5 and 3' noncoding regions of Japanese encephalitis virus genomic RNA. J. Virol. 71:3466-3473.
  • HEV hepatitis E virus
  • RNA transcripts from Sindbis virus cDNA clones mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 61 :3809-19.
  • HEV Hepatitis E virus
  • ORF3 protein of HEV is a phosphoprotein that associates with cytoskeleton. .1. Virol.
  • VYGVSPGLVHNLIGMLQAVA DGKAHFTESVKPVLDLTNSILCRVE.
  • NQGWRSVETSGVAEE EATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFAL

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Abstract

La présente invention concerne un clone d'acide nucléique génétiquement modifié du virus de l'hépatite E (VHE) qui comprend les gènes structuraux (ORF2 et ORF3) et non structuraux (ORF1) complets, c'est à dire la séquence codante complète, du VHE, flanquée par les régions non codantes 5` et 3` avec une queue polyadénylée 3`dans la région non codante 3`, le clone précité étant capable d'infecter des cellules hépatiques en culture et de répliquer, transcrire et assembler le VHE qui s'y trouve. L'invention se rapporte également à un procédé permettant de synthétiser ce clone et à l'expression par ce dernier de protéines virales aptes à être utilisées pour détecter la présence d'anticorps dirigés contre le VHE dans des prélèvements biologiques. Le clone et les protéines virales exprimées de l'invention peuvent également être utilisés dans une trousse d'essai comprenant les protéines virales précitées, qui permet de diagnostiquer la présence d'une infection par VHE, dans un vaccin anti-VHE qui comprend des particules non infectieuses de VHE produites à partir d'une mutation du clone de l'acide nucléique du virus de l'hépatite E précité, et dans un système d'analyse qui permet de tester rapidement un médicament anti-VHE.
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US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003063679A2 (fr) * 2001-11-09 2003-08-07 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Clones du virus de l'hepatite e et leurs procedes d'utilisation
WO2003063679A3 (fr) * 2001-11-09 2003-12-04 Us Gov Health & Human Serv Clones du virus de l'hepatite e et leurs procedes d'utilisation
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

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